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WORKS  OF 
PROFESSOR  J.  A.  MANDEL 

PUBLISHED   BY 

JOHN  WILEY  &  SONS,  INC. 


TRANSLATIONS 
A  Text-book  of  Physiological  Chemistry. 

By  Olof  Hammarsten,  Emeritus  Professor  of  Medi- 
cal and  Physiological  Chemistry  in  the  University 
of  Upsala,  with  the  Collaboration  of  S.  C.  Hedin, 
Professor  of  Medical  and  Physiological  Chemistry 
in  the  University  of  Upsala.  Authorized  translation 
from  the  Author's  enlarged  and  revised  8th  German 
edition,  by  John  A.  Mandel,  Sc.D.,  Professor  of 
Chemistry  in  the  New  York  University  and  Bellevue 
Hospital  Medical  College.  viii+1026  pages.  6  by 
9.      Cloth,  $4.00  net. 

A  Compendium  of  Chemistry,    Including  General, 
Inorganic,  and  Organic  Chemistry. 

By  Dr.  Carl  Arnold,  Professor  of  Chemistry  in  the 
Royal  Veterinary  School  of  Hannover.  Authorized 
translation  from  the  eleventh  enlarged  and  revised 
German  edition,  by  John  A.  Mandel,  Sc.D.  xii  + 
627  pages.     51  by  8.     Cloth,  $3.00  net. 


A  TEXT-BOOK 


OF 


PHYSIOLOGICAL  CHEMISTS! 


BY 

OLOF    HAMMARSTEN 

EMERITUS    PROFESSOR    OP    MEDICAL    AND    PHYSIOLOGICAL    CHEMISTRY~IN    THE   UNIVERSITY    OP    UPSALA 


WITH  THE  COLLABORATION  OF 

S.  G.  HEDIN 

PROPE890R   OP    MEDICAL    AND    PHYSIOLOGICAL    CHEMISTRY    IN    THE    UNIVERSITY    OP    UPSALA 


JVutljoriseb  translation 

FROM  THE  A  UTHOR'S  ENLARGED  AND  REVISED 
EIGHTH  GERMAN  EDITION 

BY 

JOHN  A.  MANDEL,  ScD. 

PBOPE8SOR    OP    CHEMISTRY    IN    THE    NEW    YORK    UNIVERSITY    AND    BELLEVUE  HOSPITAL  MEDICAL  COLLSGX 


SEVENTH  EDITION 
TOTAL    ISSUE,    THIRTEEN"    THOUSAND 


NEW  YORK 

JOHN  WILEY  &  SONS,  Inc. 

London:  CHAPMAN  &  HALL,  Limited 


Copyright.  1893.  1898,  1900,  1904,  1908,  1911,  1914, 

BY 

JOHN  A.   MANDEL 


¥¥ 


\3\A- 


PRISB    or 

BRAUNWORTH    l     CO. 

BOOK    MANUFACTURER* 

BROOKLYN,     N.     V. 


PREFACE   TO   THE   EIGHTH   GERMAN   EDITION 


The  revision  of  this  edition  has  been  accomplished  with  the  collabora- 
tion of  Professor  S.  G.  Hedin,  and  the  work  has  been  divided  so  that 
Hedin  has  revised  Chapters  I,  III,  VIII,  XII  and  XVI,  besides  the  Index 
of  Authors,  while  Hammarsten  has  revised  Chapters  II,  IV,  V,  VI,  VII, 
IX,  X,  XI,  XIII,  XIV,  XV,  and  XVII,  besides  the  General  Index. 
The  numerous  recent  developments  in  physiological  chemistry  have  made  a 
thorough  revision  and  reconstruction  necessary  in  all  the  chapters,  and  in 
order  to  prevent  a  noticeable  increase  in  the  size  of  the  work,  it  was  also 
necessary  to  change  the  arrangement  of  the  foot-notes  more  economically 
The  number  of  chapters  in  this  edition  is  XVII  instead  of  XVIII  as  in  the 
seventh  edition,  because  for  several  reasons  it  was  found  advisable  to 
combine  the  first  two  chapters  of  the  seventh  edition  into  one  chapter,  and 
at  the  same  time  certain  parts  of  the  first  chapter  have  been  incorporated 
into  other  chapters,  thus  for  example  the  oxidation  processes  have  been 
introduced  into  Chapter  XVI  (on  respiration  and  oxidation).  In  general 
the  plan  of  the  work  remains  unchanged. 

Olof  Hammarsten. 

Upsala,  September,  1913. 

iii 


TRANSLATOR'S   PREFACE   TO   THE   SEVENTH 
AMERICAN   EDITION 


Workers  in  Biochemistry  are  to  be  congratulated  on  the  appearance 
of  a  new  edition  of  Hammarsten's  "  Physiologischen  Chemie."  At  this 
time,  when  so  many  new  and  important  biochemical  facts  are  being 
published  and  when  so  many  older  theories  and  deductions  are  found 
more  or  less  erroneous,  due  to  recent  investigations  using,  new  methods, 
it  is  very  fortunate  that  we  have  this  complete  and  critical  compilation 
from  the  master  hand  of  Professor  Hammarsten,  now  in  his  73d  year. 
We  all  owe  him  a  great  debt  of  gratitude  for  his  painstaking  work  for  so 
many  years. 

I  take  great  pleasure  in  expressing  my  indebtedness  to  my  assistant, 
Dr.  A.  O.  Gettler,  for  the  help  he  has  given  me  in  revising  the  proof  and  for 
making  the  Indexes. 

John  A.  Mandel. 
New  York,  June,  1914. 

v 


CONTENTS 


CHAPTER   I 

PAGE 

General  and  Physico-chemical 1 

CHAPTER   II 
The  Proteins 77 

CHAPTER   III 
The  Carbohydrates 196 

CHAPTER  IV 
Animal  Fats  and  Phosphatides 232 

CHAPTER  V 
The  Blood 250 

CHAPTER  VI 
Chyle,  Lymph,  Transudates  and  Exudates 345 

CHAPTER  VII 
The  Liver 381 

CHAPTER  VIII 

PlGESTION , 451 

vii 


Tin  CONTENTS. 


CHAPTER  IX 

PAGE 

Tissues  of  the  Connective  Substance 514 


CHAPTER  X 
The   Muscles 565 

CHAPTER  XI 
Brain  and  Nerves 604 

CHAPTER  XII 
Organs  of  Generation 620 

CHAPTER  XIII 
The  Milk 643 

CHAPTER  XIV 
The  Urine 672 

CHAPTER  XV 
The  Skin  and  its  Secretions 837 

CHAPTER  XVI 
Respiration  and  Oxidation 850 

CHAPTER  XVII 
Metabolism 878 

Index  to  Authors 945 

General  Index 983 


PHYSIOLOGICAL  CHEMISTRY. 


CHAPTER   I. 
GENERAL  AND   PHYSICO-CHEMICAL. 

I.     OSMOTIC   PRESSURE. 

When  certain  substances  are  placed  in  contact  with  water  they 
dissolve  therein  and  finally  a  liquid  is  obtained  which  contains  an  equal 
quantity  of  the  dissolved  substance  in  each  unit  volume.  There  exists 
between  the  water  and  the  soluble  body  a  certain  attractive  force.  Upon 
this  force  depends  also  the  so-called  diffusion,  which  manifests  itself  when 
two  different  solutions  of  the  same  or  different  substances  are  brought 
into  immediate  contact  with  each  other.  The  dissolved  molecules  and 
the  water  intermingle  with  each  other  so  that  finally  the  dissolved 
bodies  are  equally  divided  in  the  entire  quantity  of  water.  Imagine 
a  cane-sugar  solution  in  contact  with  pure  water;  the  equilibrium  or  the 
homogeneity  of  the  system  can  then  be  brought  about  in  two  ways; 
namely,  the  sugar  molecule  can  migrate  in  part  into  the  water,  and  sec- 
ondly, the  water  can  pass  into  the  solution.  If  the  two  fluids  at  the 
beginning  are  in  immediate  contact  with  each  other  then  the  two  proc- 
esses take  place  simultaneously. 

The  conditions  change  when  the  two  liquids  are  separated  from 
each  other  by  a  membrane,  which  allows  of  the  passage  of  water  but 
not  of  the  dissolved  substance  (in  this  case  cane-sugar) .  In  the  presence 
of  such  a  so-called  semipermeable  membrane  the  equilibrium  can  only 
be  established  by  the  water  passing  into  the  cane-sugar  solution.  Semi- 
permeable membranes  have  been  artificially  prepared,  and  they  also 
occur  in  nature,  or  conditions  exist  which  give  results  like  those  of  the 
membranes.  To  the  first  group  belong  Tratjbe's  so-called  precipitation 
membranes.1  Such  a  membrane,  for  example  can  be  produced  by  care- 
fully dropping  a  concentrated  solution  of  copper   sulphate  into  a  dilute 

1  Arch.  f.  (Anat.  u.)  Physiol.,  1867,  pages  87  and  129. 


2  GENERAL  AND  PHYSICO-CHEMICAL. 

solution  of  potassium  ferrocyanide.  Thereby  the  drop  of  copper  sulphate 
is  surrounded  by  a  membrane  of  copper  ferrocyanide,  which  is  imper- 
vious to  copper  sulphate  as  well  as  to  potassium  ferrocyanide,  but  allows 
water  to  pass.  The  drops  retain  their  blue  color  in  the  yellow  solution 
but  increase  in  volume,  due  to  the  taking  up  of  water,  until  the  tension  of 
the  membrane  prevents  the  further  increase  in  size.  If  the  difference  in 
concentration  of  the  two  solutions  is  great  enough,  the  membrane  is 
ruptured  by  the  pressure. 

In  order  to  give  the  copper-ferrocyanide  membrane  a  greater  rigidity, 
Pfeffer  has  suggested  forming  the  precipitate  on  a  porous,  rigid  wall.1 
For  this  purpose  he  makes  use  of  a  small,  porous  earthenware  cell  which, 
after  careful  cleaning,  is  treated  with  copper  sulphate  and  potassium 
ferrocyanide  so  that  the  membrane  is  precipitated  on  the  inner  wall 
of  the  cell.  The  membrane  thus  obtained  is  impervious  to  the  cane- 
sugar.  If  the  cell  is  filled  with  a  cane-sugar  solution  and  then  placed 
in  pure  water,  no  sugar  leaves  the  cell,  while  water  passes  into  the  cell, 
and  this  continues  until  the  opposite  pressure  produced  prevents  the 
further  passage  of  water.  If  the  cell  is  completely  closed  and  in.  con- 
nection with  a  manometer,  then  on  the  establishment  of  an  equilibrium 
the  manometer  indicates  the  force  with  which  the  inclosed  solution 
attracts  water. 

As  the  sugar  is  attracted  with  the  same  force  by  the  water  as  the  water 
is  by  the  sugar  and  also  as  the  sugar  cannot  pass  through  the  membrane 
therefore  the  sugar  exerts  a  pressure  upon  the  membrane  equal  to  the 
pressure  indicated  by  the  manometer.  This  pressure  is  called  the 
osmotic  pressure  of  the  enclosed  solution.  For  dilute  cane-sugar  solutions 
Pfeffer's  determinations  show  that  the  osmotic  pressure  is  approx- 
imately proportional  to  the  concentration  and  slowly  rises  with  the 
temperature. 

Experiments  with  other  semipermeable  membranes  have  also  been 
carried  out  by  de  Vries,  and  these  will  be  discussed  on  page  5.  De 
Vries'  experiments  have  led  to  the  following  result:  Solutions  of  analo- 
gously constructed  bodies  having  the  same  molecular  concentration  give  the 
same  osmotic  pressure. 

Van't  Hoff  first  called  attention  to  the  analogy  which  exists  between 
the  laws  of  osmotic  pressure  of  a  dissolved  substance  and  of  gases,2 
namely,  that  the  osmotic  pressure  is  proportional  (or  inversely  propor- 
tional to  the  volume  of  the  solution)  to  the  concentration,  and  corre- 
sponds completely  with  Boyle-Mariotte's  law  on  the  relation  between 
the  volume  and   pressure  of  gases.     Also,  that  equimolecular  solutions 


1  Osmotische  Untersuchungen,  Leipzig,  1877. 

2  Zeitschr.  f.  physik.  Chem.,  1,  481  (1887). 


OSMOTIC  PRESSURE.  3 

have  the  same  osmotic  pressure,  corresponds  to  Avogadro's  law,  that 
equal  volumes  of  different  gases  under  the  same  pressure  contain  the 
same  number  of  molecules. 

From  Pfeffer's  results  of  the  osmotic  pressure  of  cane-sugar  solu- 
tions van't  Hoff  has  calculated  that  it  is  the  same  as  the  pressure  exerted 
by  any  gas  of  the  same  molecular  concentration  and  temperature.  In 
general  the  following  is  true: 

Dissolved  bodies  exert  in  solution  the  same  osmotic  pressure  they  would 
exert  if  they  were  gases  at  the  same  temperature  and  in  equal  volume. 

Recently  Morse,  Frazer  and  collaborators  have  brilliantly  substan- 
tiated the  theory  of  van't  Hoff  for  solutions  of  cane-sugar  and  glucose, 
by  making  use  of  Pfeffer's  method  but  using  a  very  refined  technique.1 

From  what  has  been  given,  the  osmotic  pressure  of  a  solution,  sepa- 
rated from  the  surrounding  pure  solvent  by  a  semipermeable  membrane, 
exerts  its  effects  in  two  ways.  First  the  pure  solvent  tries  to  enter 
the  solution  and  secondly  the  dissolved  substance  presses  upon  the 
membrane  with  a  force  equal  to  the  gas  pressure.  According  to  whether 
we  consider  either  one  or  the  other  of  these  ways,  the  osmotic  pressure 
of  a  solution  can  be  considered  as  its  ability  to  attract  the  solvent,  or  as 
a  pressure  directed  toward  the  outside.  This  last  conception  seems  prob- 
ably for  the  present  to  be  the  most  acceptable,  nevertheless,  the  fact  that 
the  pure  solvent  enters  through  the  unmovable  semipermeable  membrane 
(as  in  Pfeffer's  experiments)  is  difficult  of  reconciliation  with  this  mode 
of  explanation.  Obviously,  and  for  physiological  purposes,  it  seems  best 
to  make  use  of  the  former  explanation,  in  which  the  osmotic  pressure 
is  considered  as  a  measure  of  the  force  with  which  a  solution  attracts 
the  solvent. 

Pfeffer's  above-described  method  of  directly  determining  the 
pressure  can  only  be  used  in  exceptional  cases,  first  because  the  prepara- 
tion of  the  semipermeable  membrane  is  connected  with  difficulties,  and 
second,  because  there  are  only  a  few  crystalline  bodies  for  which  imper- 
meable membranes  have  been  found.  There  are  other  quicker  and  easier 
ways  of  determining  the  osmotic  pressure. 

Solutions  of  non-volatile  substances  boil  at  a  higher  temperature 
than  the  pure  solvent.  This  is  due  to  the  fact  that  the  dissolved  sub- 
stance, because  of  the  osmotic  pressure,  holds  on  to  the  solvent  with 
a  certain  force.  As  in  boiling  a  part  of  the  solvent  is  separated  from 
the  dissolved  body,  and  as  the  osmotic  pressure  can  be  considered  as  a 
measure  of  the  attractive  power  between  the  solvent  and  the  dissolved 
substance,  then  it  is  clear  that  solutions  which  are  prepared  with  the 
same  solvent  and  have  the  same  osmotic  pressure  (isosmotic  solutions) 

1  Amer.  Chem.  Journ.,  37,  425,  558  (1907);  41,  1,  257  (1909). 


4  GENERAL  AND  PHYSICO-CHEMICAL. 

must  also  boil  at  the  same  temperature.  The  rise  in  the  boiling-point 
of  a  solution  above  the  boiling-point  of  the  solvent  (elevation  of  the 
boiling-point)  is  also,  like  the  osmotic  pressure,  for  dilute  solutions  pro- 
portional to  the  concentration. 

Solutions  have  a  lower  freezing-point  than  the  pure  solvent,  and  as 
in  dilute  solutions  the  solvent  can  be  frozen  out  from  the  dissolved  body, 
then  isosmotic  solutions  have  the  same  freezing-point.  The  depres- 
sion of  the  freezing-point  is  also  proportional  to  the  concentration. 

The  determination  of  the  elevation  of  the  boiling-point  for  the  esti- 
mation of  the  osmotic  pressure  of  animal  fluids  is  applicable  only  in 
exceptional  cases,  because  on  heating,  precipitates  often  form.  The 
determination  of  the  depression  of  the  freezing-point  has  been  found  of 
much  greater  use.  This  can  be  accomplished  in  an  easy  manner  by  aid  of 
the  apparatus  suggested  by  Beckmann.  In  regard  to  the  use  of  this 
method  we  must  refer  to  more  complete  works.1 

The  above  rule  that  equimolecular  solutions  of  different  bodies  have 
the  same  osmotic  pressure  is  only  applicable  to  non-electrolytes.  The 
electrolytes  (bases,  acids,  salts)  show  in  aqueous  solution  a  much  greater 
pressure  (i.e.,  a  much  lower  depression  of  the  freezing-point)  than  equi- 
molecular solutions  of  non-electrolytes.  As  is  known,  Arrhenius  has 
explained  this  lack  of  correspondence  by  the  assumption  that  the  mole- 
cule of  the  electrolyte  is  divided  or  dissociated  into  so-called  ions  hav- 
ing an  opposed  electric  charge.  An  ion  exerts  upon  the  osmotic  pressure 
the  same  influence  as  the  non-dissociated  molecule.  The  larger  the 
number  of  dissociated  molecules  the  more  does  the  osmotic  pressure 
of  the  solution  rise  above  the  pressure  of  an  equimolecular  solution  of  a 
non-dissociated  body.  The  osmotic  action  of  a  dissociated  body  is  equal  to 
that  of  a  non-dissociated  body  which  in  a  given  volume  contains  as  many 
molecules  as  the  dissociated  body  contains  ions  plus  non-dissociated  mole- 
cules. If  we  assume  that  a  is  the  degree  of  dissociation,  i.e.,  the  number  of 
the  molecules  that  are  dissociated,  then  1— a  is  the  number  that  is  not 
dissociated.  If  in  the  dissociation  of  a  molecule  n  ions  are  formed 
then  the  relation  of  the  molecules  present  before  the  dissociation  to  the 
ions  +  molecules  present  after  the  dissociation  is  1:(1— a-\-na)  or 
=  l:(l-r-[n  —  l]a).  The  expression  (l+[n  — l]a)  is  generally  denoted  by 
the  letter  i,  and  can  be  directly  determined  by  estimating  the  freezing- 
point  of  a  solution  of  known  molecular  concentration. 

A  gram-molecule  aqueous  solution  (one  that  contains  as  many  grams  per 
liter  as  the  molecular  weight  of  the  substance)  of  any  non-electrolyte  freezes 
at  about  - 1.86°,  or,  the  depression  of  the  freezing-point  A  is  =1.86°.     For  example, 

1  Ostwald-Luther,  Hand-  und  Hilfsbuch  zur  Ausfuhrung  physik.-chemischer 
Messung,  3  Aufl.,  1910. 


OSMOTIC  PRESSURE.  5 

if  we  find  that  A  for  a  gram  molecular  solution  of  NaCl  is  3.40°  then  we  have 
according  to  the  above  1  :  (l+[n  — l]o)  =1.86  :  3.40.  In  the  dissociation  of 
NaCl  two  ions  are  formed,  therefore  n  =  2,  and  from  the  above  equation  the  degree 
of  dissociation  can  be  calculated,  a  =0.83.  The  degree  of  dissociation  can  also 
be  calculated  from  the  electrical  conductivity.  Only  the  ions  take  part  in  the  con- 
duction of  electricity,  and  the  molecular  conductivity  (  = — i i —     — — : : — } 

*   V     molecular  concent  nit  ion/ 

is  proportional  to  the  degree  of  dissociation.  The  dissociation  increases  with  the 
dilution  and  at  infinite  dilution  all  molecules  are  dissociated  (a  =  l).  If  we  desig- 
nate with  n*>  the  limit  value  which  the  molecular  conductivity  approaches  in 
infinite  dilution  and  with  y.v  the  molecular  conductivity  at  some  definite  dilution 

v,  then  the  degree  of  dissociation  at  this  dilution  is  a  =  — . 

The  positively  charged  ions  are  called  cations,  and  the  negatively 
charged  ones  anions.  Common  for  all  acids  are  the  positively  charged 
H-ions  while  the  negatively  charged  OH-ions  are  common  for  all  bases. 

Osmotic  Experiments  with  Plant  Cells.  We  often  meet  the 
word  osmosis  in  literature  without  understanding  exactly  what  is  meant 
thereby.  As  a  rule  diffusion  streams  are  meant,  w-hich  are  modified 
by  means  of  the  permeability  conditions  of  an  inclosing  membrane. 
We  now  know  that  the  driving  force,  namely,  the  streaming,  is  brought 
about  by  the  differences  in  concentration,  i.e.,  by  difference  in  the  osmotic 
pressure  on  the  two  sides  of  the  membrane. 

After  Nageli  found  that  certain  plant  cells,  when  they  were  treated 
with  a  sufficiently  concentrated  solution  of  certain  substances,  changed 
their  appearance  so  that  the  protoplasm  retracted,1  de  Vries  studied 
this  phenomenon  further.2  He  called  it  plasmolysis.  The  most  important 
substances  for  bringing  about  plasmolysis  are  the  salts'  of  the  alkalies  and 
alkaline  earths,  varieties  of  sugars,  polyatomic  alcohols,  and  neutral  amino- 
acids.  An  indispensable  condition  for  bringing  about  plasmolysis  is  that 
the  solution  must  not  have  any  destructive  action  upon  the  cells.  Xageli 
gave  the  correct  interpretation  of  plasmolysis,  which  is  that  those  bodies 
which  plasmolyze  plant  cells  pass  through  the  cell  membrane  of  the  cell,  but 
not  through  the  protoplasmic  layer  which  follows.  Instead  of  this  the  sub- 
stance attracts  water  from  the  inner  parts  of  the  cell.  The  cell  contents 
surrounded  by  protoplasm  therefore  diminish  in  volume  and  the  protoplasm 
recedes  more  or  less  from  the  cell  membrane.  From  this  it  follows  that 
only  those  solutions  whose  power  of  attracting  water  is  greater  than  that 
of  the  cell  contents  can  bring  about  plasmolysis.  As  the  ability  to  attract 
water  (or  the  osmotic  pressure)  increases  with  concentration,  there  must 
be  a  limit  solution  for  every  substance  above  which  all  higher  concentra- 
tions plasmolyze.     The  limit  solution  is  called  isotonic  with  the  cells; 


1  Pflanzenphysiol.  Untersuch.,  1855. 

2Eine  Analyse  der  Turgorkraft,  Jahresber.  f.  Wissensch.    Botanik,  14,  427  (1884). 


6  GENEKAL  AND  PHYSICO-CHEMICAL. 

weaker  solutions  are  called  hypotonic,  and  stronger  hypertonic.  De 
Vries,  with  the  aid  of  equal  cells  (cells  of  the  epidermis  of  the  lower 
side  of  the  leaf  of  the  Tradescantia  discolor)  has,  for  various  substances, 
determined  the  concentration  of  this  limit  solution.  It  was  found  that 
the  limit  solution  of  analogously  constructed  salts  had  the  same  molec- 
ular concentration.  Thus  the  alkali  salts  of  the  type  NaCl  (haloid 
salts,  nitrate,  acetate)  plasm olyzed  at  one  molecular  concentration  and 
the  salts  of  the  type  Na2SC>4  (sulphate,  oxalate,  diphosphate,  tartrate) 
at  another  concentration.  If  the  plasmolyzing  power  of  a  molecule 
of  the  first  group  is  equal  to  3,  then  the  molecule  of  the  second  group 
equals  4.  The  concentration  of  the  limit  solution  varied  in  de  Vries' 
experiments  between  the  limits  corresponding  to  a  NaCl  solution  of 
0.6-1.3  per  cent. 

As  above  mentioned,  only  those  substances  bring  about  plasmolysis 
which  cannot  themselves  pass  through  the  protoplasm  envelope  of  the 
cell  content,  and  these  substances  only  in  the  case  that  the  concentration 
is  sufficient.  If  a  body  is  taken  up  by  the  protoplasm  it  produces  no 
plasmolysis,  because  its  tendency  to  attract  water  has  been  satisfied 
by  its  own  passage  into  the  cell.  These  substances  do  not  produce 
plasmolysis  in  any  concentration.  If  a  body  slowly  passes  in,  then 
at  first  it  causes  plasmolysis,  but  this  then  ceases  later.  The  plasmolytic 
methods  have  been  used  by  de  Vries,  and  especially  by  Overton.1 

Experiments  with  Blood  Corpuscles.  Over  a  hundred  years  ago 
Hewson  observed  that  the  blood  corpuscles  were  destroyed  in  water, 
and  that  salts  in  certain  concentrations  prevented  destruction.2  Ham- 
burger3 has  carefully  and  systematically  investigated  the  action  of 
salts  of  the  alkalies  and  alkaline  earths,  and  concludes  that  when  blood 
is  mixed  with  certain  volumes  of  solutions  of  different  concentrations 
of  the  same  salt,  all  solutions  whose  concentration  lie  below  a  certain 
limit  cause  the  exudation  of  haemoglobin.  On  comparing  the  molec- 
ular concentration  of  the  limit  solution  of  different  salts  it  was  found 
that  they  bore  the  same  relation  to  each  other  as  the  relative  figures 
found  by  de  Vries  for  the  molecular  concentration  of  the  plasmolytic 
salt  solutions.  From  this  it  probably  follows  that  the  protective  action 
of  the  salts  upon  the  blood  corpuscles  depends  upon  the  same  reason 
as  the  plasmolysis.  This  conclusion  is  also  supported  by  the  fact  that 
those  substances  which,  according  to  de  Vries,  in  proper  concentration 
cause  plasmolysis  in  living  plant  cells,  can  also  under  similar  conditions 
prevent  the  exudation  of  haemoglobin.     Those  bodies,  on  the  contrary, 


"  Vierteljahwchr.  d.  Naturf.  Gesellsch.  zu  Zurich,  40,  1  (1895);  41,  383  (1896). 

'Phil.  Trans.,  1773,  p.  303. 

1  Arch.  f.  (Anat.  u.)  Physiol.,  1888,  p.  31;  Zeitschr.  f.  Biol.,  26,  414,  (1889). 


OSMOTIC  PRESSURE.  7 

which  do  not  cause  plasmolysis,  act  in  aqueous  solution  in  the  same 
manner  upon  the  blood  corpuscles  as  pure  water.  This  has  been  espe- 
cially shown  by  the  investigations  of  Gryns.1 

Different  investigators  have  attempted  to  perform  plasmoh  tic 
experiments  with  animal  cells,  but  without  any  special  result.  With 
the  microscope  one  can  often  observe  that  the  red  blood  corpuscles 
shrink  under  the  influence  of  a  strong  salt  solution,  but  the  limit  solu- 
tion when  the  shrinking  just  begins  cannot  be  exactly  determined  because 
the  changes  in  volume  are  so  very  small.  If  we  summate  the  changes 
in  volume  of  many  corpuscles,  which  can  be  done  by  centrifuging  the 
blood  mixture  in  a  graduated  tube,  then  very  small  changes  can  be  detected. 
Such  determinations  have  been  made  by  Hedin,2  Koeppe  3  and  others. 
It  was  found  that  the  blood  corpuscles  swell  in  a  weak  salt  solution, 
shrink  in  a  stronger  solution,  and  there  is  a  certain  concentration  which 
does  not  change  the  volume.  By  determining  the  freezkig-point  Hedin 
found  that  this  concentration  for  NaCl  was  nearly  isosmotic  with  the 
serum  of  the  blood  corpuscles  used.  The  depression  of  the  freezing- 
point  was  about  0.56°  and  the  concentration  of  the  NaCl  solution  is 
0.9  per  cent,  or  about  0.15  normal. 

The  question  as  to  the  permeability  of  the  blood  corpuscles  has  been 
investigated  by  Hedin,  using  a  method  depending  upon  the  following:4 

The  depression  of  the  freezing-point  of  a  solution  is  proportional  to  its  con- 
centration. A  certain  amount  of  the  substance  to  be  tested  is  dissolved  in  blood. 
The  serum  of  this  treated  blood  freezes  at  a  lower  temperature  than  before  the 
salt  was  added.  The  depression  of  the  freezing-point  can  be  designated  as  a. 
Now  the  same  amount  of  substance  is  dissolved  in  serum  using  the  same  volume 
of  serum  as  blood  was  previously  used.  The  depression  of  the  freezing-point 
of  this  serum  can  be  designated  as  b.  From  this  it  is  evident  that  a  =6  if 
the  blood  corpuscles  take  up  just  as  much  dissolved  substance  from  the  blood 
as  an  equal  volume  of  serum.     If  the  blood  corpuscles  take  up  less  than  the  serum 

then  a>  b  or-r >  1 ,  and  when  they  take  up  more  than  the  serum  then  a  <b  or-?  <  1. 

The  result  r,  in  the  calculation  of  which  the  change  taking  place  in  the  volume 

of  the  blood  corpuscles  on  the  addition  of  the  substance  must  be  considered, 
gives  immediately  an  approximate  idea  of  the  quantity  of  substance  which  has 
passed  into  the  blood  corpuscles. 

The  results  were  as  follows: 

The  salts  of  the  fixed  alkalies  and  alkaline  earths,  neutral  amino- 
acids,  varieties  of  sugars  as  well  as  hexatomic  and  pentatomic  alcohols 
pass  into  the  blood  corpuscles  only  to  a  slight  degree.     Erythrite  (tetra- 

1  Pfluger's  Arch.,  63,  86  (1896). 

2Skand.  Arch.  f.  Physiol.,  5,  207,  238.  377  (1895). 

3  Arch.  f.  (Anat.  u.)  Physiol.,  1895,  154. 

4  Pfluger's  Arch.,  68,  229  (1897);  70,  525  (1898). 


8  GENERAL  AND  PHYSICO-CHEMICAL. 

tomic  alcohol),  passes  slowly,  and  glycerin  (triatomic)  also  passes  slowly, 
but  faster  than  erythrite.  Ethylene  glycol  (diatomic  alcohol)  passes  rather 
rapidly,  and  the  monatomic  alcohols  immediately  divide  themselves 
equally  in  the  serum  and  blood  corpuscles.  Ether,  esters,  aldehyde, 
and  acetone  divide  themselves  so  that  the  blood  corpuscles  contain  more 
than  does  an  equal  volume  of  serum.  These  bodies  are  equally  absorbed 
by  the  blood  corpuscles.  Ammonium  salts  with  univalent  anions  pass 
in  quickly  while  with  divalent  or  polyvalent  anions  the  greater  part 
remains  in  the  serum;  still  they  pass  in  to  a  greater  extent  than  do  the 
corresponding  salts  of  the  fixed  alkalies. 

Overton  had  previously  arrived  at  the  same  results,  using  plant 
cells  and  chiefly  by  making  use  of  the  plasmolytic  method.  Urea  is  prob- 
ably more  quickly  taken  up  by  the  blood  corpuscles  than  by  plant  cells, 
and  ammonium  salts  also  seem  to  pass  more  easily  into  the  blood  cor- 
puscles than  into  the  plant  cells. 

In  regard  to  other  salts  Hedin's  results  have  been  substantiated 
by  Oker-Blom,1  by  estimating  the  electrical  conductivity  of  the  blood. 

It  must  also  be  stated  that  according  to  Hedin,  only  those  bodies 
which  do  not  pass,  or  pass  slowly  into  the  cells,  can  essentially  alter 
the  volume  of  the  cells.  A  close  correspondence  exists  in  this  regard 
between  the  plant  and  animal  cells. 

Gurber  found  that  when  blood  corpuscles  are  repeatedly  washed 
with  salt  solution  until  the  wash  solution  does  not  show  any  alkaline 
reaction,  and  are  then  suspended  in  NaCl  solution  and  treated  with 
CO2,  the  alkaline  reaction  increased  while  the  blood  corpuscles  became 
richer  in  chlorine.  No  exchange  of  K  or  Na  took  place.2  Gurber 
explains  the  experiment  as  follows:  the  carbonic  acid  set  a  small  amount 
of  HO  free  from  the  salt,  and  this  HC1  was  taken  up  by  the  blood  corpuscles. 
The  Na2CC>3  formed  at  the  same  time  gave  the  alkaline  reaction  to  the 
solution.  Koeppe3  as  well  as  Hamburger  and  v.  Lier4  claim,  on  the 
contrary,  that  an  exchange  of  HC03-ions  and  Cl-ions  takes  place  between 
the  blood  corpuscles  and  the  solution,  and  Hamburger  and  v.  Lier 
claim  to  have  shown  that  the  blood  corpuscles  are  permeable  only  for 
anions,  while  the  cations  do  not  pass  in. 

Hamburger5  and  his  collaborators  have  also  found  about  the  same 
osmotic  phenomena  with  other  free  mobile  cells  such  as  leucocytes, 
spermatozoa  as  with  the  red  blood  corpuscles.  The  osmotic  relations 
have  also  been  tried  with  intact   parts  of   organs,  therefore  with  cells 

1  Pfliiger's  Arch.,  81,  167  (1900). 

2  Sitzungsber.  d.  med.  phys.  Gesellsch.  zu  Wurzburg,  1895. 
»  Pfliiger's  Arch.,  67,  189  (1897). 

<Arch.  f.  (Anat.  u.)  Physiol.,  1902,  492. 

'Osmotischer  Druck  und  Ionenlehre,  Wiesbaden,  1902,  1,  401. 


OSMOTIC  PRESSURE.  9 

in  connection  with  other  tissue  constituents.  By  investigations  on  the 
changes  in  the  weight  (instead  of  the  volume  changes  in  the  above-mentioned 
experiments  with  plant  cells  and  blood  corpuscles)  which  frog  muscles 
undergo  in  solutions,  various  experimenters,  Nasse,1  Loeb,2  and  Over- 
ton,3 have  tried  to  prove  the  ability  of  muscle  to  take  up  various  substances. 
Overton  found  that  as  long  as  the  irritability  of  the  muscle  was  retained 
the  muscle  took  up  the  same  bodies  as  the  plant  cells.  The  sarcolemma 
is  not  responsible  for  the  permeability,  but  the  outer  layers  of  the  muscle 
protoplasm  are. 

The  skin  of  amphibians  seems  according  to  Overton  to  behave  like  the  muscles4 
in  regard  to  permeability. 

Theories  of  Admissibility.  On  what  does  the  permeability  or  non- 
permeability  of  membranes  and  of  cells  for  certain  bodies  depend?  The 
discoverer  of  precipitation  membranes,  M.  Traube,  considered  the  mem- 
brane as  a  sort  of  molecular  sieve.  The  relation  of  the  size  of  the  particles 
passing  and  the  width  of  the  pores  of  the  membrane  is  important.5  This 
view  cannot  be  contested.  The  copper  ferrocyanide  membrane  may  be 
considered  to  act  in  this  way  and  the  non-permeability  of  most  mem- 
branes for  colloid  substances  depends  upon  the  fact  that  the  pores  are 
too  narrow  for  the  particles. 

The  question  as  to  the  occurrence  of  a  special  outer  limiting  layer 
of  the  cells  is  of  interest  for  the  understanding  of  the  metabolism  of  the 
cells  as  well  as  for  the  knowledge  as  to  the  manner  in  which  the  cells  take 
up  and  give  out  substances.  In  this  connection  it  must  be  recalled  that 
in  the  protoplasm  of  certain  cells  we  find  an  outer  dense  layer  or  a  true 
membrane  which  seems  to  consist  of  protein  substances.  Still,  even  in  cells 
in  which  no  special  outer  limiting  layer  can  be  seen,  the  presence  of  such  a 
limiting  layer  must  be  admitted  because  of  the  permeability  condi- 
tions of  these  cells. 

Nernst  6  has  shown,  by  special  experiments,  that  the  permeability 
of  a  membrane  for  a  certain  substance  is  essentially  dependent  upon 
the  solvent  power  of  the  membrane  for  this  substance.  This  question 
which  is  very  important  for  the  study  of  the  osmotic  phenomenon  in 
living  cells  has  been  especially  studied  by  Overton.7     From  the  behavior 

1  Pfluger's  Arch.,  2,  114  (1869). 

iIbid.,  69,  1;  71,  457  (1898). 

J  Ibid.,  92,  115  (1902);  105,  176  (1904). 

*  Verhandl.  d.  phys.  med.  Gesellsch.  zu  Wurzburg  (N.  F.),  36,  277  (1904). 

5  Arch.  f.  Anat.  Physiol,  u.  Med.,  1867,  87. 

6  Zeitschr.  f.  physikal.  Chem.,  6,  37  (1890). 

7  Vierteljahrsschr.  d.  Naturf.  Gesellsch.  in  Zurich,  44  (1899)  and  Overton,  Studien 
uber  die  Narkose,  Jena,  1901. 


10  GENERAL  AND  PHYSICO-CHEMICAL. 

of  living  cells  to  dye-stuffs,  as  well  as  the  special  ease  in  which  certain 
substances,  which  are  not  soluble  in  water  or  only  slightly  so,  but  are  readily 
soluble  in  fats  or  fat-like  bodies,  pass  into  animal  and  plant  protoplasms 
has  led  Overton  to  the  conclusion  that  the  protoplasmic  limiting  layer 
behaves  like  a  substance  layer  having  the  solvent  properties  similar  to  the 
fatty  oils.  According  to  Overton  the  protoplasmic  layer  is  probably 
impregnated  with  lipoids,  i.e.,  bodies  more  or  less  similar  to  the  fats 
in  regard  to  their  solubilities  and  their  solvent  power  upon  certain  sub- 
stances. The  lipoids  do  not  form  a  chemically  definable  class  of  bodies. 
Certain  of  them  are  still  of  an  unknown  constitution  while  others  are 
known,  especially  the  lecithins  (the  phosphatides  as  a  group)  and  the 
cholesterin  are  to  be  especially  mentioned  on  account  of  their  great 
importance. 

The  assumption  that  an  accumulation  of  lipoids  occurs,  as  a  special 
limiting  layer,  in  the  cells  is  not  sufficiently  founded  and  not  generally 
true  at  least  for  the  animal  cells.  Still  this  assumption  is  not  absolutely 
necessary  for  a  comprehension  of  the  action  of  lipoids  in  the  above 
sense.  Objections  have  been  raised  by  a  few  investigators  against 
Overton's  theory,  which  has  found  general  acceptance.1  Thus  it  fails 
to  explain  all  cases,  although  this  was  suggested  by  Overton  himself, 
for  instance  according  to  Cohnheim,  it  does  not  explain  the  absorption 
processes  in  the  intestinal  canal,  and  according  to  Moore  and  Roaf  it 
cannot  explain  certain  properties  of  the  cells,  namely  the  varied  composi- 
tion of  the  electrolytes  within  and  outside  of  the  cells,  and  the  selective 
taking  up  of  certain  soluble  substances  such  as  food  products,  drugs, 
toxins  and  antitoxins  by  the  cells.  The  investigations  of  the  last  men- 
tioned experimenters  are  based  essentially  upon  investigations  of  the 
behavior  of  mineral  substances,  and  they  show  that  the  above  theory 
offers  certain  difficulties  in  explaining  the  exceedingly  important  exchange 
of  mineral  substances  between  the  cells  and  the  external  fluid.  Also  the 
fact  that  the  cells  are  readily  permeable  for  water  is  explained  with  diffi- 
culty by  Overton's  theory. 

J.  Traube2  especially  has  put  forth  objections  to  Overton's  theory. 
According  to  him,  the  passage  of  a  substance  from  a  watery  solution 
into  the  cells,  is  in  the  first  place  due  to  its  so-called  solution  tenacity  in  the 
watery  solution.  This  solution  tenacity  is  according  to  Traube  the  attrac- 
tion bf 'tween  the  solvent  and  the  solute;  and  is  not  identical  with  the 
osmotic  pressure,  but  is  measured  by  the  surface  tension  of  the  solution. 

1  See  O.  Cohnheim,  Die  Physiologie  der  Verdauunp;  u.  Ernahrung  (1908).  J.  Loeb 
in  Oppenheimer'8  Handbuch  der.  Biochem.  Bd.  2,  105.  T.  B.  Robertson  Journ.  of 
biol.  Chem.,  I    L908).     B.  Moore  and  H.  Roaf,  Biochem.  Journ.,  3  (1908). 

M'fluger's  Arehiv.,  105,  541  (1904);  123,  419  (1908);  132,  511  (1910);  140,  109 
(1911). 


OSMOTIC  PRESSURE.  11 

It  has  been  shown  that  those  substances  which  are  not  taken  up  by  the 
cells  at  all  or  only  slightly,  do  not  lower  the  surface  tension  of  the  water 
when  dissolved  therein.  On  the  contrary,  those  substances  which  lower 
the  surface  tension,  pass  into  the  cells.  According  to  Gibbs  those  sub- 
stances, which  when  dissolved  in  water  lower  the  surface  tension,  occur 
in  greater  concentration  on  the  surface  as  compared  with  the  interior. 
Thus  according  to  Traube  the  solution  tenacity  is  less  the  lower  the 
surface  tension  of  the  watery  solution.  Otherwise  the  direction  of 
movement  of  a  substance  in  the  boundary  between  two  phases  (watery 
solution  and  cells)  is  determined  by  the  relationship  between  the  solution 
tenacity  of  the  substances  in  the  two  phases.  However,  the  solution  tenac- 
ity of  a  substance  can  only  be  directly  measured  in  the  watery  solution. 
Traube  supports  his  theory. upon  different  experiments  in  which  mem- 
bers of  the  same  homologous  series  were  dissolved  in  water  in  such  con- 
centration #as  to  have  the  same  surface  tension  and  also  showed  the 
same  ability  to  pass  into  the  cells.  The  disagreement  in  other  cases  can 
be  explained  by  the  unknown  solution  tenacity  in  the  cell  phase.  As  we  will 
show  below  Traube's  proposition  calls  to  mind  the  accepted  views  as  to 
the  origin  of  the  adsorption  phenomena  or  the  taking  up  of  dissolved  sub- 
stances by  solid  bodies.  Lowe  l  has  also  found,  in  studying  the  taking 
up  of  different  dissolved  substances  by  lipoids,  that  the  process  does  not 
take  place  as  called  for  by  Overton's  theory  according  to  Henry's  law 
of  absorption  but  rather  an  adsorption. 

Certain  substances  which  are  of  the  very  greatest  importance  for  life 
processes  and  which  probably  are  burned  to  a  great  extent  within  the  cells, 
have  according  to  the  above  experiments  only  a  limited  ability  to  enter 
the  cells.  These  bodies  are  the  sugars  and  the  amino-acids.  Also  the 
presence  of  salts  within  the  cells  is  not  easily  understood  in  view  of  the 
above  experiments.  In  consideration  of  this  it  must  be  remarked  that 
the  above  described  experiments  on  the  permeability  of  animal  cells  have 
been  carried  out  with  cells  that  were  removed  from  their  attachment  to 
the  living  animal.  Although  these  cells  are  not  considered  as  physio- 
logically dead  cells  still  it  is  very  probable  that  certain  life  functions 
have  been  arrested.  It  is  readily  conceivable  that  the  oxidation  processes, 
whereby  the  organic  substances  taken  up  within  the  cells  are  trans- 
formed into  simpler  products,  are  at  least  partly  brought  to  a  standstill 
(see  Chapter  XVI).  That,  nevertheless,  at  least  salts  and  sugar  also 
attract  water  in  the  living  organism  and  therefore  only  pass  into  the 
cells  in  small  quantities  follows  from  the  experiments  of  Heidenhain, 
according  to  whom  these  substances  are  designated  as  lymph  forming 
agents  of  the  second  order  (Chapter  VI).     This  action  is  also  explained 

1  Bioch.  Zeitschr.,  42;  150,  190,  205,  207  (1912). 


12  GENERAL  AND  PHYSICO-CHEMICAL. 

by  Heidenhain  as  being  dependent  upon  their  power  of  abstracting 
water  from  the  tissues. 

If  we  admit  that  the  cells  normally  contain  only  small  amounts  of 
sugar  and  amino-acids  at  any  one  time,  then,  if  these  substances  are  being 
continuously  burned  within  the  cells,  new  quantities  must  constantly 
be  taken  up  and  in  this  way  gradually  large  quantities  of  the  mentioned 
substance  would  be  taken  up  and  burned.  If  the  combustion  is  arrested 
no  new  quantities  are  taken  up.  The  fact  that  certain  substances 
are  only  taken  up  in  small  quantities  at  a  time  does  not  prove  that  they 
are  not  burned  within  the  cells. 

According  to  Moore  and  Roaf  1  the  salts  exist  in  the  blood  cor- 
puscles in  the  form  of  "  adsorpates;"  these  are  adsorbed  by  the  solid 
constituents  of  the  blood  corpuscles.  As  we  will  see  further  on  (page 
27)  an  adsorbing  substance  can  only  take  up  a  limited  amount  of  another 
substance.  If,  after  the  saturation  limit  is  reached,  more  of  the  adsorbed 
substance  is  added  then  practically  no  more  is  taken  up.  In  this  way 
we  can  explain  why  the  blood  corpuscles  only  take  up  very  little  of  the 
salts  added.  The  slight  ability  of  the  sugars  and  amino-acids  to  be 
taken  up  can  perhaps  be  explained  in  a  similar  manner. 

Osmotic  Pressure  of  Animal  Fluids.  As  is  apparent  from  the 
above,  a  substance  exerts  upon  living  cells  an  entirely  different  influence, 
depending  upon  whether  the  substance  is  able  to  pass  into  the  cell  or 
not,  and  whether  the  substance  which  does  not  pass  in  has  the  ability 
of  attracting  water  or  not.  Therefore  that  part  of  the  osmotic  pres- 
sure of  body  fluids  which  is  caused  by  bodies  not  passing  in  is  called 
the  effective  osmotic  pressure.  In  this  manner  therefore  the  salts  of  the 
alkalies  and  alkaline  earths  and  the  sugars  act.  As  sugar,  as  well  as  the 
bodies  which  according  to  the  just  mentioned  experiments  are  readily 
taken  up  by  the  cells,  occurs  under  ordinary  conditions  only  in  very 
small  amounts  in  the  blood,  and  also  as  the  proteins  are  practically  with- 
out influence  upon  the  osmotic  pressure,  the  normal  osmotic  pressure  of 
the  blood  is  chiefly  due  to  the  salts.  As  the  depression  of  the  freezing- 
point  is  almost  the  only  method  used  for  animal  fluids,  therefore  ordinarily 
the  freezing-point  depression  (A)  is  given  as  a  measure  of  the  osmotic 
pressure.  For  mammalian  blood  A  is  constant  with  the  exception  of  slight 
variations  flue  to  the  food  and  perhaps  also  to  other  circumstances.  It 
is  0.560,2  which  corresponds  to  a  0.90  per  cent  NaCl  solution  and  to  an 
osmotic  pressure  of  about  6|  atmospheres.  In  lower  animals  A  may 
be  slightly  lower,  for  example,  in  the  frog  A  =  0.46°.  In  invertebrate 
sea  animals  the  body  fluid  is  equal  to  the  osmotic  pressure  of  the  sur- 

1  Bioch.  Journ.,  3,  55  (1908). 

J  Hamburger,  Osmotischer  Durck  u.  Ionenlehre,  1,  456. 


colloids.  ia 

rounding  sea  water  (A  =  2.3°)  and  varies  with  the  quantity  of  salt  in  the 
water  (Bottazzi).  Irf  lower  fishes  (Selachii)  the  osmotic  pressure 
of  the  blood  is  equal  to  the  surrounding  medium,  and  in  higher  fishes 
(Teleostomi)  lower  (A  =  1.0°)  (Bottazzi).  In  Selachii  the  osmotic 
pressure  of  the  blood  is  chiefly  due  to  urea  (Schroeder).1 

In  sea  fishes  as  well  as  fresh-water  fishes,  for  example,  the  eel,  a  lower 
osmotic  pressure  (A  =  0.41°)  is  found  when  kept  in  fresh  water  than 
when  kept  in  sea  water  (A  =  0.55°)2.  In  lower  sea  animals  the  osmotic 
pressure  is  equal  to  the  surrounding  medium,  while  higher  animals  are 
independent  of  the  surroundings.  Hober  calls  attention  to  this  condi- 
tion and  points  out  the  analogy  with  the  body  heat  of  the  various 
animals.3 

If  we  pass  to  other  body  fluids  we  must  mention  that  the  lymph 
shows  a  somewhat  higher  osmotic  pressure  than  the  blood,  and  this  is 
due  to  the  lymph  taking  up  from  the  tissues  metabolic  products  hav- 
ing a  low  molecular  weight.4  Milk  and  bile  have  the  same  osmotic 
pressure  as  the  blood,5  while  saliva  has  a  lower  pressure.6  The  urine 
of  man  and  mammalia  generally  has  a  much  higher  osmotic  pressure 
than  the  corresponding  blood.7  For  human  urine  A  varies  between  1.3 
and  2.3°.  After  abundant  drinking  as  well  as  under  pathological  con- 
ditions (diabetes  insipidus)  the  osmotic  pressure  cf  the  urine  can  be  lower 
than  the  blood.  In  regard  to  the  osmetic  pressure  of  animal  fluids  under 
normal  and  pathological  conditions  we  refer  to  the  work  of  Koranyi 
and  Richter.8 

n.    COLLOIDS. 

The  word  colloid  originated  with  Graham,  who  included  in  this  name 
different  substances  which  did  not  have  the  property  of  diffusing  through 
an  animal  membrane.  In  opposition  to  this  Graham  called  those 
bodies  which  passed  through  a  membrane,  crystalloids,  because  they 
were  as  a  rule  crystalline,  a  property  which  with  few  exceptions  does 
not  belong  to  the  colloids.9     Graham  included  soluble  silicic  acid  among 

1  Bottazzi,  Archives  ital.  de  biol.  28,  61  (1897).  Schroeder,  Zeitschr.  f.  physiol. 
Chem.  14,  576  (1890). 

2  Dekhuisen,  Arch,  neerland,  10,  121  (1905);  Quinton,  Compt.  rend.  boc.  biol., 
57,  470,  513  (1904). 

3Physik.  Chem.  d.  Zelle  u.  Gewebe,  3.  Aufl.  353,  (1911). 
4Leathes,  Journ.  of  Physiol.,  19,  1  (1895). 

6  Dresser,  Arch.  f.  exp.  Path.  u.  Pharm.,  29,  303  (1892). 
.6Nolf,  Traveaux  du  lab.  de  phys.  de  Liege,  6,  225  (1901). 

7  Kordnyi,  Zeitschr.  f.  klin.  Med.,  33,  1  (1897),  34,  1  (1898). 

8  Physikalische  Chemie  und  Medizin.     Leipzig  (1907). 

s  Ann.  d.  Chem.  u.  Pharm.,  121,  1  (1862)  as  well  as  Ann.  de  chim.  et  de  Phys.  (4)„. 
3,  127  (1864). 


14  GENERAL  AND  PHYSICO-CHEMICAL. 

the  colloids  and  also  analogous  forms  of  stannic  acid,  titanic  acid, 
molybdic  acid  and  tungstic  acid,  aluminium  hydroxide  and  analogous 
metallic  oxides,  when  they  exist  in  the  soluble  form,  and  also  starch,  dex- 
trins,  the  gums,  caramel,  tannin,  albumin  and  gelatin. 

Some  colloids  are  characterized  by  the  fact  that  under  certain  con- 
ditions they  solidify  into  a  gelatinous  form  containing  considerable  water. 
In  the  case  where  water  is  the  solvent  then  Graham  called  the  soluble 
form  hydrosol  and  the  gelatinous  form  hydrogel. 

By  diffusion  through  a  membrane  (called  dialysis  by  Graham)  colloid  sub- 
stances can  be  separated  from  crystalloids.  Colloidal  silicic  acid  as  well  as 
corresponding  forms  of  certain  other  bodies  are  obtained  by  treating  the  soluble 
alkali  salt  with  hydrochloric  acid,  then  removing  the  excess  of  hydrochloric  acid 
as  well  as  of  chlorides,  by  means  of  dialysis.  Colloidal  alumina  was  obtained  by 
Graham  by  dissolving  aluminium  hydroxide  in  aluminium  chloride.  This  last  salt 
was  removed  by  dialysis  and  the  hydroxide  remained  with  more  or  less  HC1  com- 
bined in  solution. 

Various  metallic  sulphides  can  be  obtained  in  colloidal  solution.  Such  solu- 
tions of  As2S3  and  Sb2S3  can  be  obtained  by  passing  H2S  into  dilute  solutions 
of  the  respective  metallic  oxide,1  and  colloidal  CuS  can  be  prepared  by  washing 
the  precipitated  compound  with  water,  by  which  treatment  thej,CuS  finally  becomes 
soluble  in  water.2 

The  metals  can  be  obtained  as  hydrosols,  and  indeed  in  two  ways: 

1.  By  treating  a  salt  with  various  reducing  agents  (for  example  formaldehyde, 
hydrosulphurous  acid,  hydrazine,  Irydroxylamine)  the  various  metals  are  obtained 
in  colloidal  solution.3  As  the  solutions  thus  obtained  are  often  very  unstable, 
it  has  been  found  advisable  to  help  their  stability  by  the  addition  of  organic 
colloids  (gelatin).  We  will  discuss  the  mode  of  action  of  these  so-called  pro- 
tective colloids  on  page  23. 

2.  Bredig  4  has  discovered  a  method  which  makes  possible  the  production 
of  pure  metallic  sols  by  the  cathode  spraying  of  metallic  wires  under  water. 
Svedbeug  5  prevents  the  heating  of  the  fluid  in  this  spraying  by  using  the 
induction  current.  This  makes  the  spraying  also  possible  under  organic  fluids 
and  sols  of  the  light  metals  have  also  been  prepared.  Practically  sols  of  all 
metals  and  metalloids  can  be  prepared  in  this  way. 

Among  those  bodies  which  can  be  obtained  in  the  colloidal  state 
we  have  acids  as  veil  as  bases,  and  the  chemical  elements  are  also  known 
as  colloids,  as  well  as  bodies  of  more  complex  molecular  structure  like 
the  proteins  and  starches.  The  colloid  bodies,  therefore,  have  from  a 
chemical  standpoint  nothing  in  common.  More  likely  the  colloidal  con- 
dition is  due  to  physical  properties,  and  this  follows  from  the  researches 
of  Graham.  The  crystalloids  and  the  colloids  are  therefore  not  to 
be  considered  as  chemically  different  classes  of  bodies,  but  rather  only 
as  different  physical  conditions  of   matter   and  the  boundary   between 

1  II.  Schulze,  Journ.  prakt.  Chem.  (N.F.),  25,  431  (1882),  and  27,  320  (1883). 
•Spring,  Ber.  d.  d.  chem.  Gesellsch.,  16,  1142  (1883). 
'  Miillr-r,  Allg.  Chemie  d.  KoHoide.    Leipzig  (1907),  6. 

*  A&arganiflcfae  l  ermente.  Leipzig  (1901),  24. 

*  Ber.  d.  d.  chem.  GeBellach.,  38,  3616  (1905);  39,  1705  (1906). 


COLLOIDS.  15- 

these  two  conditions  is  often  very  indefinite.  Certain  chemically  definable 
classes  of  substances,  such  as  proteins,  occur  only  or  chiefly  in  the  col- 
loidal condition  while  others,  such  as  the  inorganic  salts,  occur  as  crys- 
talloids. Finally  we  find  others  that  can  occur  in  both  forms,  namely 
the  soaps  (page  17).  In  short  the  difference  between  the  crystalloid  and 
colloidal  condition  may  be  considered  in  that  the  crystalloids  occur  in 
solution  as  molecules  of  medium  size  while  the  colloids  are  either  very  large 
molecules,  molecular  aggregations  or  at  least  particles  of  a  larger  spacial 
volume  than  the  crystalloids.  According  to  such  a  conception  many 
properties  of  the  colloids  can  be  explained. 

In  order  to  give  a  better  review  we  will  give  a  classification  of  the 
colloids  which  seems,  for  the  present,  to  be  rather  universally  accepted. 
This  was  first  suggested  by  Perrin1  and  later  accepted  by  Hober,2 
A.  Muller,3  and  Wo.  Ostwald,4  although  different  authors  use  different 
names  for  the  two  classes.  The  classifications  of  Hardy  5  and  Zsig- 
mondy6  have  also  much  in  common  with  the  classification  given  below. 

One  of  the  two  groups  of  colloids  is  called  hydrophile  colloids  (emul- 
sion colloids,  emulsoides)  because  in  the  aqueous  solution  a  certain  rela- 
tion still  exists  between  the  dissolved  substance  and  the  solvent  which 
is  evident  especially  by  a  certain  viscosity  of  the  solution.  The  hydro- 
phile colloids  often  gelatinize  on  cooling,  the  gel  is  again  soluble  in 
water  (reversible),  and  in  general  the  hydrophile  colloids  are  separated 
from  their  solution  by  electrolytes  wnth  greater  difficulty  than  the  col- 
loids of  the  second  group.  Bodies  of  the  greatest  importance  for  phys- 
iological chemistry  like  the  proteins,  starch,  glycogen,  and  soaps  in 
watery  solution  belong  to  the  hydrophile  colloids. 

Contrary  to  the  hydrophile  colloids,  the  colloids  of  the  colloidal  metal 
type  are  called  suspension  colloids  (suspensoids)  as  they  must  be  con- 
sidered as  suspended  solid  particles  in  a  solvent  and  have  no  close 
relation  to  the  solvent.  The  viscosity  of  the  solution  does  not  differ 
much  from  that  of  the  pure  solvent;  besides  this,  the  suspension  col- 
loids do  not  gelatinize,  do  not  swell  up,  and  are  readily  precipitated 
by  electrolytes.  To  this  group  belong  the  metallic  sols,  the  colloidal 
metallic  sulphides,  and  certain  typical  suspensions  obtained  by  dissolving 
water-insoluble  substances  in  another  liquid  (alcohol,  acetone)  and  then 
pouring  this  solution  into  a  large  volume  of  water.  In  this  way  the 
substance  is  precipitated  in  a  finely  divided  condition.     Such  suspensions 


1  Journ.  de  Chimie  phy.,  3,  84  (1905). 

2  Physik.  Chem.  d.  Zelle  u.  Gewebe,  2  Aufl.  (1906),  208. 
s  Allg.  Chemie  d.  Kolloide  (1907),  187. 

*  Zeitschr.  f.  Chem.  u.  Ind.  d.  Koll.,  1,  331  (1907). 

5  Proc.  Roy.  Soc,  66,  95  (1899). 

6  Zur  Erkenntnis  d.  Koll.  (1905),  16. 


16  GENERAL  AND  PHYSICO-CHEMICAL. 

behave  in  many  respects  like  suspension  colloids.     Suspensions  of  mastic,1 
colophony,2  and  cholesterin3  belong  to  this  class. 

The  hydrophile  colloids  stand  closer  to  the  crystalloids  than  do  the 
suspension  colloids,  and  the  transition  between  the  crystalloids  and  the 
hydrophile  colloids  is  only  gradual.  At  the  boundary  we  find  the  pep- 
tones and  proteoses  which  belong  to  the  proteins,  but  at  the  same  time 
dialyze  rather  well.  On  the  other  hand,  we  also  have  colloids  which  to  a 
certain  extent  form  intermediary  steps  between  the  hydrophile  colloids 
and  suspension  colloids.  Finally,  there  are  also  numerous  intermediary 
members  between  the  suspension  colloids  and  the  finely  divided  substances 
suspended  in  water  (kaolin). 

Osmotic  Pressure.  As  above  stated,  the  osmotic  pressure  of  solu- 
tions of  crystalloids  can  be  determined  only  in  exceptional  cases  by 
means  of  the  semipermeable  membrane,  because  it  is  very  difficult  to 
prepare  membranes  which  are  impermeable  for  crystalloids.  As  pre- 
viously stated,  most  membranes  are  impermeable  for  colloids,  and  the 
osmotic  pressure  of  the  colloids  can  be  best  directly  determined  by  the 
aid  of  a  membrane  in  a  so-called  osmometer.  As  shown  bjr  Moore  and 
Roaf,  in  such  an  apparatus  changes  in  pressure  can  be  determined  which 
are  not  detectable  by  the  determination  of  the  freezing-point.4 

Equimolecular  solutions  of  various  non-electrolytes  give  the  same 
osmotic  pressure.  From  this  it  follows  that  when  different  non-elec- 
trolytes exist  in  solutions  with  the  same  percentage  concentration,  the 
osmotic  tension  of  these  solutions  must  be  in  inverse  proportion  to  their 
molecular  weights.  Certain  colloids  which  will  be  discussed  in  another 
connection  (proteins,  glycogen,  etc.)  must  have  a  very  large  molecule. 
From  this  it  follows  that  these  bodies  must  exert  a  very  low  osmotic 
pressure.  The  proteins  always  contain  a  small  amount  of  salts  which 
«xist  either  in  a  sort  of  combination  with  the  colloids  or  are  to  be  con- 
aidered  as  contaminations  which  are  difficult  to  remove.  For  this  reason 
it  has  been  repeatedly  stated  that  these  salts  are  responsible  for  the 
small  differences  in  the  osmotic  pressure.  By  carefully  washing  crys- 
stalline  proteins  from  serum  and  egg-white,  Reid  was  able  to  prepare 
bodies  which  gave  finally  no  osmotic  pressure  in  the  osmometer.5  In 
opposition  to  this,  Mooue  and  Roaf  as  well  as  Lillie  call  attention  to 
the  fact  that  the  osmotic  pressure  of  protein  solutions  is  influenced  by 
the    tn-atmcnt    which    the    protein    received   before    the    determination. 


1  Zeitsc.hr.  f .  physik.  Chem.,  57,  47  (1906). 

2  Ibid.,  38,  385  (1901). 

■  Bioch.  Zeitschr.,  7,  152  (1908). 

*  Bioch.  Journ.,  2,  34  (1906). 

•  Journ.  of  Physiol.,  31,  438  (1904). 


COLLOIDS.  17 

Starling,1  Moore  and  Parker,2  Moore  and  Roaf3  and  Lillie,4  using 
protein  preparations  which  had  not  been  exposed  to  any  strong  treat- 
ment before  use  (serum  proteins,  ovalbumin),  as  well  as  Reid  5  (with 
haemoglobin),  have  been  able  to  detect  a  low  osmotic  pressure  and 
indeed  by  the  aid  of  osmometric  methods.  According  to  Starling, 
the  proteins  of  the  serum  correspond  to  a  pressure  of  30-40  mm.  Hg. 
and  Reid6  found  a  pressure  of  3-4  mm.  Hg.  for  a  1  per  cent  haemoglobin 
solution. 

Th©  influence  of  added  bodies  upon  the  osmotic  pressure  has  been  tested  by 
Lillie  by  adding  the  substance  to  be  tested  in  the  same  percentage  concentration 
to  the  inner  and  outer  fluids.  It  was  found  that  non-electrolytes  were  without 
action  while  acid  and  alkalies  increased  the  osmotic  pressure  of  gelatin  solutions, 
while  salts  lowered  the  pressure  of  gelatin  as  well  as  ovalbumin  solutions.  Adam- 
son  and  Roaf  7  arrived  at  similar  results  in  regard  to  alkalies  and  acids.  Besides 
this,  Lillie  found  that  the  osmotic  pressure  was  dependent  upon  the  past  history 
of  the  colloid.  Warming  as  well  as  shaking  the  solutions  seems  to  change  the 
aggregate  condition,  which  returns  very  slowly  or  not  at  all.  The  changes 
in  the  osmotic  pressure  produced  by  salts,  Lillie  explains  by  a  change  in  the 
aggregate  condition  of  the  colloid,  by  the  addition  of  salts  it  is  brought  closer 
to  its  precipitation  point  and  is  probably  united  in  large  aggregations.  In  this 
way  the  number  of  particles  is  diminished  and,  as  this  number  must  be  important 
for  the  osmotic  pressure,  this  pressure  is  lowered.  In  agreement  with  this 
the  above  mentioned  influence  of  acids  and  alkalies  upon  the  osmotic  pressure  of 
gelatin  can  be  explained  by  an  increase  in  the  particles.8 

As  we  have  seen  above  the  determination  of  the  elevation  of  the  boil 
ing-point  or  the  depression  of  the  freezing-point  is  the  simplest  way 
for  estimating  the  osmotic  pressure  of  a  crystalloid  substance  in  solution. 
If  such  determinations  are  made  with  a  colloidal  solution  then  unmeas- 
urable  results  are  found  for  the  elevation  of  the  boiling-point  or  the  depres- 
sion of  the  freezing-point.  This  indicates,  as  above  stated,  that  the 
molecules  or  the  particles  must  be  very  large.  F.  Kraft9  found  no 
elevation  of  the  boiling-point  for  soaps  in  watery  solution  but  obtained 
values  which  correspond  to  the  calculated  molecular  weights  when  the 
soaps  were  dissolved  in  alcohol.  Therefore  the  soaps  are  colloidal  in 
watery  solution  and  crystalloidal  bodies  in  alcoholic  solution. 

Filterability.  Large  particles  suspended  in  a  liquid  can  be  removed 
from  the  fluid  by  filtering.     The  finer  the  suspended  particles  are  the 

1  Journ.  of  Physiol.,  19,  322  (1896). 

2  Amer.  Journ.  of  Physiol.,  7,  261  (1902). 

3  Bioch.  Journ.,  2,  34  (1906). 

*  Amer.  Journ.  of  Physiol.,  20,  127  (1907). 

6  Journ.  of  Physiol.,  33,  12  (1905). 

8  Bioch.  Journ.,  3,  422  (1908).  / 

'Ibid. 

"Pauli,  Koll.  Zeitschr.,  7,  241  (1900). 

•Ber.  d.  d.  chem.  Gesellsch.,  29,  1328  (1896);  32,  1584  (1899). ' 


18  GENERAL  AND  PHYSICO-CHEMICAL. 

closer  must  the  filter  be  Extensive  experiments  on  the  filtering  of 
colloids  have  been  carried  out  by  Bechhold.1  He  used  paper  filters 
which  were  impregnated  with  collodion  dissolved  in  glacial  acetic  acid. 
According  to  the  concentration  of  the  collodion  solution  filters  of  dif- 
ferent porosity  were  obtained.  The  colloid  solutions  were  pressed 
through  the  filter  by  a  pressure  up  to  five  atmospheres.  It  was  shown 
that  all  colloid  solutions  contained  particles  of  various  sizes.  Never- 
theless for  every  solution  a  filter  could  be  prepared  whose  pores  were 
small  enough  to  retain  all  the  particles.  In  this  manner  Bechhold  was 
able  to  classify  the  colloids  in  a  series  according  to  the  size  of  the  smallest 
particles.  He  found  that  in  general  the  inorganic  colloids  (Prussian 
blue,  platinum,  iron  oxide,  gold,  silver)  form  larger  particles  than  the 
organic  colloids  (gelatin,  haemoglobin,  seralbumin,  proteoses,  dextrin). 
Still  it  must  be  remarked  that  according  to  Zsigmondy2  the  size  of  the 
particles  of  the  same  colloid  are  larger  in  one  preparation  than  in  another 
and  that  the  size  can  change  on  keeping. 

On  filtering  proteose  solutions  through  filters  of  unequal  thickness 
Bechhold  was  able  to  show  that  the  larger  the  particles  of  the  proteoses, 
the  easier  are  they  precipitable  by  ammonium  sulphate. 

Diffusion.  We  have  already  seen  that  the  osmotic  pressure  of  a 
colloid  solution  is  very  small  and  also  that  the  osmotic  pressure  of  a  solu- 
tion is  the  cause  for  the  diffusion  of  the  particles,  therefore  it  is  evident 
that  the  diffusion  ability  of  colloids  can  only  be  very  slight.  This  is 
not  only  true  for  the  free  diffusion  but  also  for  the  diffusion  through  a 
membrane.  Both  of  these  was  first  studied  by  Graham.  The  first  was 
found  very  slight  but  measurable  in  several  cases  while  the  fact  that 
the  colloids  did  not  diffuse  through  membranes  (non-dialysable)  was 
given  as  the  most  constant  difference  between  colloids  and  crystalloids. 
Nevertheless,  there  does  not  exist  any  sharp  boundary  and  dialysis  depends 
principally  upon  the  size  of  the  particles  as  well  as  upon  the  character  of 
the  membrane. 

Internal  Friction.  By  the  internal  friction  of  a  fluid  we  mean  the 
force  whiph  resists  the  displacement  of  the  particles  of  the  fluid  among 
one  another.  The  internal  friction  is  therefore  an  expression  for  the 
great  thickness  or  viscosity  of  the  fluid. 

For  physiological  purposes  the  internal  friction  is  determined  by 
measuring  the  time  which  a  given  volume  of  the  fluid  requires  to  flow 
through  a  capillary  tube  under  a  pressure  of  its  own  weight. 

It  is  generally  accepted  that  the  internal  friction  of  suspension  col- 


'  ZeilM  hr.  f.  physik.  Chem.,  60,  257  (1907). 

2  Zur  Erkenntnis  d.  Koll.,   (1905),   104  as  well  aB  Zeitschr.  f.  Elektrochem.,    12, 
681     Ifl 


COLLOIDS.  19 

loids  is  equal  to  that  of  the  pure  solvent  or  differs  from  it  only  slightly. 
On  the  contrary  hydrophile  colloids  are,  in  proper  concentration,  very 
viscous  which  is  probably  the  reason  that  they  gelatinize  under  certain 
circumstances.  Pauli  as  well  as  Pauli  and  Handovsky1  have  inves- 
tigated strongly  dialyzed  serum  in  regard  to  its  internal  friction.  The 
addition  of  a  little  salt  (to  0.05  normal)  causes  a  lowering  of  the  internal 
friction  below  that  of  a  pure  albumin  solution,  while  acids  and  alkalies 
in  small  amounts  cause  a  powerful  rise  in  the  viscosity. 

Optical  Properties.  Colloidal  solutions  are  opalescent  by  reflected 
light,  which  depends  upon  the  fact  that  the  ligljt  is  reflected  by  the  sus- 
pended particles.  The  reflected  light  is  partly  polarized.  This  phenom- 
enon, called  Tyndall's  phenomenon,  depends  upon  the  presence  of 
small  particles  in  the  liquid,  and  is  considered  as  a  test  for  colloid  solu- 
tions. Still  there  are  colloid  solutions  (certain  gold  solutions,  Zsig- 
mondy),  which  do  not  give  Tyndall's  phenomenon,  and  on  the  other  hand 
we  also  have  solutions  of  certain  high  molecular  crystalloids  (cane 
sugar,  rafnnose),  which  produce  this  phenomenon.2 

With  the  aid  of  the  ultramicroscope  of  Siedentopf  and  Zsigmondy, 
it  has  been  made  possible  to  see  the  colloidal  particles  directly.3  In 
this  apparatus  the  colloidal  particles  are  strongly  illuminated  by  direct 
light,  so  that  no  ray  of  light  falls  directly  into  the  eye  of  the  observer. 
The  particles  are  hereby  made  visible  on  account  of  the  formation  of 
diffraction  disks  which  are  visible  through  the  miscroscope.  In  colloidal 
solutions  where  the  particles  are  close  together,  a  more  or  less  intense, 
homogeneous,  polarized  sphere  of  light  is  seen  in  the  microscope  where 
the  individual  particles  cannot  be  distinguished  from  each  other.  This 
is  possible  on  diluting  the  solution.  Those  particles  which  are  only 
made  visible  by  dilution  are  called  submicrons,  while  those  that  gradually 
disappear  on  dilution  are  called  amicrons. 

The  investigations  of  Zsigmondy  and  others  upon  the  growth  of  colloidal 
metallic  particles  are  also  interesting.  Thus  the  reduction  of  gold  chloride  by 
formaldehyde,  whereby  colloidal  gold  is  formed,  is  accelerated  by  the  addition  of 
colloidal  gold,  and  the  added  particles  indeed  grow  at  the  cost  of  the  newly 
reduced  gold.4  In  a  similar  manner  the  reduction  of  silver  nitrate  with  ammonia 
and  formaldehyde  is  helped  by  the  addition  of  colloidal  gold  when  the  reduced 
silver  precipitates  upon  the  gold  particles.5  In  such  processes  the  amicrons  can 
enlarge  so  that  they  can  be  observed  by  the  ultramicroscope   (submicrons). 


'Pauli,  Koll.  Zeitschr.,  3,  5  (1908);    Pauli  and    Handovsky,  Biochem.  Zeitschr., 
18,  340  (1909);  24,  239  (1910). 

2  Lobry  de  Bruyn  and  Wolff,  Rec.  trav.  chim.  des  Pays-Bas.,  23,  155  (1904). 

3  Zsigmondy,  Colloids  and  the  Ultramicroscope,  translated  by  Alexander,  New  York, 
1909. 

*  Zsigmondy,  Zeitschr.  f.  physik.  Chem.,  56,  65  (1906). 
5  Zsigmondy  and  Lottermoser,  ibid.,  56,  77  (1906). 


20  GENERAL  AND  PHYSICO-CHEMICAL. 

According  to  the  manner  of  preparation  the  colloids  may  have  particles  of  different 
sizes.     (See  page  00.) 

Submicrons  have  also  been  detected  in  solutions  of  organic  colloids.  The 
work  of  Gatix-Gruzewska  and  Biltz,  *  who  used  a  specially  pure  glycogen, 
must  be  especially  mentioned.  They  found  that  the  aqueous  solution  of  glycogen 
contained  amicrons  as  well  as  easily  recognizable  submicrons,  whose  presence 
was  only  evident  by  a  homogeneous  sphere  of  light,  but  on  the  addition  of  alcohol, 
conglomerate  into  detectable  submicrons. 

Molecular  Movement.     R.  Brown2  first  found  that  small  particles 

suspended  in  water  showed  a  quivering  motion,  and  this  phenomenon 

has  been  called,  from  its  discoverer,  Brownian  molecular  motion,  although 

the  particles  in  no  manner  are  to  be  considered  as  molecules.     This 

phenomenon  has  been  observed  since  then  by  many  investigators  in 

fluids  having  suspended  solid  particles  as  well  as  in  substances  dissolved 

in  colloidal  condition. 

The  Brownian  movement  is  considered  by  some  as  a  manifestation  of  a  general 
molecular  movement  of  matter.  According  to  this  view  it  is  comparable  with 
the  supposed  motion  of  gas  molecules  according  to  the  kinetic  theory  of  gases. 
Perrix  as  well  as  Svedberg  3  claim  that  the  law  of  gases  also  holds  for  very 
dilute  colloidal  solutions. 

Electrical  Transportation  of  Suspended  Particles.  A  not  too  weak 
electric  current  has  the  power  of  causing  motion  in  small  quantities 
of  fluid  enclosed  in  a  capillary  tube  or  in  a  porous  diaphragm.  The 
particles  suspended  in  a  fluid  also  wander  under  the  influence  of  the 
electric  current,  and  indeed  to  the  anode  or  cathode,  according  to  the 
nature  of  the  fluid  and  the  particles.  This  phenomenon  is  called  cata- 
phoresis.  Such  movements  have  also  been  found  in  colloidal  solutions. 
According  to  Biltz,4  in  dialyzed  aqueous  solution,  the  colloidal  metallic 
hydroxides  wander  to  the  cathode,  and  the  other  colloids  (metals, 
metallic  sulphides,  acids)  wander  to  the  anode.  The  colloidal  particles 
in  water  are  therefore  probably  electrically  charged,  hence  the  nega- 
tively charged  wander  to  the  anode  and  the  positively  charged  to  the 
cathode.  Dialyzed  protein  solutions  show  according  to  older  investiga- 
tions no  cataphoresis.  The  addition  of  acid  or  alkali  gives  to  the  pro- 
tein a  positive  or  negative  charge  respectively,  hence  an  alkaline  solu- 
tion wanders  to  the  anode  and  an  acid  solution  to  the  cathode  (Hardy,5 
Pal  li6).     According  to   Michaelis7    the  proteins  in   perfectly   neutral 

1  Pfliiger's  Arch.,  105,  115  (1904). 

»Edinb.  Phil.  Journ.,  5,  358  (1828);  8,  41  (1830). 

*  Perrin,  Colloid-chem.  Beihefte,  1,  221  (1910).  Svedberg,  KoU.  Zeitschr.,  7,  1 
(1910). 

*  Ber  d.  d.  chem.  Gesellsch.,  37,  1095  (1904). 

*  Joum.  of  Physiol.,  24,  288  (1899). 

«  Hofrneister's  Beitrage,  7,  531  (1906). 

'Biochem.  Zeitschr.,  16,  81  (1909);  19,  181  (1909);  24,  79;  27,  (38;  28,  193; 
29,  439  (1010);   33,  456  (1911);   41,  373  (1912). 


COLLOIDS.  21 

solution  wander  to  the  anode  in  the  case  when  the  experiment  is  so 
carried  out  that  the  formation  of  acid  or  alkali  is  prevented  at  the  anode 
or  cathode  respectively.  If  the  neutral  protein  solution  is  treated  with 
a  trace  of  acetic  acid  then  the  particles  wander  to  the  cathode.  With 
a  certain  very  slight  degree  of  acidity  the  direction  of  the  wandering  of 
the  particles  is  reversed.  With  this  reaction  no  wandering  or  a  double- 
sided  wandering  of  the  protein  bodies  can  be  detected.  This  so-called 
isoelectric  point  has  been  determined  by  Michaelis,  Rona  and  their 
collaborators  for  different  protein  substances.1  Michaelis  and  Rona 
claim  to  have  found  in  the  isoelectric  point  the  most  favorable  reaction 
for  the  heat  coagulation  of  the  protein  substances,  while  Sorensen  and 
Jurgensen  consider  the  reaction  which  the  pure  protein  substance 
gives  to  pure  water  as  the  optimal  precipitation  reaction.2 

According  to  Gatin-Grtjzewska3  pure  glycogen  wanders  distinctly 
to  the  anode. 

Precipitation  of  the  Colloids. 

The  colloids  can  be  separated  from  their  solutions  in  various  ways. 
Many  colloidal  solutions  are  so  unstable  that  they  flock  out  after  a 
time  without  the  addition  of  anything  (silicic  acid,  metallic  hydroxides). 
Certain  colloids  appear  as  flocculent  precipitates  on  heating  their  solu- 
tions (certain  proteins,  see  Chapter  II).  Others  solidify  on  cooling 
from  hot  concentrated  solutions,  as  semisolid  forms,  so-called  jellies 
or  hydrogels,  containing  considerable  water  (glue,  starch,  agar). 

On  evaporating  the  hydrosols  at  ordinary  temperature  we  obtain 
a  residue  which  Zsigmondy  divides  into  reversible  and  irreversible  col- 
loids, according  whether  they  are  again  soluble  in  water  or  not.4  Accord- 
ing to  this  definition  starch,  dextrin,  agar,  gum,  and  proteins  belong  to  the 
reversible  colloids  while  colloidal  silicic  acid,  stannic  acid,  colloidal  metallic 
hydroxides  and  sulphides,  and  the  pure  colloidal  metals  belong  to  the 
irreversible  colloids.  The  former  are  relatively  non-sensitive  toward 
the  addition  of  electrolytes,  while  the  latter  flock  out  on  the  addition 
of  the  smallest  quantity  of  electrolyte,  and  indeed  again  in  an  irreversible 
form.  This  classification  stands  in  accord  with  what  was  given  above 
(page  15),  as  the  reversible  colloids  coincide  in  a  measure  with  the 
hydrophile   colloids   and  the   irreversible   with   the   suspension   colloids. 

Electrolyte  Precipitation  of  Suspension  Colloids.  It  must  be 
remarked  that  for  every  precipitating  electrolyte  a  certain  minimal  con- 

1  See  page  74. 

*  See  Ergebnisse  d.  Physiologie,  12,  506  which  also  gives  the  literature. 

J  Pfliiger's  Arch.,  403,  287  (1904). 

4  Zur  Erkenntnis  d.  Koll.,  page  21. 


22  GENERAL  AND  PHYSICO-CHEMICAL. 

cent  rat  ion  is  necessary  to  bring  about  flocking.  In  comparing  the 
precipitation  ability  of  various  electrolytes  the  concentration  of  that 
solution  which  is  just  sufficient  to  cause  a  visible  cloudiness  is  given  in 
millimolls  (  =  roVir  gram-molecule)  per  liter. 

Hardy  1  has  also  found  that  colloids  which  wander  to  the  anode 
are  chiefly  flocked  out  by  the  cations  of  the  precipitating  electrolyte, 
and  colloids  wandering  to  the  cathode  are  chiefly  flocked  out  by  the  anions. 
H.  Schultze  2  has  proven  that  the  precipitating  ability  is  influenced 
greatly  by  the  valence  of  the  precipitating  ions,  as  the  divalent  ions  act 
much  stronger  than  the  monovalent  and  the  trivalent  are  still  more 
active  than  the  divalent.     This  rule  has  been  substantiated  by   Hardy.3 

This  valence  rule  becomes  clear  by  the  following  experiment  of  Freundlich.4 
The  figures  give  the  lowest  precipitation  concentration  expressed  in  millimolls 
per  liter.  The  hydrosol  was  As2S3  (negative)  and  the  valence  of  the  cations  is 
applicable  chiefly  for  the  precipitating  action. 

KgSOj  __  _  MgCl2 0.717 

~2~ b5b  MgSO-4 0.810 

KC1 49.5  CaCl2 0.649 

KNO, 50.0  SrCl2 0.635 

NaCl 51.0  BaCl2 .0.691 

LiCl 58.4  Ba(N03)2 0.687 

H*SOi  ^"Cl2 0.685 

— 2~ 30.1  U02(N03)2 0.642 


HC1 30.8 


AICI3 0.0932 

A!(NOs)3 0.0982 


The  precipitating  action  of  anions  upon  a  positive  hydrosol  (Fe[OH]3)  is  shown 
in  the  following  experiment  of  Freundlich  : 

KCJ 9.03  K2SO4 0.204 

KNO, 11.90  T12S04 0.219 

NaCl 9.25  MgS04 0.217 

BaGk 


.9.64  K2Cr207 0.194 


Freundlich  has  extended  the  valence  rule  by  the  fact  that  with  a  negative 
sol,  II  ions,  the  ions  of  the  heavy  metals,  as  well  as  organic  cations  in  weaker  con- 
centration, have  a  greater  precipitating  action  than  other  cations;  OH  ions  as  well 
SB  organic  anion-  act  against  the  precipitating  action  of  the  cations.  The  reverse 
own  with  a  positive  sol;  OH  ions  and  organic  anions  of  smaller  precipitation 
concentration  than  corresponds  to  their  valence;  H  ions  and  organic  cations 
gainst  the  precipitating  properties  of  the  anions. 

Certain  above-mentioned  suspensions  (mastic),  as  well  as  other  particles 
suspended  in  water,  act  the  same  as  suspension  colloids.  Schulze  5  has  found 
thai  cloudiness  due  to  'lav  particles  on  the  addition  of  clarifying  bodies  (alum, 
lime)  give  a  voluminous  deposition.     Schloessing  •  found  that  clay  suspensions 

1  Zeitschr.  f.  physik.  Chem.,  33,  385  (1900). 

'Journ.  prakt.  Chem.  (2),. 25,  431  (1882). 

■Proc.  Roy.  80c.,  66,  110  (1899). 

4  Zeitschr.  f.  Chem.  u.  Ind.  d.  Koll.,  1,  323  (1907). 

•Ann.  Phys.  (2),  12'.),  366  (1866).  , 

•Compt.  rend.,  70,  1345  (1870). 


COLLOIDS.  23 

which  do  not  settle  after  months  are  precipitated  in  24-48  hours  by  a  minimum 
quantity  of  lime  or  magnesia.  Be  also  calls  attention  to  the  essential  role  which 
the  salts  of  sea  water  must  play  in  the  sedimentation  of  the  cloudy  fresh  water 
flowing  into  the  sea  (delta  formation). 

In  consideration  of  the  conditions  just  mentioned,  under  which  the 
suspension  colloids  are  precipitated  by  electrolytes,  the  mutual  precipita- 
tion ability  of  suspension  colloids  is  of  considerable  interest.  Accord- 
ing to  what  lias  been  stated  previously,  the  colloids  are  considered  as  carriers 
of  electricity,  and  it  has  been  proved  that  the  oppositely  charged  col- 
loids can  act  precipitatingly  upon  each  other.  This  rule  was  first  pro- 
posed by  Linder  and  Picton,1  and  has  subsequently  been  substantiated 
by  many  investigators.  Biltz  2  has  made  especially  systematic  investiga- 
tions on  this  subject  and  finds  that  colloids  carrying  'the  same  kind  of 
charge  do  not  precipitate  each  other.  For  the  mutual  complete  precipita- 
tion of  opposed  electrically  charged  colloids,  a  certain  quantitative  rela- 
tion is  necessary.  On  the  action  of  two  colloids  with  opposite  charges  in 
variable  quantities  an  optimum  of  the  precipitation  action  is  noticed;  while 
on  overstepping  the  desirable  precipitation  conditions  in  both  directions 
no  precipitation  occurs  at  all. 

In  analogy  with  the  mutual  precipitation  ability  of  the  colloids,  Biltz  believes 
that  the  especial  great  ability  of  most  salts  of  the  heavy  metals  to  precipitate 
colloids  lies  in  the  hydrolytically  split  and  colloid-dissolving  metallic  hydroxides. 

Protective  Colloids.  Certain  hydrophile  colloids,  which  are  precip- 
itated with  difficulty  by  electrolytes,  have  the  power  of  protecting 
suspension  colloids  against  the  precipitating  action  of  electrolytes.  Meyer 
and  Lottermosser  3  have  found  with  silver  hydrosol  that  the  presence 
of  protein  prevented  the  flocking  out  by  electrolytes.  Zsigmondy  4 
has  investigated  the  relative  action  of  the  protective  colloids  and  has 
found  considerable  differences.  The  figure  in  milligrams  of  colloid  which 
is  just  insufficient  to  protect  10  cc.  of  gold  solution  (0.0053-0.0058 
per  cent)  against  the  action  of  1  cc.  10  per  cent  NaCl  solution  is  called  the 
gold  equivalent  for  the  respective  colloid.  Gelatin  offers  the  best  pro- 
tection, then  comes  isinglass,  casein,  ovalbumin,  gum  arabic,  Irish  moss, 
dextrin,  starch.  The  colloidal  sulphides  (As2^3.  Sb2S3,  CdS)  are  also 
protected  in  the  same  manner  against  the  influence  of  electrolytes  (A. 
Miller  and  Artmaxx5).     Inorganic  colloids  may  also  act  as  protective 


1  Journ.  chem.  Soc,  71,  572  (1897). 

2  Ber.  d.  d.  chem.  Gesellsch.,  37,  1095  (1904). 

3  Journ.  prakt.  Chem.  (2),  56,  241  (1897). 

4  Zeitschr.  analyt.  Chem.,  40,  697  (1901). 
5Oester.  Chem.  Ztg.,  7,  149  (1904). 


24  GENERAL  AND  PHYSICO-CHEMICAL. 

colloids.  Thus  according  to  Biltz  l  zirconium  hydroxide  protects  gold 
better  than  does  gelatin. 

By  the  addition  of  organic  protective  colloids,  the  inorganic  colloids 
which  on  evaporation  otherwise  become  irreversible,  are  made  reversible, 
in  that  the  dry  residue  is  soluble  in  water  again.  On  this  depends  the 
use  of  the  protective  action  in  the  preparation  of  permanent  inorganic 
hydrosols,  and  this  is  of  importance  in  many  cases. 

According  to  Bechhold2  the  filterability  of  suspension  colloids  through 
collodion  filters  is  increased  by  the  addition  of  organic  colloids.  It  is  also 
well  known  that  certain  finely  divided  substances  (carbon)  pass  more 
easily  through  a  filter  in  the  presence  of  protein  than  without  protein. 

The  action  of  the  protective  colloids  is  ordinarily  explained  accord- 
ing to  the  theor3r'of  Quincke3  on  the  mutual  surface  tension  of  the 
active  bodies,  and  the  process  belongs  accordingly  to  the  adsorption 
phenomenon  which  will  be  discussed  later.  According  to  this  theory 
the  protective  colloid  under  certain  conditions  spreads  like  an  envelope 
around  the  particles.  In  this  wise  the  entire  mass  takes  the  properties 
of  the  protective  colloid  and  is  therefore  not  precipitated  by  the  elec- 
trolyte any  more  than  the  protective  colloid  itself.  In  filtration  the  pro- 
tective colloid  acts  to  a  certain  extent  like  a  lubricant.  This  theory  of 
colloid  envelope  has  recently  received  support  by  experiments  of  Michaelis 
and  Pincussohn.4  They  found  that  when  suspensions  of  indophenol 
and  mastic  were  mixed  together  the  number  of  particles  visible  in  the 
ultramicroscope  diminished;  after  mixing,  the  physical  properties  of 
the  indophenol  (pseudofluorescence,  positive  cataphoresis)  were  not 
evident. 

Electrolyte  Precipitation  of  Hydrophile  Colloids.  The  salts  of  the 
alkalies  precipitate  the  suspension  colloids  even  in  low  concentrations. 
The  alkali  salts  behave  differently  toward  the  hydrophile  colloids.  This 
may  in  part  be  due  to  the  fact  that  hydrophile  colloids  have  much  less 
of  a  certain  electric  charge  than  the  suspension  colloids.  For  this  reason 
the  hydrophile  colloids  are  often  precipitated  from  their  solution  by  alkali 
salts.  For  this  purpose,  firstly,  certain  concentrations  are  necessary; 
secondly,  the  precipitates  of  the  hydrophile  colloids  are  again  solul  le 
in  water  (reversible)  in  opposition  to  those  of  the  suspension  colloids. 
In  regard  to  the  ability  of  different  alkali  salts  to  act  precipitatingly 
certain  laws  have  been  formulated,  but  they  cannot  be  arranged  in  a 
general  rule. 


1  Ber.  d.  d.  chem.  Gesellsch.,  35,  4431  (1902). 

Zeitechr.  f.  physik.  Chem.,  60,  301  (1907). 
'  Ann.  I'liys.  (3),  3.r>.  580  (1888). 
1  Bioch.  Zeitechr.,  2,  251  (1907). 


COLLOIDS.  25 

On  comparing  the  concentration  of  various  salts  just  sufficient  for  precipita- 
tion, where  at  one  time  the  same  anion  with  different  cations  was  tested  and 
another  time  the  same  cation  with  different  anions,  Pauli  has  arranged  the  cations 
and  anions  in  the  following  order  in  increasing  precipitation  ability: 

CNS  <I  <Br  <XO, <C1  <OCO.CH3  <HPO< <S04 
XH4<K<Xa<Li. 

The  protein  used  in  these  experiments  was  white  of  egg.  According  to  Pauli 
certain  ions  have  a  precipitating  action  and  others  a  solvent  action.  The  action 
of  a  salt  corresponds  to  the  algebraic  sum  of  the  action  of  the  ions.1  Pauli  has 
attempted  to  associate  the  precipitation  ability  of  the  salts  in  relation  to  their 
action  upon  the  coagulation  temperature,  but  without  any  positive  results.2 

Nevertheless  Spiro  !  has  shown  that  the  kind  of  protein  as  well  as  its  con- 
centration are  of  importance  for  the  precipitation  action,  and  Hober  *  has  recently 
shown  that  the  series  I<Br<Cl<S04  and  Li<Xa<K  <Rb  <Cs  is  valid  in 
alkaline  reaction,  but  that  the  series  is  reversed  in  acid  reaction.  In  nearly 
neutral  reaction  irregularities  in  the  ion  series  occur  which  can  be  considered  as 
a  transition  series  between  the  two  just-mentioned  series.  That  the  reaction  must 
be  of  great  importance  in  the  precipitation  of  proteins  seems  very  probable  in 
consideration  of  the  fact  that  the  proteins  take  a  decided  electric  charge  on  the 
addition  of  acid  or  alkali.  In  regard  to  the  precipitation  by  salts  of  the  heavy 
metals,  the  hydrophile  colloids  do  not  seem  to  differ  essentially  from  the  suspension 
colloids.5 

On  boiling  a  protein  solution  the  protein  suffers  an  irreversible  change 
and  under  certain  circumstances  flocks  out.  Boiled  but  not  flocked 
egg-white  behaves  with  precipitating  substances,  like  a  suspension  colloid.6 
In  regard  to  the  precipitation  of  proteins  see  Chapter  II. 

Theories  of  Precipitation  Phenomena. 

At  least  for  the  suspension  colloids  there  is  no  question  that  they 
are  flocked  out  by  ions  which  carry  an  electric  charge  opposite  to  the 
colloid  particles,  and  also  by  other  colloids  having  an  opposite  charge. 
This  fact  follows  from  Hardy's  theory,  according  to  which  the  flocking 
out  is  a  neutralization  process  in  which  the  charge  of  the  colloid  is 
just  neutralized  and  the  colloid  therefore  precipitates.7  The  mixture 
formed  on  precipitation  has  been  showm  to  be  electrically  neutral  (iso- 
electric) as  the  precipitated  particles  show  no  cataphoresis.  In  this 
manner  it  is  easily  understood  that  polyvalent  ions  have  a  stronger 
precipitating  action  than  monovalent,   as  the  electrical   charge  in,   for 

1  Hofmeister's  Beitrage,  3,  225  (1902). 

2  Pfluger's  Arch.,  78,  315  (1899). 

3  Spiro,  Hofmeister's  Beitrage,  4,  300  (1903). 
*  Ibid.,  11,35  (1908). 

5  Pauli,  Ibid.,  6,  233  (1905). 

6  Hardy,  Proc.  Roy.  Soc,  66,  110  (1900). 

7  Zeitschr.  f.  physik.  Chem.,  33,  385  (1900). 


26  GENERAL  AND  PHYSICO-CHEMICAL. 

example,  a  trivalent  ion  is  three  times  greater  than  in  a  monovalent  ion. 
Otherwise  greater  precipitation  ability  of  polyvalent  ions  can  also  be 
explained  by  a  greater  hydrolytic  cleavage  of  the  salts  (page  23). 

The  mechanism  of  the  precipitation  of  the  isoelectric  solution  accepted 
in  Hardy's  theory  is  explained  by  Bredig  x  as  follows:  At  the  boundary 
between  suspended  particles  and  solvent  a  certain  surface  tension  exists 
which  tries  to  diminish  the  total  contact  surface  between  the  two  media, 
which  can  happen  by  the  small  particles  uniting  to  form  larger  ones, 
when  nocking  is  brought  about.  The  electrical  charge  of  the  particles 
acts  against  the  surface  tension  so  that  equally  charged  particles  repel 
each  other.  If  the  electrical  charge  is  discharged,  as  takes  place  in  the 
isoelectric  point,  then  the  surface  tension  reaches  its  highest  value  and 
the  precipitation  may  occur. 

The  correctness  of  Hardy's  claim  that  precipitation  occurs  just  in 
the  isoelectric  fluid  is  disputed  on  special  grounds  by  Billitzer.  He 
believes  that  the  ions  have  a  much  greater  charge  than  the  colloid  par- 
ticles. An  ion  collects  the  oppositely  charged  colloid  particles  around 
itself,  and  during  these  neutralization  processes  it  may  occur,  that  the 
entire  complex  may  become  so  large  as  to  become  visual  and  on 
account  of  the  gravity  it  precipitates  out. 

In  general  it  can  be  stated  that  the  stability  of  a  colloid  is  greater  the  smaller? 
cet.  par.,  the  particles  are;  as  the  probability  that  the  number  of  particles  sufficient 
for  the  precipitation  is  then  less.  With  equal  size  of  particles  the  stability  of  a 
colloid  is  dependent  upon  the  size  of  the  charge  which  the  particles  carry.  Too 
weak  and  very  strongly  charged  colloids  are  relatively  more  stable;  the  first 
because  of  the  large  number  which  must  collect  around  an  ion  when  flocking  takes 
place  and  the  second  because  the  number  of  particles  required  for  the  neutrali- 
zation is  perhaps  too  small,  so  that  the  necessary  size  of  the  complex  for  precipita- 
tion is  not  attained.2 

The  findings  of  Linder  and  Picton  3  that  when  colloidal  AS2S3  is 
precipitated  with  BaCk>  the  solution  becomes  acid,  and  a  small  quantity 
of  barium  remains  in  the  precipitate,  corresponds  to  Billitzer's  theory. 
This  quantity  of  barium  cannot  be  removed  by  water,  but  can  be  replaced 
by  the  corresponding  cation  by  washing  with  a  solution  of  another  salt. 
According  to  Billitzer  in  the  mutual  precipitation  of  colloids  a  quan- 
tity relation  exists  which  is  dependent  upon  the  electrical  charges4  (see 
also   page  22). 

The  fact  that  the  precipitation  of  colloids  is  a  manifestation  of 
processes  which  occur  in  a  homogeneous  medium,  makes  the  understand- 
ing of  these  especially  difficult.     If,  as  is  generally  accepted,  we  consider 

1  Anorganische  Fermente  (1901),  15. 

Zeitechr.  f.  physik.  Chem.  Soc,  45,  327  (1904);  51,  129  (1905). 
Mourn.  Chem.  Soc,  67,  63  (1895). 
•Zeitechr.  f.  physik.  Chem.,  51,  141  (1905). 


COLLOIDS.  27 

the  colloid  BOlution  as  a  homogeneous  fluid  of  suspended  solid  or  fluid 
particles,  then  in  the  "  solution  "  there  occur  at  least  two  special  con- 
stituents] separated  from  each  other — the  colloid  particles  and  the  sol- 
vent. This  is  expressed  as  follows:  the  system  contains  two  phases. 
The  solvent  is  often  more  correctly  called  the  dispersion  means  and  the 
colloid  particles  called  the  disperse  phase.  If  to  such  a  system  a  new 
sul  stance  is  added,  then  the  reaction  which  follows,  depends  essentially 
upon  the  division  of  the  new  substance  between  the  two  phases.  In 
regard  to  the  possible  division  two  cases  will  be  presented: 

1.  The  process  can  be  similar  to  the  division  of  a  soluble  substance 
between  two  solvents.  If  a  substance  is  brought  in  contact  with  two 
solvents  at  the  same  time,  then  it  divides  itself  so  that  the  relation 
between  the  concentration  in  the  two  solvents  remains  the  same  but 
independent  of  the  total  quantity  of  the  dissolved  substance.  If  the 
quantity  of  substance  in  each  100  cc.  of  the  two  solutions  1  and  2  is 

Cl 

designated  bv  ci  and  c-2,  then  it  follows  that  — =k  where  k  is  a  constant.1 

C-2 

The  first  example  where  this  law  was  shown  to  be  correct  was  the  divi- 
sion of  succinic  acid  between  water  and  ether  (Berthelot  and  Jung- 
fleisch -'.).  This  law  was  also  shown  to  be  true  for  the  division  of 
a  gas  between  a  gaseous  and  a  fluid  phase,  i.e.,  for  the  absorption  of  a 
gas  in  a  fluid  (Henry's  law  of  absorption).  The  conditions  for  the  cor- 
rectness of  this  law  are  that  the  temperature  remains  the  same  in  experi- 
ments with  different  quantities  of  substance  as  well  as  that  the  substance 
has  the  same  molecular  size  in  the  two  phases. 

2.  In  those  cases  where  finely  divided  solids  take  up  dissolved  sub- 
stances or  gases  the  division  is  generally  not  independent  of  the  total 
quantity  of  the  dissolved  substance  or  of  the  gas.  This  is  often  called 
adsorption.3  For  example,  if  we  are  dealing  with  the  adsorption  of  a 
dissolved  substance  by  a  finely  divided  solid  occurring  in  a  solution, 
then  a  greater  percentage  is  taken  up  from  a  dilute  solution  than  from  a 
concentrated  one.  On  increasing  concentration  the  adsorbed  fraction 
becomes  continuously  less  so  that  the  absolute  quantity  taken  up  reaches 
a  maximum  which  corresponds  to  the  greatest  adsorption  ability  of  the 
solid  body. 

This  is  expressed  by  the  formula  —  =  k,  where  Ci  and  c2  indicate  the  concentra- 
tion of  the  solid  body  and  in  the  solution;  n  and  k  are  constants  and  indeed,  n  is 

'Nenut,  Zeitschr.  f.  physik.  Chem.,  8,  110  (1891). 

2  Ann.  Chim.  phys.  (4>.  26.  396  (1872). 

3  It  must  be  remarked  that  in  the  older  literature  oftentimes  no  difference  was 
made  between  adsorption,  and  absorption,  in  which  case  both  processes  were  included 
under  the  name  absorption. 


28  GENERAL  AND  PHYSICO-CHEMICAL. 

always  >  1.     (If  n  =  \  then  the  formula  would  be  —=k  and  we  would  be  dealing 
with  a  so-called  solid  solution.) 

Appleyard  and  Walker  l  have  studied  the  adsorption  of  organic 
acids  from  aqueous  and  alcoholic  solutions  by  means  of  silk;  the  divi- 
sion was  found  to  correspond  to  the  above  formula  for  adsorption. 
Freundlich  2  has  also  carefully  tested  the  adsorption  of  crystalloids 
by  carbon.  From  these  experiments  it  was  shown  that  the  equilibrium 
could  be  quickly  attained  from  both  sides,  i.e.,  that  the  process  was  readily 
reversible.  The  above-given  formula  was  found  sufficiently  accurate 
for  the  case  wrhere  only  the  total  quantity  of  the  dissolved  (to  adsorb) 
substance  varied.  The  series  in  which  the  organic  acids  were  adsorbed 
by  silk,  as  found  by  Appleyard  and  Walker,  were  pratically  the  same 
as  with  carbon.     The  influence  of  temperature  was  slight. 

According  to  Kuster,3  the  combination  between  starch  and  iodine 
is  to  be  considered  as  an  adsorption  compound,  and  Biltz  4  finds  for  the 
division  of  AS2O3  between  iron  hydroxide  (1)  and  water  (2)  the  for- 
mula—  =  0.631. 

The  theoretical  foundations  for  the  adsorption  phenomenon  are 
not  especially  clear.  Generally  the  adsorption  is  considered  as  con- 
nected with  segregation  and  surface  tension  phenomenon.  At  the  con- 
tact surface  between  a  solid  body  and  solution  a  surface  tension  exists 
which  is  considered  as  positive,  i.e.,  this  attempts  to  diminish  the 
contact  surface.  The  surface  energy  used  thereby  tends  to  be  a  min- 
imum potential  energy.  As  the  product  from  size  of  surface  and  surface 
tension  are  the  same,  and  as  the  first  cannot  change,  the  surface  energy 
can  only  be  diminished  by  a  reduction  of  the  tension.  If,  therefore, 
the  tension  is  diminished  by  increasing  the  concentration  of  a  sub- 
stance dissolved  in  a  fluid,  then  this  substance  tries  to  collect  itself 
at  the  surface  in  greater  concentration  than  in  other  parts  of  the  fluid 
(Ostwald,5  Freundlich  6).  In  regard  to  the  surface  tension  of  solid- 
fluid  we  only  know  that  it  is  positive,  but  can  otherwise  show  great 
differences  (Ostwald,7  Hulett8).  According  to  this  theory  the  facts 
are  that  certain  solid  substances  possess  the  ability  of  adsorbing  dis- 

1  Journ.  Chem.  Soc,  69,  1334  (1896). 

2  Debar  die  Adsorption  in  Losungen,  Leipzig  (1906). 
»  Ann.  d.  Chem.  u.  Pharm.,  283,  360  (1894). 

*  Ber.  d.  d.  chem.  Gesellsch.,  37,  3138  (1904). 

•Lehrh.  d.  allg.  Chem.,  2.  Aufl.,  2.  Bd.,  3.  Teil,  237  (1906). 

8  Ueber  Adsorption  in  Losungen,  50-51. 

7  Zeitschr.  f.  physik.  Chem.,  34,  495,  1900. 

•Ibid.,  37,  385  (1901). 


COLLOIDS.  29 

solved  bodies,  and  for  this  reason  the  ads  >rbed  substance  lowers  the 
surface  tension  of  the  solid-fluid,  and  indeed,  the  more  the  greater  con- 
centration in  which  it  occurs.  That  especially  carbon  and  colloid  sub- 
stances are  adsorption  bodies  lies  in  the  fact  that  they  have  an  especially 
large  surface  due  to  their  finely  divided  state  or  porosity,  which  there- 
fore, cet.  par.,  must  give  them  a  great  surface  energy. 

That  proteins,  on  precipitation,  carry  down  other  bodies  with  avidity 
is  well  known;  inorganic  hydrogels  also  take  up  dissolved  substances 
with  energy.  The  curves  obtained  for  the  latter  process  by  van  Bem- 
melen  x  show  a  close  analogy  with  the  characteristic  curves  for  the 
adsorption  compounds.     It  often  occurs  that  the  body  taken  up  homo- 

geneouslv  saturates  the  hydrogel,   in  which  case  —  =  k,  and  a  sort  of 

solid  solution  is  the  result.  In  certain  cases,  undoubtedly,  chemical 
combinations  with  quite  positive  conditions  are  formed. 

The  precipitation  of  colloids  by  electrolytes  has  also  been  discussed 
by  Freundlich  2  from  the  standpoint  of  the  adsorption  hypothesis. 
Thus,  for  the  precipitation  ability  of  an  electrolyte,  the  electric  charge 
of  the  precipitating  ion  comes  first  into  consideration  and  secondly,  the 
ability  of  the  precipitating  colloid  to  adsorb  the  same.  According  to 
Moore  and  Roaf3  the  salts  of  the  red  corpuscles  are  retained  as  adsorp- 
tion compounds  (adsorpates)  by  the  proteins. 

Thus  far  only  the  adsorption  of  crystalloids  has  been  considered. 
Colloids  are  also  taken  up  by  solid  substances  or  by  other  colloids.  Still  in 
these  cases  the  conditions  are  more  complicated  than  in  the  above- 
mentioned  adsorption  phenomena,  as  the  combinations  formed  are  in  special 
cases  irreversible  or  gradually  become  irreversible.  It  is  well  known  that 
carbon  takes  up  colloidal  colored  substances,  and  we  have  numerous  exam- 
ples of  the  combination  of  dissolved  colloids  with  solid  colloids  in  technology. 
Biltz  4  has  been  able  to  show  that  many  dyeing  processes  are  to  be 
considered  as  adsorption  phenomena,  and  later  Freundlich  and  Losev  5 
have  measured  the  adsorption  of  basic  and  acid  pigments  by  carbon 
and  also  by  fibers  (wool,  silk,  cotton),  and  have  shown  the  correspondence 
of  the  two  processes.  With  the  basic  pigments,  which  were  used  as 
salts,  a  splitting  occurred  into  a  pigment  base,  which  was  taken  up  by 
the  fibers  as  well  as  by  carbon,  and  an  acid  which  quantitatively  remained 
behind.  This  is  similar  to  the  cleavage  which  precipitating  electrolytes 
undergo  in  the  precipitation  of  the  suspension  colloids   (see  page  26). 

1  Zeitschr.  anorg.  Chem.,  23,  111,  321  (1900). 

2  Zeitschr.  f.  Chem.  u.  Ind.  d.  Koll.,  1,  321  (1907). 

3  Bioch.  Journ.,  3,  55  (1908). 

4Ber.  d.  d.  chem.  Gesellsch.,  37,  1766  (1904);  3S,  2963,  2973,  4143  (1905). 
5  Zeitschr.  f.  physik.  Chem.,  59,  284  (1907). 


30  GENERAL  AND   PHYSICO-CHEMICAL. 

Tanning  is  also  brought  about  by  adsorption  processes,  as  the  prepared  skins 
adsorb  the  tanning  substance.1 

The  precipitation  of  portein  by  adding  finely  divided  solids  (carbon* 
kaolin 2)  or  by  suspended  solids  (mastic 3)  precipitated  in  the  liquid, 
as  well  as  the  action  of  protective  colloids  as  already  mentioned  are  also 
due  to  adsorption  processes.  The  precipitation  of  protein,  which  occurs 
on  shaking  the  protein  solution  with  liquids,  in  which  the  protein  is 
not  soluble,  is  also  to  be  considered  as  a  surface  tension  action  (Ramsden4). 

Bechhold,3  in  his  above-mentioned  experiments  on  the  filtration  of 
colloids,  has  observed  conditions  which  he  considers  as  adsorption  phe- 
nomena. Under  certain  circumstances  a  colloid  can  prevent  the  filtra- 
tion of  another  colloid.  A  filter  which  was  permeable  for  colloidal  AS2S3, 
but  retained  colloidal  Prussian  blue,  did  not  allow  a  clear  mixture  of 
the  two  to  pass  through.  The  particles  of  AS2S3,  were  adsorbed  by  the 
particles  of  Prussian  blue,  and  could  therefore  not  pass  through  the 
filter. 

Gels.  We  have  often  mentioned  gels  or  jellies  (page  14).  Only 
certain  colloids  can  occur  in  the  form  of  gels.  Certain  gels  are  spon- 
taneously formed  in  sufficiently  concentrated  solutions  (silicic  acid, 
certain  metallic  hydroxides)  and  these  do  not  redissolve  in  water.  Other 
gels,  like  gelatin  and  agar,  are  formed  on  cooling  of  the  hot,  concentrated 
solutions,  and  are  again  soluble  in  water. 

According  to  Hardy  6  the  gel  formation  of  gelatin  is  to  be  considered 
as  a  segregation  process  whereby  a  separation  into  two  fluids  occurs, 
one  of  which  solidifies.  The  two  phases  are  only  differentiated  by  the 
microscope,  and  the  chemical  testing  of  the  theory  fails  because  of  the  cir- 
cumstances that  the  two  phases  cannot  be  analyzed  separately.  In  opposi- 
tion to  this  Pauli  claims  that  the  gel  passes  through  all  of  the  intermediary 
steps  into  the  corresponding  sol  and  is  therefore  homogenous  in  the  same 
sense  as  these.7 

When  gels  are  freed  from  water  by  evaporation  or  in  other  ways, 
they  show  a  special  ability  to  take  up  water,  which  is  brought  about 
by  different  processes  which  are  included  in  the  ordinary  term  imbibition. 
The  views  on  this  imbibition  are  indefinite.  Surface  phenomena  play 
a  role  here.     According  to  van  Bemmelen  8  the  water  is  not  chemi- 


1  See  Zeitschr.  f.  Chem.  u.  Ind/d.  Koll.,  2,  257  (1908). 

2  Bioch.  Zeitschr.,  5,  365,  1907. 

•  Ibid.,  2,  219  (1906);  3,  109  (1906). 

•  Zeitschr.  f.  physik.  Chem.,  47,  343  (1904). 
1  Ibid.,  60,  299  (1907). 

•  Ibid.,  33,  326  (1900). 

■>  Bioch.  Zeitschr.,  18,  367  (1909). 

'Zeitschr.  anorg.  Chem.,  13,  233  (1896);  20,  185  (1899). 


COLLOIDS.  31 

cally  combined  in  definite  proportions,  hut  the  quantity  continually 
changes  with  the  temperature  and  the  vapor  pressure.  On  the  other 
hand,  the  imbibition  stands  in  close  relation  to  the  osmotic  pressure 
which  is  evident,  if  we  define  the  osmotic  pressure  of  a  substance  as 
its  ability  to  attract  water.  The  relation  between  imbibition  and 
osmotic  pressure  is  still  closer  in  those  cases  when  the  Substance  finally 
is  dissolved  in  water. 

If  a  hydrogel  is  placed  in  a  salt  solution  instead  of  in  pure  water, 
the  imbibition  phenomena  essentially  change.  This  was  first  studied 
by  Hofmeister,1  using  gelatin  plates.  The  process  is  rather  com- 
plicated, as  salt  is  taken  up  by  one  side  of  the  gelatin  plate  and  water 
by  the  other,  and  the  taking  up  of  water  is  influenced  by  the  quantity 
of  salt  taken  up.  It  has  also  been  found  that  when  gelatin  plates  are 
treated  with  solutions  of  increasing  concentration  of  the  same  salt, 
the  taking  up  of  salt  increases  at  first  with  the  salt  concentration,  then 
becomes  slower,  and  attempts  to  reach  a  maximum  and  then  remains 
almost  stationary.  As  long  as  the  taking  up  of  salt  increases,  the  quan- 
tity of  water  passing  into  the  gelatin  also  increases;  when  the  salt  fails 
to  pass  then  the  water  also  ceases  to  pass.  It  has  also  been  found  that 
the  maximum  of  salt  absorption  for  sulphate,  tartrate  and  citrate  can 
be  attained  with  much  lower  molecular  concentrations  than  with  chloride, 
nitrate  and  bromide.  From  this  it  follows  that  the  sulphate,  tartrate 
and  citrate  have  a  retarding  action  upon  imbibition  within  certain  limits 
of  concentration,  while  the  chloride,  nitrate  and  bromide  have  an 
accelerating  action. 

Pauli  2  has  investigated  the  influence  of  salt  solutions  upon  the  solid- 
ification and  melting-point  of  gelatin.  If  the  salts  are  arranged  in  the 
order  of  their  ability  to  lower  the  solidification  point  of  gelatin  we 
come  to  the  series  sulphate,  citrate,  tartrate,  acetate  (water),  chloride, 
chlorate,  nitrate,  bromide,  iodide.  This  series  corresponds  well  with 
that  of  Hofmeister. 

Acids  and  alkalies  exert  a  special  influence  upon  gelatin,  as  they 
both,  in  very  dilute  solutions,  strongly  accelerate  imbibition  (Spirq,3 
Wo.  Ostwald4).  From  the  previously  mentioned  investigations  of 
Lillie,  on  the  osmotic  tension  of  gelatin  solutions,  it  was  found  that  the 
addition  of  acids  and  alkalies  increased  it  (page  17). 

Since  Graham's  fundamental  experiments  it  was  believed  that  col- 
loidal sols  could  not  diffuse  into  gels  while  crystalloids  could  pass  just 


1  Arch.  f.  exp.  Pathol,  u.  Pharm.,  28,  210  (1891). 

2  Pfluger's  Arch.,  71,  333  (1898). 

3  Hofmeister's  Beitriige,  5,  276  (1904). 

4  Pfluger's  Arch.,  108,  563  (1905). 


32  GENERAL  AND  PHYSICO-CHEMICAL. 

as  quickly  into  gels  as  into  pure  water.  Nevertheless,  Spiro  *  has  observed 
that  dissolved  ovalbumin  as  well  as  haemoglobin  could  pass  into  gelatin 
plates.  On  the  other  hand  K.  Meyer  2  as  well  as  Bechhold  and  Zeigler  3 
have  found  that  the  distance  passed  by  a  crystalloid  in  gelatin  may  be 
much  shorter  than  in  pure  water.  In  such  experiments  no  doubt  adsorp- 
tion processes  must  be  considered. 

m.     CATALYSIS. 

When  two  bodies  which  can  act  chemically  upon  each  other  are 
brought  together  the  reaction  generally  takes  place  so  fast  that  it  can- 
not be  measured.  In  other  cases,  by  special  means,  we  can  observe 
how  the  reaction  gradually  proceeds.  When  cane-sugar  is  inverted 
by  weak  acid,  the  decrease  in  the  rotation  of  the  solution  can  be  fol- 
lowed with  the  polariscope;  and  when  an  ester  is  decomposed  by  alkali 
the  quantity  of  still  free  alkali  can  be  determined  by  titration.  The 
quantity  of  substance  measured  in  gram-molecule  per  liter  (mole) 
which  is  decomposed  in  the  unit  of  time,  is  called  the  reaction  velocity 
of  the  system.  The  so-called  law  of  mass  action,  as  proposed  by  Gtjld- 
berg  and  Waage,  states  that  the  reaction  velocity  is  every  moment 
proportional  to  the  molecular  concentration  of  the  reacting  bodies.  A 
mixture  cf  alcohol  and  acetic  acid  is  transformed  into  acetic  ether  and 
water,  especially  in  the  presence  of  some  mineral  acid.  If  the  molec- 
ular concentration  of  the  alcohol  and  acid  be  designated  by  CA  and  Cs, 
then  according  to  the  law  of  mass  action  the  reaction  velocity  is 
vi=ki.CA-Cs,  where  &i  indicates  a  constant  which  is  independent  of 
the  quantity  of  reacting  substances  and  the  time  limit  is  so  short  that 
the  concentration  can  be  considered  as  constant.  This  reaction,  like 
many  others,  is  reversible,  #i.e.,  two  reactions  occur  simultaneously: 
one  between  the  alcohol  and  acetic  acid,  producing  acetic  ether  and 
water,  and  second,  between  acetic  ether  and  water,  re-forming  alcohol 
and  acetic  acid.     This  is  expressed  as  follows: 

C2H5.()H+HO.CO.CH3^C2H5.O.CO.CH3+H20. 

The  velocity  of  reaction  when  it  passes  from  left  to  right  is  called 
V\.  If  the  velocity  in  the  reverse  reaction  is  called  v<z  and  the  molecular 
concentration  of  the  acetic  ether  and  water  is  called  CB  and  Cw, 
then  we    obtain  V2  =  ko- Ce-Cw.     At  the  beginning  when  CE  as  well  as 


1  Hofmeister's  Beitriige,  5,  294  (1904). 

2  Ibid.,  7,  393  (1905). 

1  Zeitschr.  f.  physik.  Chem.,  56,  105  (1906). 


CATALYSIS.  33 

Cw  =  0,  the  velocity  of  the  ester  formation  is  expressed  by  the  formula 
Vi  =  ki-CA-Cs\  afterward  it  is  expressed  by  the  difference  v\  —  V2  or 
ki'CA'Cs—k2-CE-Cw.  Of  the  two  reaction  velocities  V\  and  V2  at  the 
begining  vi  always  diminishes  while  V2  increases.  When  k\-CA-Cs  = 
A*2"Ce-CV  is  attained,  then  the  velocity  of  both  reactions  is  the  same; 
no  measurable  decomposition  occurs  and  the  system  is  in  equilibrium. 
The  equilibrium  condition  is  the  same  irrespective  of  whether  we  start 
from  alcohol +acetic  acid  or  from  the  corresponding  quantity  of  acetic 
ether + water.     On  equilibrium  it  is 

h.CA.Cs  =  h.CE.Cw  or  ^^-J^  =  K. 
t       Le-^w     m 

K  is  called  the  equilibrium  constant;  as  is  apparent  it  can  be  determined 
in  two  ways — either  from  the  concentration  of  the  reacting  bodies  when 
equilibrium  is  present  or  from  the  velocity  coefficient  k\  and  fo  as  deter- 
mined in  a  manner  given  below. 

In  the  above-mentioned  transformation  of  alcohol  and  acetic  acid 
these  two  bodies  are  simultaneously  used  up.  The  reaction  is  therefore 
called  bimolecular,  and  a  reaction  is  called  mono-,  bi-,  tri-,  etc.,  molecular 
according  to  the  number  of  the  kinds  of  molecules  which  diminish  their 
concentration  thereby.1 

Berzelius  2  found  that  certain  bodies  by  their  mere  presence,  and 
not  by  their  affinity,  have  the  power  of  awakening  the  dormant  affinity 
at  a  certain  temperature,  i.e.,  the  power  of  starting  a  reaction.  These 
phenomena  were  called  catalytic  by  Berzelius. 

According  to  Ostwald  3  catalysis  is  the  acceleration  (or  retardation) 
of  a  slow-proceeding  chemical  change  by  the  presence  of  a  foreign  body. 
That  body  which  influences  a  reaction  in  this  manner  is  called  a  catalyst. 
It  does  not  itself  undergo  any  appreciable  change  by  the  reaction. 

Catalytic  reactions  have  been  studied,  especially  by  Wilhelmy.4 
van't  Hoff,5  Ostwald,6  Arrhenius  7  and  Bredig.8  Of  all  other  sub- 
stances the  acids  and  alkalies  seem  to  act  most  catalytic.     A  well-known 


1  It  is  assumed  here  that  of  every  kind  of  molecule  one  molecule  of  each  takea 
part  in  the  reaction. 

o 

2  Berzelius,  Arsberattelse  om  framstegen  i  Fysik  och  Kemi.,  13,  p.  245  (1836). 

3  Lehrb.  d.  aUg.  chem.  2.  Aufl.  II.,  1,  515. 

4  Poggendorff's  Ann.,  81,  413  (1850). 

5  Etudes  de  dynam.  chim.  (1884). 

'  6  Lehrb.  d.  aUg.  Chem.,  2.  Auf.  II,  2,  199. 
7Zeitschr.  f.  physik.  Chem.,  4,  226  (1889). 
8  Anorganische  Fermente  (1901);  Bioch.  Zeitschr.,  6,  283  (1907). 


34  GENERAL  AND  PHYSICO-CHEMICAL. 

example  is  the  inversion  of  cane-sugar  by  means  of  acid.  This  reac- 
tion is  monomolecular  because  only  the  cane  sugar  is  consumed.  If 
the  concentration  of  the  cane-sugar  at  the  beginning  is  C  moles,  and  if 
x  moles  are  transformed  in  t  time,  then  at  that  time  there  are  (C  —  x) 
moles  remaining.     If   dx  indicates  the   quantity  which  is   transformed 

dx 
in   dt   time,   then  the   reaction  velocity  is  — .     According  to   the  law 

dt 

of  mass  action  this  is  at  every  moment  proportional  to  the  concentration 

of  the  decomposing  substance,  or 

~=k.(C-x) '.     .     .     (1) 

dt 

For  practical  use  this  equation  is  integrated  into  the  following: 

A,-  =  -nat.  log. -— (2) 

t  C  —  x 

If  the  theoretical  considerations  upon  which  this  formula  is  based 

are  correct,  then  the  x  values  determined  by  the  polariscope  after  various 

times  must  give  the  same  figure  for  k.     This  is  indeed  the  case.1     k  is 

called  the  velocity  coefficient  (also  velocity  constant  or  specific  reaction 

velocity).     If  in  the  equation  (1)  C  —  x  or  the  concentration  of  the  still 

dx 
undecomposed  cane-sugar  =1,   then  the  equation  becomes  ~r  =  k,    from 

which  it  follows  that  k  indicates  the  reaction  velocity  if  the  concentra- 
tion of  the  substrate  could  be  kept  the  entire  time  at  =1. 

In  these  experiments  k  retains  the  same  value.  If  in  different  experi- 
ments the  quantity  of  catalyst  (acid)  varies,  then  the  obtained  value 
for  k  is  proportional  to  the  concentration  of  the  H  ions.  This  is  so 
prominent  that  the  catalytic  action  of  acids  is  due  to  the  H  ions  (Ar- 
rhenius  2) .  Still  irregularities  occur  as  the  anions  of  acids  as  well  as 
of  salts  present  can  under  certain  circumstances  influence  the  action  of 
H  ions  (see  page  70). 

Frankel  3  has  recently  studied  the  decomposition  of  diazoacetic  ether  under 
the  influence  of  different  acids.     The  reaction  is  as  follows: 

Na  :  HC.CO.O.C2H&+H20=HO.CH2.CO.O.C2H5+i\2. 

1  See  Poggend.  Ann.,  81,  413  and  499  (1850). 
2Zeitschr.  f.  physik.  Chem.,  4,  226  (1889). 
3  Ibid.,  60,  202  (1907). 


CATALYSIS. 


35 


The  progress  of  the  reaction  can  be  determined  by  measuring  the  nitrogen 

set  free.     The  following  figures  explain  the  results: 


Acid. 

Cone,  of  the 

Acid  in  Mol. 

per  Liter. 

'On- 

Cone,  of  the 

H  ions  liy 

Electric 

Conductivity. 

K 

Velocity 
Coefficient. 

K 

Nitric  acid 

0.001820 

0 . 000909 
I)  000909 
0 . 000364 
0 . 009900 
0.003640 
0.009090 
0.018200 

0  001820 
0.000909 
0.000909 

0.000364 
0.001680 
0.001460 
0.C00724 
0.000563 

0 . 0703 
0.0346 

0.0356 
0.0140 
0 . 0632 
0.0571 

0 . 0285 
0.0218 

38.7 

Picric  acid 

w-Nitrobenzoic  acid 

Fumaxic  acid 

38.0 
39.2 
38.3 
37.7 
39.1 

Succinic  acid 

38.5 

Acetic  acid 

38.7 

7.' 

As  77-for  the  different  acids  and  different  quantities  of  acid  is  the  same,  then 
the  velocity  coefficient  is  here  also  proportional  to  the  concentration  of  the  H  ions. 

As  the  catalytic  action  of  acids  is  caused  by  the  H  ions,  so  are  the 
catalytic  properties  of  bases  due  to  the  OH  ions.  The  first  determined 
case  of  this  kind  was  the  transformation  of  byoscyamine  into  the  stable 
atropine.1 

Koelichen  2  has  studied  a  specially  pretty  case  of  the  catalytic  action  of 
OH  ions  in  the  decomposition  of  diacetonalcohol  into  acetone : 

CH3.CO.CH,.C(CH3)2.OH  =  2CH3.CO.CH3. 

The  reaction  is  reversible,  and  from  the  following  table  it  is  seen  that  the 
velocity  constant  for  various  concentrations  of  the  same  catalyst  remains  the  same 
as  well  as  by  using  different  bases. 

Catalyst.  Cone,  of  the  Velocity 

Catalyst.  Constant. 

Piperidine 0.1090  0.038 

Triethylamine 0 .  4900  0 .  036 

Ammonia 0.5500  0.038 

Tetraethvlammonium           /  0.0760  0.037 

hydroxide \  0.0076  0.037 

«  j.        ,     ,       •  ,  /  0.0725  0.036 

Sodium  hydroxide |  Q  m2  Q  Q35 

By  this  a  rule  which  van't  Hoff  and  Ostwald  3  proved  by  thermo- 
dynamic means,  is  substantiated,  namely,  that  the  equilibrium  at  con- 
stant temperature  does  not  change  with  the  quantity  and  kind  of  catalyst 
when  the  catalyst  is  not  changed  by  the  reaction. 

Among  other  kinds  of  ions  which  act  as  catalysts  we  must  mention  (1)  iodine 
ions,  which  decompose  H202  in  proportion  to  their  concentration,4  and  (2)  cyan- 


1  Ber.  d.  d.  chem.  Gesellsch.,  21,  2777  (1888). 
2Zeitschr.  f.  physik.  Chem.,  33,  129  (1900). 

3  Van't  Hoff,  Vorlesungen,  1,  211. 

4  Walton,  Zeitschr.  f.  physik.  Chem.,  47,  185,  1904. 


36  GENERAL  AND  PHYSICO-CHEMICAL. 

ions,   which  transform  benzaldehyde  into   benzoin  according  to  the  following 
equation: 

2C6H5.COH  =C6H6.CO.CH(OH).C6H5.1 

If  those  bodies  which  accelerate  a  reaction  are  to  be  considered  as  catalysts, 
then  certainly  the  solvents  must  belong  to  the  catalytes.  Attention  must  be 
called  to  the  enormous  influence  which  the  solvent  can  exert  upon  the  velocity 
of  a  reaction  under  otherwise  equal  conditions.  Thus  Menschutkin  2  found  for 
the  reaction 

(C2H5)3.N+C2H5.I  =  (CJBi)4.N.I.f 

the  following  velocity  in  different  solvents: 

Hexane 0.00018 

Heptane 0 .000235 

Xylene 0.00287 

Benzene 0.00584 

Ethyl  alcohol 0.03660 

Benzyl  alcohol 0. 13300 

Recently  Bredig  and  Fajans  3  have  been  able  to  show  that  an  optically  active 
solvent  can  help  in  the  decomposition  of  optical  antipodes  to  a  varying  extent. 
Of  the  optical  antipodes  of  campho-carboxylic  acid,  the  d-form  is  17  per  cent 
more  quickly  decomposed  than  the  Z-form,  when  they  are  dissolved  in  nicotine 
or  when  nicotine  is  present,  dissolved  with  the  catalyte,  while  in  an  optically 
indifferent  solvent  and  without  any  nicotine  the  catalyte  decomposes  both  forms 
with  equal  rapidity.  The  reaction  proceeds  differently  with  or  without  catalyst, 
and  hence  the  catalyst  brings  about  changes  in  reaction  other  than  those  of  velocity. 
It  is  apparent  that  this  does  not  conform  with  Ostwald's  definition  of  a  catalyst 
(page  33).  It  must  be  mentioned  that  Bredig  and  Fiske  have  been  able  to 
perform  the  asymmetric  synthesis  of  benzaldehyde  and  hydrocyanic  acid  by  means 
of  quinine  and  quinidine  as  catalysts  (page  60). 

Catalysis  in  Heterogeneous  Systems.  The  above-treated  catalytic 
processes  all  occur  in  homogenous  systems,  i.e.,  the  systems  which  by 
mechanical  means  cannot  be  separated  into  different  constituents.  In 
heterogeneous  systems  with  phases  which  can  be  separated  from  each 
other  by  mechanical  means,  catalytic  reactions  can  also  occur,  and 
indeed,  in  such  cases  the  substances  taking  part  [in  the  reaction  and 
the  catalyst  occur  in  different  phases.  Such  a  reaction  is  the  union  of 
detonating  gas,  the  synthesis  of  SO3  (from  SO2  and  O),  and  the  decom- 
position of  H2O2  by  platinum.  In  case  the  system  is  two-phased,  and 
the  reaction  takes  place  only  at  the  boundary  between  both  phases,  or 
in  the  one  we  can  differentiate  two  simple  limits: 

1.  The  accumulation  of  the  bodies  which  are  necessary  for  the 
reaction  at  the  proper  place  takes  such  a  short  time  that  in  comparison 


1  Stern,  ibid.,  50,  513  (1905). 

2  Ibid.,  6,  41  (1890). 

8  Ber.  d.  d.  chem.  Gesellach.,  41,  752  (1908). 


BNZYME8.  37 

with   the  real   chemical   reaction   it    can   be   neglected.     In  these   cases 
the  reaction  velocity  behaves  similarly  to  a  homogeneous  system.1 

2.  The  chemical  reaction  occurs  at  a  rate  which  in  comparison  with 
the  time  necessary  for  the  accumulation  can  be  neglected.  In  this 
case  the  time  necessary  can  be  generally  compared  with  a  diffusion 
process.2 

The  catalytic  processes  in  heterogeneous  systems  have  excited 
interest  since  Bredig  3  showed  that  the  colloidal  metals  prepared  by 
him  showed  catalj'tic  properties.  The  best-studied  process  is  the  decom- 
position of  H2O2  by  colloidal  platinum,  gold,  and  other  metals  or  oxides 
(Mn02,  Pb02).  Attention  must  be  called  to  the  small  quantity  of 
catalyst  sufficient  to  decompose  H2O2.  The  action  of  1  gram  atom 
platinum  in  70  million  liters  of  reaction  mixture  has  been  detected.  The 
decomposition  of  H0O2  by  platinum  catalyst  in  nearly  neutral  or  faintly 
acid  solution  has  been  shown  to  be  a  monomolecular  reaction. 

Still  certain  differences  occur  from  the  conditions  formed  in  the 
homogeneous  catalysis.  At  one  time  in  certain  experiments  the  value 
for  A;  rises  considerably  during  the  catalysis,  and  secondly,  k  is  not 
proportional  to  the  ferment  concentration,  but  rises  more  quickly  than 
this. 

In  connection  with  these  experiments  Bredig  has  expressed  the 
view  that  an  analogy  exists  between  the  catalytic  processes  of  the  inor- 
ganic world  and  the  enzyme  action  of  the  organic. 

The  following  important  facts  give  support  to  Bredig's  view: 

1.  In  both  cases  we  are  dealing  with  catalytic  processes;  the  metallic  sol  and 
the  enzyme  are  active  in  very  small  quantities  and  during  the  reaction  they  do 
not  undergo  any  appreciable  change. 

2.  In  the  decomposition  of  H202  by  platinum  sols  or  by  the  enzyme  haemase, 
the  reaction  is  monomolecular. 

3.  The  action  of  metallic  sols  as  well  as  enzymes  is  paralvzed  by  certain 
poisons  (HCN,  H2S). 

4.  Both  classes  of  bodies  are  colloid  substances  and  possess  an  enormous 
surface  upon  which  their  catalytic  action  depends. 

According  to  Neilson,4  ethyl  butyrate,  salicin  and  amygdalin  are  decom- 
posed by  platinum  black  as  well  as  by  enzymes. 

IV.     ENZYMES. 

Chemical  Processes  in  Plants  and  Animals.  It  follows  from  the  law 
of  the  conservation  of  matter  and  of  energy  that  living  beings,  plants 
and  animals,  can   produce  neither   new  matter   nor  new  energy.     They 

1  Goldschmidt.  Zeitschr.  f.  physik.  Chem.,  31,  235  (1899). 

2  Nernst  and  Brunner,  ibid.,  47,  52  and  56  (1904). 

3  Anorganische  Fermente,  Leipzig,  42  (1901). 

4  Amer.  Journ.  of  Physiol.,  10,  191  (1904);  15,  148  (1906). 


38  GENERAL  AND   PHYSICO-CHEMICAL. 

are  only  called  upon  to  appropriate  and  assimilate  material  already  exist- 
ing and  to  transform  it  into  new  forms  of  energy. 

Out  of  a  few  relatively  simple  combinations,  especially  carbon 
dioxide  and  water,  together  with  ammonium  compounds  or  nitrates, 
and  a  few  mineral  substances,  which  serve  as  its  food,  the  plant  builds 
up  the  extremely  complicated  constituents  of  its  organism — proteins, 
carbohydrates,  fats,  resins,  organic  acids,  etc.  The  chemical  work 
which  is  performed  in  the  plant  must,  therefore,  in  the  majority  of  cases, 
consist  in  syntheses;  but  besides  these,  processes  of  reduction  take 
place  to  a  great  extent.  The  radiant  energy  of  the  sunlight  induces 
the  green  parts  of  the  plant  to  split  off  oxygen  from  the  carbon  dioxide 
and  water  and  this  reduction  is  generally  considered  as  the  starting- 
point  in  the  syntheses  that  follow.  According  to  a  hypothesis  suggested 
by  A.  Baeyer,1  formaldehyde  is  first  produced,  C02+H20  =  CH20+02, 
which  by  condensation  is  transformed  into  sugar.  From  the  sugar  other 
bodies  can  then  be  built  up. 

With  the  aid  of  the  silent  electric  discharge  W.  Loeb  2  has  succeeded 
in  obtaining  from  carbon  dioxide  and  water,  formaldehyde,  and  as  a 
product  of  polymerization,  also  glycolaldehyde,  CH2OH.CHO,  from 
which  sugar  can  be  readily  produced.  Still  the  conditions  under  which 
these  bodies  were  formed  cannot  be  applied  to  the  conditions  in  the 
plants.  The  investigations  of  Usher  and  Pristley3  are  of  greater 
interest  in  that  they  show  the  formation  of  formaldehyde  in  the  photo- 
lytic  decomposition  of  moist  carbonic  acid  in  the  presence  of  chloro- 
phyll. These  investigations  also  do  not  seem  to  be  entirely  free  from 
exception.  The  conception  as  to  the  formation  of  sugar  from  formalde- 
hyde is  also  often  different  from  that  explained  by  v.  Baeyer's  idea, 
and  his  view  as  to  the  assimilation  of  carbonic  acid  constitutes  a  hypoth- 
esis which  requires  further  proof.  The  essentials  of  this  hypothesis, 
namely,  a  formation  of  formaldehyde  with  a  subsequent  sugar  formation 
by  condensation  of  the  aldehyde  groups,  is  still  very  generally  accepted 
as  probably  correct.  Independent  of  the  ways  and  means  of  how  the 
assimilation  processes  in  the  plants  originate,  it  is  obvious  that  the  free, 
radiant  energy  of  the  sun  is  hereby  bound  and  stored  in  a  new  form,  as 
chemical  energy,  in  the  combinations  formed  by  the  syntheses. 

In  animal  life  the  conditions  are  not  the  same.  Animals  are  depend- 
ent either  directly,  as  the  herbivora,  or  indirectly,  as  the  carnivora, 
upon  plant-life,  from  which  they  derive  the  three  chief  groups  of  organic 
nutritive  matter — proteins,  carbohydrates,  and  fats.  These  bodies, 
of  which  the  protein  substances  and  fats  form  the  chief  mass  of  the 

1  Ber.  d.  d.  chem.  Gesellseh.,  3. 
*Zeitschr.  f.  Electrochem.,  12. 

1  Proc.  Roy.  Soe.  London,  78,  Series  B. 


ENZYMES.  39 

animal  body,  undergo  within  the  animal  organism  a  cleavage  and  oxi- 
dation, and  yield  as  final  products  exactly  the  above-mentioned  chief 
components  in  the  nutrition  of  plants,  namely,  carbon  dioxide,  water, 
and  ammonia  derivatives,  which  are  rich  in  oxygen  and  have  little  energy. 
The  chemical  energy,  which  is  partly  represented  by  the  free  oxygen 
and  partly  stored  up  in  the  above-mentioned  more  complex  chemical 
compounds,  is  transformed  into  other  forms  of  energy,  principally  heat 
and  mechanical  work.  While  in  the  plant  we  find  chiefly  reduction 
processes  and  syntheses,  which  by  the  introduction  of  energy  from 
without  produce  complex  compounds  having  a  greater  content  of  energy, 
we  find  in  the  animal  body  the  reverse  of  this,  namely,  cleavage  and  oxi- 
dation processes,  which,  as  we  used  to  state,  convert  chemical  tension 
into  living  force. 

This  difference  between  animals  and  plants  must  not  be  overrated, 
nor  must  we  consider  that  there  exists  a  sharp  boundary  line  between 
the  two.  This  is  not  the  case.  There  are  not  only  lower  plants,  free 
from  chlorophyll,  which  in  regard  to  chemical  processes  represent  inter- 
mediate steps  between  higher  plants  and  animals,  but  the  difference 
existing  between  the  higher  plants  and  animals  is  more  of  a  quantitative 
than  of  a  qualitative  kind.  Plants  require  oxygen  as  peremptorily  as 
do  animals.  Like  the  animal,  the  plant  also,  in  the  dark  and  by  means 
of  those  parts  which  are  free  from  chlorophyll,  takes  up  oxygen  and 
eliminates  carbon  dioxide,  while  in  the  light  the  oxidation  processes  going 
on  in  the  green  parts  are  overshadowed  or  hidden  beneath  the  more  intense 
reduction  processes.  As  in  the  animal,  Ave  also  find  a  heat  production 
in  fermentation  produced  by  plant  organisms;  and  even  in  a  few  of  the 
higher  plants — as  the  aroidcce  when  bearing  fruit — a  considerable  develop- 
ment of  heat  has  been  observed.  On  the  other  hand,  in  the  animal 
organism,  besides  oxidation  and  splitting,  reduction  processes  and  syn- 
theses also  take  place.  The  contrast  which  seemingly  exists  between 
animals  and  plants  consists  merely  in  that  in  the  animal  organism  the 
processes  of  oxidation  and  splitting  are  predominant,  while  in  the  plant 
chiefly  those  of  reduction  and  synthesis  have  thus  far  been  studied. 

Wohler  l  in  1824  was  the  first  to  observe  an  example  of  the  syn- 
thetical processes  within  the  animal  organism.  He  showed  that 
when  benzoic  acid  is  introduced  into  the  stomach,  it  reappears  as  hippuric 
acid  in  the  urine  after  combining  with  glycocoll  (aminoacetic  acid). 
Since  the  discovery  of  this  synthesis,  which  may  be  expressed  by  the 
following  equation : 

CoH5.COOH+XH2.CH2.COOH  =  NH(C6H5.CO).CH2.COOH+H20, 

Benzoic  acid  Glycocoll  Hippuric  acid 


1  Berzelius,  Lehrb.  d.  Chemie,  iibersetzt   von  Wohler,  4,  p.  356,  Abt.  1,  Dresden 
(1831). 


40  GENERAL  AND  PHYSICO-CHEMICAL. 

and  which  is  ordinarily  considered  as  a  type  of  an  entire  series  of  syn- 
theses occurring  in  the  body,  where  water  is  eliminated,  the  number  of 
known  syntheses  in  the  animal  kingdom  has  increased  considerably. 
Many  of  these  syntheses  have  also  been  artificially  produced  outside 
of  the  organism,  and  numerous  examples  of  animal  syntheses  of  which 
the  course  is  absolutely  clear  will  be  found  in  the  following  pages.  Besides 
these  well-studied  syntheses,  there  also  occur  in  the  animal  body  similar 
processes  unquestionably  of  the  greatest  importance  to  animal  life,  but 
of  which  we  know  nothing  with  positiveness.  We  enumerate  as  examples 
of  this  kind  of  synthesis  the  re-formation  of  the  red-blood  pigment  (the 
haemoglobin),  the  formation  of  the  different  proteins  from  simpler  sub- 
stances, and  the  production  of  fat  from  carbohydrates.  This  last- 
mentioned  process,  the  formation  of  fat  from  carbohydrates,  is  also  an 
example  of  reduction  processes  which  occur  to  a  considerable  extent  in 
the  animal  body. 

Certain  reactions,  which  are  either  not  reproduceable  with  dead  ma- 
terial or  are  only  possible  under  conditions  which  destroy  the  cells,  belong 
to  the  chemical  decompositions  going  on  within  the  living  organism. 
Thus  the  synthesis  of  glycogen  or  of  protein  has  not  been  accomplished 
outside  of  the  organism  or  without  the  aid  of  agents  prepared  by  the 
cells.  On  the  other  hand  proteins  and  starches  can  be  split  into  simpler 
products  without  these  agents,  but  for  this  purpose  the  action  of  acids  or 
alkalies  of  a  concentration  which  would  kill  the  cells  is  necessary.  In 
certain  cases  it  is  possible  to  bring  about  such  reactions  outside  of  the 
organism  without  any  injurious  effect  upon  the  cells.  This  is  accomplished 
by  the  aid  of  substances  which  are  formed  within  the  cells  but  have  the 
power  of  being  active  after  they  have  left  the  cells.  These  substances 
have  been  called  enzymes  or  ferments. 

Enzymotic  Processes.  We  must  now  mention  a  group  of  reactions 
which  are  more  or  less  related  to  enzyme  action. 

In  the  first  place  the  so-called  hydrolytic  cleavage  processes  in  which 
complex  substances  are  divided  into  simpler  substances  with  the  simul- 
taneous decomposition  of  water  and  the  taking  up  of  its  constituents. 
These  processes  are  of  the  greatest  importance  in  the  digestion  of  the 
food-stuffs  and  for  making  them  of  value  but  they  are  also  important  for 
the  metabolic  processes  in  general.  As  examples  of  such  cleavages 
we  will  mention  the  division  of  proteins  into  simpler  products,  the  trans- 
formation of  starch  into  sugar  and  the  cleavage  of  neutral  fats  into  the 
corresponding  fatty  acid  and  glycerin: 

(•,,n5^i.sH,,502),+3H20  =  C3F5rOH)34-3C18H3o02 

Tristearin  Glycerin  Stearic  Acid. 

The  importance  of  the  hydrolytic  cleavage  processes  for  digestion 
will  be  discussed  in  detail  in  Chapter  VIII. 


ENZYMES.  41 

Other  cleavage  processes  are  certain  so-called  fermentation  processes, 

which  are  connected  with  the  presence  of  living  organisms,  fungi  and 
bacteria  of  various  kinds.  Among  these  we  include  chiefly  the  alcoholic 
fermentation  and  butyric  acid  fermentation  of  carbohydrates.  Accord- 
ing to  the  view  based  upon  Pasteur's  investigations  it  has  been  gen- 
erally considered  that  these  processes  are  phases  of  the  life  of  these 
organisms  and  the  name  organized  ferments  or  ferments  have  been  given 
to  such  organisms,  especially  to  the  ordinary  yeast  fungus. 

A  ferment,  according  to  this  view,  is  a  living  organism.  By  the  name 
enzyme,  as  introduced  by  Kuhne,  we  mean  a  product  of  the  chemical 
processes  in  the  cells,  which  is  active  without  the  life  of  the  cell  and  which 
can  be  separated  from  the  cell.  The  decomposition  of  invert  sugar  into 
carbon  dioxide  and  alcohol  in  fermentation  is  considered  as  a  fermentative 
process  closely  connected  with  the  life  of  the  yeast  fungus.  The  inver- 
sion of  cane-sugar  previous  to  fermentation  is  on  the  contrary,  an  enzymotic 
process  which  is  brought  about  by  a  body  or  mixture  of  bodies  which 
are  formed  in  the  fungus  and  which  can  be  removed  from  the  fungus 
and  are  still  active  after  the  death  of  the  fungus.  Consequently  fer- 
ments and  enzymes  are  capable  of  manifesting  a  different  behavior  toward 
certain  chemical  reagents.  Thus  there  exist  a  number  of  substances, 
among  which  we  may  mention  arsenious  acid,  phenol,  toluene,  salicylic 
acid,  boracic  acid,  sodium  fluoride,  chloroform,  ether,  and  protoplasmic 
poisons,  which  in  certain  concentration  kill  ferments,  or  at  least  retard 
their  action,  but  which  do  not  noticeably  impair  the  action  of  the  enzymes. 

The  above  view  as  to  the  difference  between  ferments  and  enzymes 
has  lately  been  essentially  shaken  by  the  researches  of  E.  Buchner  : 
and  his  pupils.  He  has  been  able  to  obtain  from  beer-yeast,  by  grind- 
ing and  strong  pressure,  a  cell-fluid  rich  in  protein,  and  which  when  intro- 
duced into  a  solution  of  a  fermentable  sugar  caused  a  violent  fermenta- 
tion. The  objections  raised  from  several  sides  that  the  fluid  expressed 
still  contained  dissolved  living  cell  substance  has  been  so  successfully 
answered  by  Buchner  and  his  collaborators  that  there  is  at  present 
no  question  that  alcoholic  fermentation  is  caused  by  a  special  enzyme  or 
mixture  of  enzymes  called  zymase,  which  is  formed  in  the  yeast-cell. 

As  from  the  yeast-cells  so  also  from  ether   lower  organisms,  indeed 


lE.  Buchner,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30  and  31;  E.  Buchner  and  Rapp, 
ibid.,  31,  32,  34;  H.  Buchner,  Stizungsber.  d.  Gesellsch.  f.  Morphol.  u.  Physiol,  in 
Munehen,  13  (1897),  part  1,  which  also  contains  the  discussion  on  this  topic.  See  also 
E.  and  H.  Buchner  and  M.  Hahn,  Die  Zymasegarung,  Munehen  (1903);  Stavenhagen, 
Ber.  d.  deutsch.  chem.  Gesellsch.,  30;  Albert  and  Buchner,  ibid.,  33;  Buchner.  ibid. 
33;  Albert,  ibid.,  33;  Albert,  Buchner,  and  Rapp,  ibid.,  35;  in  regard  to  the  opposed 
views  see  Macfadyen,  Morris,  and  Rowland,  ibid.,  33;  Wroblewski,  Centralbl.  f.  Physiol., 
13,  and  Journ.  f.  prakt.  Chem.  (N.  F.),  64. 


42  GENERAL  AND  PHYSICO-CHEMICAL. 

from  the  lactic-acid  bacilli  and  beer  vinegar  bacteria,  it  is  possible  to 
separate  the  specific  fermentative  principle  of  these  organisms  from  the 
living  organism  and  to  bring  about  changes  with  the  dead  organism 
(E.  Buchner,  and  Meisenheimer  and  Gaunt,  Herzog  x)  .  The  ques- 
tion whether  there  exist  ferment  processes  which,  in  Pasteur's  sense, 
are  the  result  of  the  biological  phenomena  connected  with  the  metab- 
olism of  the  micro-organism  and  which  we  can  directly  identify  with  the 
life  processes,  is  very  difficult  to  answer;  hence  for  the  present  we  have 
no  foundation  for  a  sharp  differentiation  between  the  organized  ferments 
and  enzymes.  The  metabolic  processes  of  the  living  organisms  which 
we  recognize  as  fermentation  phenomena  must  as  a  rule  be  ascribed  to 
enzymes  acting  within  the  cell.  If  such  processes  are  closely  connected 
with  the  life  of  the  cell,  then  this  is  explained  in  part  by  the  fact  that 
this  special  enzyme  is  produced  only  by  living  cells  and  in  part  by  the 
fact  that  it  cannot  be  separated  from  the  living  cells  or  that  it  is  readily 
destroyed  on  the  death  of  the  cell.  The  names  enzyme  and  ferment  are 
now  generally  used  in  the  same  sense. 

Formerly  the  view  was  generally  accepted  that  animal  oxidation 
takes  place  in  the  fluids,  while  to-day  we  are  of  the  opinion,  derived 
from  the  investigations  of  Pfluger  and  his  pupils,2  that  it  is  connected 
with  the  form-elements  and  the  tissues.  The  question  as  to  how  this 
oxidation  in  the  form-elements  is  induced  and  how  it  proceeds  cannot 
be  answered  with  certainty.  On  the  other  hand,  it  is  accepted  that  the 
living  protoplasm  in  some  manner  or  other  takes  part,  in  which  case  the ' 
oxidation  processes  must  cease  with  the  life  of  the  cells  while  ion  the 
other  hand  it  has  been  found  that  certain  oxidative  processes  can  be 
brought  about  by  means  of  catalyts  in  the  ordinary  sense  as  well  as  by 
enzymotic  substances.  If  in  the  latter  case  the  oxygen  of  the  air  is 
directly  transported  to  the  oxidizable  substance  then  we  call  this  a  direct 
oxidation.  Ordinarily  the  oxidative  processes  take  place  in  the  follow- 
ing way.  First  a  peroxide  is  formed  by  taking  up  oxygen,  like  in 
the  formation  of  hydrogen  peroxide,  H-O-O-H,  which  then  transfers 
oxygen  to  the  oxidizable  substance  by  aid  of  the  mentioned  substances. 
In  these  cases  the  oxidation  is  indirect.  All  these  oxidative  processes 
will  be  treated  in  detail  in  Chapter  XVI.  At  the  same  time  also  other 
enzymes  will  be  discussed  which  decompose  hydrogen  peroxide,  with 
the  setting  free  of  oxygen,  without  oxidizing  at  the  same  time.     These 


1  E.  Buchner  and  J.  Meisenheimer,  Ber.  d.  d.  chem.  Gesellsch.,  36;  634  (1903); 
and  Annal.  d.  Chem.  u.  Pharm.,  349;  with  Gaunt,  ibid.,  349;  Herzog,  Zeitschr.  f. 
plivMol.  f.'hern.,  37. 

2  Pfliiger,  Pfiuger's  'Archiv,  6  and  10;  Finkler,  ibid.,  10  and  14;  Oertmann,  ibid., 
14  and  15;  Hoppe-Seyler,  ibid.,  7. 


ENZYMES.  43 

have  been  called  catalases.  Also  reduction  processes  will  be  mentioned 
which  seem  to  be  brought  about  by  enzymes. 

Besides  these  processes  just  mentioned  the  following  processes, 
namely,  autolysis  and  putrefaction,  are  to  be  considered  as  due  to  enzyme 
action  entirely  or  in  part. 

If  an  animal  organ  is  kept  in  water  at  37°  C.  under  conditions  so 
that  no  micro-organisms  are  active  then  the  organ  gradually  dissolves 
in  great  part  under  the  influence  of  the  contained  enzymes.  This 
process  is  called  autodigestion  or  aidohjsis.  The  action  of  micro- 
organisms can  be  prevented  either  by  removing  the  organ  under  strictly 
aseptic  conditions  or  by  allowing  the  digestion  to  take  place  in  the 
presence  of  antiseptic  substances  (toluene,  chloroform,  etc.).  As  the 
animal  organs  consist  chiefly  of  protein  substances  the  autolysis  con- 
sists chiefly  in  the  action  of  enzymes  which  dissolve  proteins.  Autol- 
ysis was  first  observed  and  studied  by  Salkowski  and  his  pupils 
with  liver,  muscle  and  supra-renal  capsule.1  Jacoby  then  showed 
that  the  enzymes  active  in  autolysis  do  not  orginate  in  the  digest- 
ive tract  and  are  not  identical  with  trypsin  or  pepsin.2  Biondi  found 
that  hydrochloric  acid  had  a  favorable  influence  upon  the  autolysis 
of  the  liver  while  Hedin  and  Rowland  3  observed  that  the  organic 
acids  accelerate  the  autolysis  of  nearly  all  organs.  This  has  been  sub- 
stantiated by  several  authors  (Wiener,  Arinkin4).  The  findings  of 
Lane-Claypton  and  Schryver  5  that  the  autolysis  of  the  liver  and 
kidneys  begins  only  after  a  latent  period  of  from  two  to  four  hours 
when  the  post  mortem  formation  of  acid  is  at  its  height,  substantiates 
the  influence  of  the  acid  reaction. 

The  autolysis  is  retarded  to  a  great  extent  by  an  alkaline  reaction. 
This  is  shown  by  the  experiments  of  Schwienig  with  the  liver  as  well 
as  those  of  Hedin  and  Rowland  with  several  other  organs.  Hedin 
has  also  shown  by  experiments  with  various  organs  that  a  preliminary 
treatment  with  acetic  acid  markedly  helps  the  autolysis  in  alkaline  reac- 
tion, which  for  the  spleen  at  least  is  explained  by  a  destruction  through 
the  treatment  of  acetic  acid  of  a  substance  which  has  an  inhibiting  action 
in. alkaline  solution.     Such  an  inhibiting  substance  destroved  by  acetic 


^eitschr.  f.  klin.  Med.,  1890,  Suppl.,  Schwiening,  Yirchow's  Arch.,  136,  444  (1894), 
Biondi,  ibid,  144,  373  (1896). 

2  A  complete  summary  of  the  literature  of  intracellular  enzymes  and  autolysis  may 
be  found  in  Jacoby,  Ueber  die  Bedeutung  der  intrazellularen  Fermente,  etc.,  Ergeb- 
nisse  der  Physiologie,  Jahrg.  1,  Abt.  1,  1902. 

3Zeitschr.  f.  physiol.  chem.,  32,  341,  531  (1901). 

4  Wiener,  Centralbl.  f.  Physiol.,  19,  349  (1905);  Arinkin,  Zeitschr.  f.  physiol.  Chem., 
53,  192  (1907). 

6  Journ.  of  Physiol.,  31,  169  (1904). 


44  GENERAL  AND   PHYSICO-CHEMICAL. 

acid  is  also  found  in  serum.1  The  serum  also  inhibits  the  autolysis  of 
the  liver  (Baer,  Longcope  and  others)2  and  also  the  thymus  under 
certain  circumstances  (Rhodin3). 

Experience  has  shown  that  the  post-mortem  autolytic  process  may 
also  be  influenced  by  many  other  bodies  and  indeed  in  various  ways. 
For  example,  according  to  Hess  and  Saxl,  arsenious  acid  exerts  a 
retarding  action  on  the  first  stages  of  autolysis,  while  phosphorus  accel- 
erates it.  Izar  as  well  as  Laqueur  and  Ettinger  4  obtained  with  small 
quantities  of  different  arsenic  preparations  an  acceleration  of  the  autol- 
ysis and  with  larger  amounts  a  retardation.  Laqueur  5  obtained  a 
retardation  with  oxygen  and  an  acceleration  with  carbon  dioxide. 
Ascoli  and  Izar  6  have  thoroughly  investigated  the  action  of  inorganic 
colloids  upon  autolysis.  Radium  rays  as  well  as  radium  emanations 
accelerate  post-mortem  autolysis  of  normal  as  well  as  carcinoma  tissues 
(Wohlgemuth,  Neuberg,  Lowenthal  and  Edelstein7). 

The  products  of  the  activity  of  the  different  enzymes  dissolving  pro- 
teins in  autolysis  have  been  studied  by  Hedin  and  his  collaborators,  by 
studying  the  action  of  organ  press  juices  upon  protein  added,  or  upon 
the  protein  contained  in  the  juice.  The  same  cleavage  products  were 
found  as  in  the  deep-seated  cleavage  of  proteins  in  the  digestive  canal.  8 
Similar  investigations  have  also  been  carried  on  by  Levene  and  Jones  9 
who  chiefly  considered  the  decomposition  of  the  nuclein  substances. 
The  combined  action  of  various  enzymes  in  autolysis  also  explains  to  us 
why,  as  especially  shown  by  Levene  and  by  Jones,  the  products  obtained 
by  the  hydrolytic  cleavage  of  an  organ  by  means  of  an  acid  are  some- 
what different  from  those  products  produced  on  autolysis.  In  autol- 
ysis we  are  not  only  dealing  with  the  cleavage  of  the  proteins,  but  other 
enzymotic  processes  also  occur  such  as  the  splitting  of  fats  and  carbo- 
hydrates, the  splitting  off  of  NH2  groups  from  amino-acids,  oxidations, 
reductions  and  perhaps  also  syntheses. 

1  Hammarsten's  Festschr.,  1906. 

2  Baer,  Arch.  f.  expt.  Path.  u.  Pharm.,  56,  68  (1906);  Longcope,  Journ.  nied. 
Research,  13,  45  (1908). 

3  Zeitschr.  f.  physiol.  Chem.,  75,  197  (1911). 

4  Hess  and  .Saxl,  Zeitschr.  f.  expt.  Path.  u.  Therapie,  5  (1908);  Izar,  Bioch.  Zeitschr., 
21,  46  (1909);  Laqueur  and  Ettinger,  Zeitschr.  f.  physiol.  Chem.,  79,  1  (1912). 

5  Zeitschr.  f.  physiol.  Chem.,  79,  82  (1912). 
8  Bioch.  Zeitschr.,  17,  361  (1909). 

7  Wohlgemuth,  Berl.  klin.  Wochenschr.,  26,  704;  Neuberg,  Zeitschr.  f.  Krebsfor- 
schung,  2,  171  (1904);   Lowenthal  and  Edelstein,  Bioch.  Zeitschr.,  14,  484  (1908). 

8  Leathes,  Journ.  of  Physiol.,  28,  360  (1902);  Dakin,  ibid.,  30,  84  (1904);  Hedin,. 
ibid.,  30,  155  (1904);   Cathcart,  ibid.,  32,  299  (1905). 

8  Levene,  Amer.  Jour,  of  Physiol.,  10,  11,  12  (1904);  Jones,  Zeitschr.  f.  physiol.. 
Chem.,  42,  35  (1904). 


ENZYMES.  45 

It  is  at  present  impossible  to  state  what  part  autolytic  processes 
take  in  life  under  physiological  conditions,  and  we  can  have  only  con- 
jectures on  this  subject.  In  the  autolysis  of  a  removed  organ  or  of  one 
through  which  the  blood  is  not  flowing,  the  conditions  in  many  ways  are 
quite  different  from  the  conditions  in  life.  The  products  which  appear 
after  weeks  or  months  of  autolysis,  sometimes  in  very  small  quantities, 
do  not  give  any  clue  to  the  nature  of  the  vital  processes,  and  conclusions 
must  be  drawn  very  carefully  from  these  results. 

For  the  present  it  is  impossible  to  judge  of  the  importance  of  the 
enzymes  active  in  autolysis  for  physiological  conditions,  but  this  does 
not  exclude  the  possibility  that  in  normal  cell  life  the  enzymes  play  a 
very  important  role.  Numerous  observations  show  this  to  be  true,  and 
we  tend  more  and  more  toward  the  view  that  the  chemical  transforma- 
tions in  the  living  cells  are  brought  about  by  enzymes,  and  that  these 
latter  are  to  be  considered  as  the  chemical  tools  of  the  cells  (Hofmeister 
and  others.1). 

From  this  standpoint  the  enzymes  are  of  especial  interest  because 
to-day  it  is  the  general  belief  that  nearly  all  chemical  processes  of  great 
importance  do  not  occur  in  the  animal  fluids,  but  on  the  contrary  in  the 
cells,  which  are  the  real  chemical  workshops  of  the  organism.  It  is  also 
chiefly  the  cells,  which  by  their  more  or  less  active  efficiency  regulate 
the  extent  of  the  chemical  processes  and  thereby  also  the  intensity  of 
the  general  metabolism.  The  folloAving  will  be  given  as  special  examples 
of  the  action  of  such  enzymes  under  pathological  conditions.  The 
changes  of  the  liver  and  blood  in  acute  phosphorus  intoxication  and 
in  acute  yellow  atrophy  of  the  liver,  where  we  find,  in  the  urine,  the 
enzymotic  decomposition  products  of  the  proteins.2  Another  example 
is  the  solution  of  pneumonic  infiltrations  by  the  enzymes  of  the  migrated 
and  inclosed  leucocytes  as  studied  by  Fr.  Muller,3  and  this  is  at  the  same 
time  an  example  of  heterolysis,  i.e.,  of  a  solution  or  a  destruction  in  an 
organ  by  enzymes  not  belonging  therein  but  introduced  from  without. 
An  autolysis,  although  not  very  marked,  occurs  in  those  organs  or  parts 
of  organs  which  have  not  been  normally  nourished  because  of  a  dis- 
turbance in  the  circulation,  and  they  are  gradually  consumed  by  this 
action.  The  part  injured  undergoes  solution,  while  the  healthy  part 
remains  unattacked.  By  this  solvent  action  as  well  as  by  the  forma- 
tion of  bactericidal  bodies,  as  observed  by  Conradi,4  and  of  antitoxins 


1  F.  Hofmeister,  Die  chemische  Organisation  der  Zelle,  Braunschweig,  1901. 

2  Jacoby,  Zeitschr.  f.  physiol.  Chera.,  30,  174  (1900). 

3  Verhandl.  d.  naturforsch.  Gesellsch.  zu  Basel,  1901.      See  also  O.  Simon,  Deutsch 
Arch.  f.  klin.  Med.,  1901. 

4  Hofmeister's  Beitrage,  1. 


46  GENERAL  AND  PHYSICO-CHEMICAL. 

(Blum  !)  by  means  of  autolysis,  we  can  consider  this  autolysis  as  a 
remedy  and  perhaps  also  as  a  protective  agent  for  the  animal  body. 
In  this  connection  the  investigations  of  Billard  2  must  be  mentioned 
where  the  autolytic  fluid  from  the  pig  liver  was  strongly  antitoxic 
toward  viper  poison,  cobra  poison,  tetanus  toxin  and  also  toward 
cocaine,  curare  and  strychnin. 

As  above  stated,  the  chemical  processes  in  animals  and  plants  do 
not  stand  in  opposition  to  each  other;  they  offer  differences  indeed, 
but  still  they  are  of  the  same  kind  from  a  qualitative  standpoint.  Pflu- 
ger  believes  that  there  exists  a  blood-relation  between  all  living  cells 
of  the  animal  and  vegetable  kingdoms,  and  that  they  originate  from  the 
same  root.  The  animal  body  is  a  complexity  of  cells,  hence  study  of  the 
chemical  processes  must  not  only  be  made  upon  higher  plants,  but  also 
upon  unicellular  organisms  in  order  that  we  get  a  proper  explanation 
of  the  chemical  processes  in  the  animal  organism.  Although  a  bio- 
chemical study  of  the  micro-organisms  is  very  important,  we  must  bear 
in  mind  also  the  important  role  played  by  such  organisms  in  animal 
life,  chiefly  as  exciters  of  disease;  hence  the  study  of  the  conditions  of 
life  of  these  micro-organisms  and  the  chemical  investigation  of  the  prod- 
ucts produced  by  them  must  be  of  infinite  importance. 

If  in  the  autolysis  of  animal  tissues  micro-organisms  are  added  and 
if  no  antiseptic  is  present  which  prevents  their  development,  then  they 
increase  abundantly  because  of  the  favorable  conditions  for  development. 
At  the  same  time  the  enzymes  are  also  formed  to  a  great  extent  and  by 
whose  aid  the  exchange  of  matter  takes  place  in  the  bacteria.  It  follows 
that  many  chemical  processes  occur  depending  upon  the  kind  of  bacteria 
present  and  which  are  foreign  to  bacteria-free  autolysis.  The  entire 
process  has  been  called  putrefaction.  Among  the  products  formed  we 
will  mention  the  sulphureted  hydrogen,  indol  and  skatol  which  chiefly 
give  the  odor  to  putrefying  proteins.  In  regard  to  other  putrefactive  prod- 
ucts we  refer  to  Chapter  VIII.  Under  ordinary  circumstances  compounds 
of  a  basic  nature  may  also  be  produced  by  putrefaction.  To  this  class 
belong  the  cadaver  alkaloids  called  ptomaines,  first  found  by  Selmi  in 
human  cadavers  and  then  specially  studied  by  Brieger  and  Gautier.3 
Certain  of  these  are  poisonous,  designated  as  toxines,  while  others  are 
non-poisonous.  They  all  belong  to  the  aliphatic  compounds  and  gen- 
erally do  not  contain  oxygen.     As  an  example  of  these  basic  substances 

■Hofmeister's  Beitrage,  5,  p.  142. 

2Cornpt.  rend.  8oc.  Biol.,  70,  623  (1911). 

:'  Selmi,  Sulle  ptomaine  od  alcaloidi  cadaverici  e  loro  importanza  in  tossicologia, 
Bologna,  1878;  Ber.  d.  d.  chem.  Gesellsch.,  11,  Correspond,  by  H.  Schiff;  Brieger, 
Ueber  Ptomaine,  Parte  1,  2,  and  3,  Berlin,  1885-1886;  A.  Gautier,  Traits  de  chimie 
ppliquee  a  la  physiologic,  2,  1873,  and  Compt.  rend.,  94. 


ENZYMES.  47 

Ave  must  mention  the  two  diamines,  cadaverine  or  pentametbylenediamine, 
C5H14X2,  and  putrescine  or  tetramethylenediamine,  C4H12N2,  which 
have  awakened  speeial  interest  because  they  occur  in  the  contents  of  the 
intestine  and  in  the  urine  in  certain  pathological  conditions,  especially 
in  cholera  and  cystinuria.1  The  putrefaction  bases  marcitine,  CsHigNa, 
putrine,  C11H26X2O3,  and  riridinine,  C8H12N2O3,  isolated  by  AcKER- 
mann,  also  belong  to  this  group.  Of  special  interest  is  the  bacterial 
poison  isolated  by  Faust,2  called  sepsine,  C8H14N2O2,  which  is  the  sub- 
stance producing  the  characteristic  toxic  action  of  putrefactive  masses. 
Sepsine  was  prepared  by  Faust  as  a  crystalline  sulphate  which,  on  repeated 
evaporation  of  its  solution,  was  readily  converted  into  cadaverine  sulphate. 

Of  especially  great  interest  are  the  toxines  which  are  found  in  the 
higher  plants  and  animals,  like  the  jequirity-bean  and  castor-seed,  in 
the  poison  of  snakes  and  spiders,  in  blood-serum,  etc.,  and  particularly 
those  produced  by  pathogenic  micro-organisms  have  an  unmistakable 
relation  to  the  enzymes.  A  closer  study  of  these  various  bodies,  lysines, 
agglutinines,  toxines,  etc.,  as  well  as  of  the  antitoxines  and  the  theory 
of  immunity,  does  not  lie  within  the  scope  of  this  work,  but  on  account 
of  the  great  importance  of  the  subject  it  will  be  briefly  discussed  on 
page  66. 

Classification  of  the  Enzymes.  If  we  exclude  those  processes  which 
are  the  result  of  several  enzymotic  reactions  (i.e.  autolysis,  putrefaction) 
then  the  most  important  enzymotic  processes  studied  so  far  are  the  fol- 
lowing : 

1.  Hydrolytic  cleavage  processes. 

2.  Cleavages  of  another  variety  (fermentation). 

3.  Oxidations. 

We  have  no  general  chemical  reaction  in  the  ordinary  sense  which 
is  common  to  all  enzymes  or  ferments  and  each  enzyme  is  characterized 
by  its  action  and  by  the  conditions  under  which  this  action  is  developed. 
As  the  action  of  an  enzyme  upon  a  substance,  or  related  substances,  or 
groups  is  limited  therefore  these  substances  or  groups  are  called  the 
substrate  of  the  enzyme. 

In  regard  to  the  terminology  it  must  be  remarked  that  an  enzyme 
is  often  named  after  the  substrate  (amylase,  protease,  lipase);  in  other 
cases  the  kind  of  action  determines  the  name  (oxidase,  reductase)  and 
finally  one  of  the  products  produced  by  its  action  forms  the  basis  for 
the  name  (alcoholase). 


^ee  Brieger,  Berlin,  klin.  Wochenschr.,  1887;  Baumann  and  Udransky,  Zeitschr. 
f.  physiol.  Chem.,  13  and  15;   Brieger  and  Stadthagen,  Berlin,  klin.  Wochenschr..  1889. 

2  Faust,  Arch.  f.  exp.  Path.  u.  Pharm.,  51;  Ackermann,  Zeitschr.  f.  phvsiol.  Chem., 
54  and  57. 


48  GENERAL  AND  PHYSICO-CHEMICAL. 

Of  the  above  mentioned  enzymotic  reactions  the  hydrolytic  cleavage 
processes  have  been  best  studied,  and  the  general  properties  of  the 
enzymes  which  will  be  given  apply  chiefly  to  the  hydrolytically  splitting 
enzymes.     Among  these  the  following  are  to  be  mentioned  especially: 

1.  Enzymes  which  split  fats  and  other  esters  with  the  formation  of 
the  corresponding  alcohol  and  acid.     They  are  called  lipases  or  esterases. 

2.  Enzymes  which  split  complex  carbohydrates  with  the  formation 
of  simpler  ones.     To  these  belong: 

a.  Disaccharide  splitting  enzymes  for  instance  saccharase  (invertase, 
invertin),  maltase,  lactase  which  act  upon  the  corresponding  disaccharide 
saccharose  (cane-sugar)  maltose  and  lactose  (milk  sugar); 

b.  Polysaccharide  splitting  enzymes  such  as  amylase,  ptyalin.  The 
name  diastase  is  often  used  to  designate  all  the  enzymes  of  this  group. 
In  close  relation  to  these  enzymes  stand  the  glucoside  splitting  enzymes 
which  occur  especially  in  higher  plants  and  the  best  known  of  which  is 
amygdalase  (emulsin)  occurring  in  the  almond. 

3.  Enzymes  which  act  upon  the  proteins  or  their  related  cleavage 
products.     Of  these  we  have : 

a.  Peptidases  and  erepsin  which  split  polypeptides  or  peptones; 

b.  Proteases  which  act  upon  proteins  as  substrate  (pepsin,  trypsin, 
autolytic  enzymes). 

Among  the  hydrolytic  enzymes  of  the  animal  kingdom  we  also 
include  the  arginase,  which  splits  arginine  into  urea  and  ornithin  and  the 
histozym,  which  splits  hippuric  acid.  The  two  following  groups  also 
belong  here,  namely,  the  nucleases  which  split  nucleic  acids  and  which 
will  be  discussed  in  Chapter  II,  and  the  coagulating  enzymes,  rennin 
and  thrombin,  which  are  probably  active  as  proteases.  The  deamidizing 
enzymes  which  split  off  the  NH2  group  from  amino  combinations  are, 
at  least  in  certain  cases,  to  be  classed  as  hydrolytic  enzymes.  This  is 
for  example  the  case  with  the  adenase  and  guanase  which  splits  off  ammonia 
from  the  two  bodies  adenine  and  guanine  converting  them  into  hypoxan- 
thine  and  xanthine  respectively.  The  urease  which  splits  urea  also  belongs 
to  this  group. 

General  Properties  of  the  Enzymes.  When  possible  we  make  use 
of  watery  solutions  of  enzymes  in  experimentation.  In  case  they  are 
insoluble  in  water  (certain  lipases)  we  use  them  in  the  form  of  more  or 
less  purified  powders  or  together  with  the  tissue  where  they  are  formed. 
We  have  no  general  method  for  preparing  enzyme  solutions.  In  certain 
cases  they  are  contained  in  secretions  (gastric  and  pancreatic  enzymes); 
in  others  they  are  prepared  from  the  cells  by  crushing  and  pressing  out 
the  cell  juice  (zymase,  organ  enzymes),  and  finally,  most  enzymes  can 
be  extracted  from  the  cells  with  water  or  glycerin,  and  as  this  last  gives 
permanent  solutions  it  has  found  great  use  as  an  extraction  medium. 


ENZYMES.  49 

The  aqueous  solutions  can  be  kept  at  low  temperatures  for  a  long  time 
after  the  addition  of  toluene  or  chloroform. 

In  all  these  cases  the  enzymes  are  obtained  strongly  contaminated 
with  other  bodies,  especially  by  proteins.  Only  in  exceptional  cases  is 
it  possible  to  free  the  enzyme  solution  from  protein  so  that  the  solution 
does  not  give  the  ordinary  protein  reactions.  This  is  true  for  the  solu- 
tion of  saccharase  obtained  from  yeast  by  treatment  with  water;  if 
this  is  shaken  with  kaolin  the  protein  is  adsorbed  by  the  kaolin  while  the 
solution  contains  the  enzymes.1 

No  enzyme  has  thus  far  been  obtained  in  a  perfectly  pure  form, 
and  the  chemical  constitution  as  well  as  structure  is  therefore  unknown. 
The  enzymes  probably  belong  to  the  colloids;  if  they  themselves  are  not 
colloids,  they  occur  at  least  with  colloids,  from  which  they  may  be  sepa- 
rated only  with  difficulty,  if  at  all.  The  enzymes  are  characterized  by 
the  fact  that  they  are  readily  taken  up  by  finely  divided  substances 
(inorganic  precipitates,  carbon,  kaolin,  infusorial  earth  and  other  col- 
loids such  as  alumina,  iron  hydroxide,  proteins 2) .  This  process  may 
act  selectively,  as  from  a  solution  certain  enzymes  can  be  taken  up  and 
others  not  at  all,  or  only  to  a  slight  extent  (Hedin,3  Michaelis  and 
Ehrenreich4).  The  adsorption  process  is  more  or  less  irreversible 
and  differs  in  this  from  the  adsorption  of  crystalloid  substances.  Still 
the  trypsin  and  rennin  adsorbed  by  charcoal  can  be  to  a  slight  extent 
expelled  from  the  charcoal  by  means  of  other  adsorbable  substances  such 
as  casein  and  albumin  (Hedin).5  Rennin  taken  up  by  charcoal  can  to 
a  very  slight  extent  be  set  free  by  the  addition  of  glucose  (Hedin)  and 
saccharase  adsorbed  by  charcoal  can  be  set  free  by  cane-sugar  (Eriks- 
son).6 As  we  will  learn  below,  the  adsorbed  enzyme  is  inactive.  The 
so-called  shaking  inactivation  of  enzymes  or  the  loss  in  activity  of  enzymes, 
which  occurs  on  shaking  their  solution  seems  to  be  due  to  an  adsorp- 
tion of  the  enzyme  when  it  is  either  taken  up  by  the  precipitate 
formed  on  shaking  (Abderhalden  and  Guggenheim)  or  is  concentrated 
at  the  surface  between  the  solution  and  the  froth  (S.  and  S.  Schmidt- 
Nielsen).7  These  latter  found  the  inactivation  of  rennin  by  shaking 
was  regained  if  the  froth  was  allowed  to  subside. 

All  enzymes  lose  their  specific   action  on  sufficiently  heating  their 

1  Michaelis,  Bioch.  Zeitschr.,  7,  488  (1907). 

2  Dauwe,  Hofmeister's  Beitrage,  6,  426  (1905). 

3  Bioch.  Journ.,  2,  112  (1907). 

« Bioch.  Zeitschr.,  10,  283  (1908). 

6  Bioch.  Journ.,  2,  81  (1906);   Zeitschr.  f.  physiol.  Chem.,  63,  143  (1909). 

6  Hedin,  ibid.,  63,  143  (1909);  Ericksson,  ibid.,  72,  313  (1911). 

7  Abderhalden  and  Guggenheim,  Zeitschr.  f.  physiol.  Chem.,  54,  352  (1907);   S.  and 
S.  Schmidt-Nielsen,  ibid.,  68,  317  (1910)  which  also  contains  the  literature. 


50  GENERAL  AND   PHYSICO-CHEMICAL. 

aqueous  solutions,  and  even  at  ordinary  temperature  the  enzymes  are 
gradually  decomposed.  In  general  the  enzymes  lose  their  activity  by 
heating  for  a  short  time  to  70°  C.  Madsen  and  Walbum  have  followed 
this  process  at  different  temperatures  and  found  that  the  decomposition 
of  trypsin,  pepsin  and  rennin  at  given  temperatures  proceeds  mono- 
molecularly,  i.e.,  that  the  velocity  of  reaction  at  every  moment  is  pro- 
portional to  the  concentration  of  the  enzyme  (page  34). l  The  readiness 
with  which  an  enzyme  is  decomposed  is  nevertheless  to  a  great  extent 
dependent  upon  the  presence  of  other  bodies  (page  54). 

Certain  enzymes  are  also  sensitive  to  light.  According  to  Schmidt- 
Nielsen  2  rennin  is  injured  by  light  and  in  particular,  by  the  ultra-violet 
rays.  The  experiments  of  Jodlbauer  and  Tappeiner3  with  invertin 
have  led  to  the  same  results;  the  visible  rays  can  also  in  some  cases 
(peroxidase,  haemase)  in  the  presence  of  oxygen  or  certain  fluorescent 
substances  exert  an  injurious  action.4 

According  to  Schmidt-Nielsen  5  the  weakening  in  the  rennin  under 
the  influence  of  light  proceeds  like  a  monomolecular  reaction. 

Experiments  on  the  cataphoresis  of  enzymes  have  been  made  by 
Bierry,  Henry  and  Schaeffer  6  as  well  as  by  Michaelis.  .  These 
investigators  found  that  saccharase  migrates  to  the  anode.  Michaelis7 
found  that  the  migration  direction  of  other  enzymes  was  dependent  upon 
the  reaction,  namely  in  faintly  acid  reaction  they  moved  to  the  cathode 
and  in  faintly  alkaline  reaction  to  the  anode.  Recently  Pekelharing 
and  \Y.  E.  Ringer  8  have  observed  that  the  migration  direction  of  pig 
pepsin  was  very  materially  influenced  by  the  addition  of  small  amounts 
of  proteoses.  From  what  was  previously  stated  (page  20)  the  saccharase 
must  therefore  have  a  negative  charge.  As  Michaelis,9  has  on  the  other 
hand,  found  that  the  saccharase  can  be  adsorbed  by  the  positively 
charged  aluminium  hydrate  and  not  by  the  negatively  charged  kaolin, 
he  concludes  that  the  formation  of  adsorption  compounds,  at  least  in 
certain  cases,  depends  upon  an  opposed  electric  charge  of  the  two  com- 
ponents. 


1  Arrhenius,  Immunochemie,  Leipzig,  1907,  58. 

2  Hofmeister's  Beitrage,  5,  355(1904);  8,481(1906);  Zeitschr.  f.  physiol.  Chem.,  58, 
233  (1908). 

Arch.  f.  klin.  Med.,  87,  373  (1906). 

*  Bioch.  Zeitschr.,  8,  61  and  84  (1908).     See  also  Agulhon,  Compt.  rend.,  153,   979 
(1911;. 

5  Zeitschr.  f.  physiol.  Chem.,  58,  232  (1908). 

•  Compt.  rend.  boo.  biol.,  68,  226  (1907). 

7  Bioch.  Zeitschr.,  16,  SI,  486;  17,  231  (1909). 
8Zeitsfhr.  f.  physiol.  Chem.,  75,  282  (1911).      . 
9  Bioch.  Zeitchr.,  10,  299  (1908). 


ENZYMES.  51 

Like  the  colloids  the  enzymes  only  diffuse  very  slowly  and  the  dif- 
fusion through  membranes  does  not  occur  in  most  cases;  only  certain 
membranes  such  as  collodion  tubes  allow  certain  enzymes  to  pass 
through.  The  collodion  tubes  can  be  impregnated  in  such  a  way  with 
lecithin  or  cholesterin  that  the  diffusion  is  very  slight.  The  same 
applies  to  the  filtration  through  collodion  membranes  (Bierry  and 
S(  iiaeffer).1  It  must  not  be  forgotten  in  such  experiments  that  the 
membrane  can  adsorb  a  considerable  part  of  the  enzyme  (Bechhold).2 

Just  as  it  is  difficult  to  prepare  an  enzyme  free  from  non-enzymotic 
contaminations,  so  also  is  it  difficult  to  exclude  the  possibility  that  a 
so-called  enzyme  is  not  a  mixture  of  several  related  enzymes.  In  fact 
the  several  enzymotic  processes  proceed  step  by  step,  and  it  is  possible 
that  the  various  steps  are  caused  by  different  enzymes.  Thus  the 
decomposition  of  protein  into  amino-acids,  with  proteoses,  peptones, 
and  polypeptides  as  intermediary  products,  may  be  the  result  of  the 
activity  of  several  enzymes  which  are  active  one  after  another  or  are 
parallel  with  one  another  in  activity.  Erepsin  does  not  attack  genuine 
proteins,  but  completes  the  decomposition  which  has  been  begun  by 
other  enzymes  (pepsin,  trypsin). 

The  enzymes  are  formed  within  the  living  cells.  In  certain  cases 
the  cells  do  not  secrete  the  complete  enzyme,  but  substances  which  are 
transformed  first  outside  of  the  cells  into  active  enzymes.  These  pre- 
liminary steps  or  mother  substances  of  the  enzymes  have  been  called 
proenzymes  or  zymogens.  These  under  certain  conditions  are  changed 
into  enzymes  and  in  certain  cases  this  is  brought  about  by  the  inter- 
action of  special  but  not  well  known  substances  which  have  been  called 
kinases  (see  Chapters  V  and  VIII).  In  other  cases  the  transformation 
of  the  zymogen  into  the  active  enzyme  is  brought  about  by  well  defined 
chemical  substances.  Thus  the  proenzymes  of  pepsin  and  of  rennin  are 
activated  by  acids  (see  below  on  the  retardation  of  enzyme  action  and 
also  Chapter  VIII). 

In  certain  other  cases  the  presence  of  bodies  which  resist  temperature 
and  are  dialyzable  and  therefore  not  enzymes,  are  necessary  or  helpful 
besides  the  real  organic  enzyme.  Thus  the  presence  of  an  acid  is  neces- 
sary for  the  action  of  pepsin  and  hydrocyanic  acid,  according  to  Mendel 
and  Blood,3  favors  to  a  high  degree  the  action  of  papain  (a  plant  pro- 
tease). R.  Magnus4  has  been  able  to  separate  by  dialysis,  from  a  solu- 
tion of  liver-lipase,  a  body  which  is  necessary  for  the  action  upon  amyl 


^ompt.  rend.  soc.  biol.,  62,  723  (1907). 

2  Zeitschr.  f.  physik.  Chem.,  60,  257  (1907). 

3  Journ.  of  biol.  Chem.,  8,  177  (1910). 

4  Zeitschr.  f.  physiol.  Chem.,  42,  149  (1904). 


52  GENERAL  AND  PHYSICO-CHEMICAL. 

salicylate.  Enzymes  made  inactive  by  dialysis  can  be  activated  again 
by  the  addition  of  boiled  enzyme  or  the  concentrated  dialysate.  Harden 
and  Young  l  on  filtering  yeast-press  juice  through  earthenware  filters 
impregnated  with  gelatin,  have  found  different  constituents  of  the  zymase 
on  the  filter  and  in  the  filtrate.  The  true  enzyme  remains  on  the  filter. 
This  alone  is  inactive,  but  becomes  active  when  the  other  part  which 
has  passed  through  the  filter,  and  which  is  dialyzable  and  resistant  to 
temperature,  is  added.  This  part  is  consumed  during  fermentation 
and  therefore  the  enzyme  becomes  inactive.  After  the  addition  of,  best, 
boiled  press-juice  to  this  the  fermentation  begins  again  (see  also  Chapter 
III).  Certain  of  the  just-mentioned  substances  which  are  resistant 
to  heat,  whose  presence  are  necessary  for  the  action  of  certain  enzymes, 
are  ordinarily  called  co-enzymes.  As  they  are  not  to  be  classified  with 
the  enzymes,  they  are  more  correctly  called  activators,  as  suggested  by 
Etjler.2  Their  action  is  probably  different  in  different  cases,  and 
differs  also  from  the  activating  action  of  the  kinases. 

Many  enzymes  are  secreted  by  the  cells  as  such  or  as  proenzymes. 
They  act  outside  of  the  cells  in  which  they  were  formed,  or  they  act  after 
having  been  transformed  into  the  enzyme,  and  hence  are  called  secre- 
tion enzymes  or  extracellular  enzymes.  Besides  these  extracellular 
enzymes  we  also  have  another  group  which  acts  within  the  cells,  hence 
are  intracellular  and  therefore  are  called  intracellular  enzymes  or  endo- 
enzymes.  To  this  group  belongs,  beside  the  yeast  zymase,  numerous 
enzymes  such  as  oxidases  and  hydrolytic  enzymes. 

Formation  and  Secretion  of  Enzymes.  The  investigations  of  Pawlow  3 
and  his  pupils  upon  the  formation  and  secretion  of  the  enzymes  active 
in  the  alimentary  tract  are  very  important.  According  to  these  investiga- 
tions the  amount  of  secretion  of  the  glands  and  the  behavior  of  the  enzymes 
contained  in  the  secretion  are  dependent  upon  the  amount  and  com- 
position of  the  food  taken  and  in  such  a  manner  that  the  kinds  and 
amounts  of  enzymes  are  appropriate  for  the  digestion  of  the  food- 
stuffs (see  Chapter  VIII).  Similar  results  were  also  obtained  by  Wein- 
land4  who  found  that  the  pancreas  does  not  normally  contain  any 
lactase  but  did  contain  this  enzyme  after  feeding  the  animal  with 
milk  or  milk  sugar.  This  has  been  substantiated  by  Bainbridge.5 
Analogous  experiments  have  been  made  with  salivary  ptyalin  by  Neil- 


^roc.  Physiol.  Soc,  32  (1904);    Proc.  Chem.  Soc,  21,  189    (1905);    Proc.  Roy. 
Soc,  77  (ser.  B),  405  (1906);  ibid.,  78,  369  (1906).      . 

2  Zeitschr.  f.  physiol.  Chem.,  57,  92  (1908). 

3  Arbeit  der  Verdauungsdriisen,  Wiesbaden,  1898,  s.  51. 
♦Zeitschr.  f.  Biol.,  38,  607  (1899);  40,  386  (1900). 

6  Journ.  of  Physiol.,  31,  98  (1904). 


ENZYMES.  53 

son  and  Lewis  1  with  the  same  results.  On  the  other  hand  the  cor- 
rectness of  these  observations  is  disputed  by  Bierry,2  Plim.mer,3  Wohl- 
gemuth 4  and  Popielski  5  as  they  could  not  find  any  accommodation. 
Mendel6  and  his  co-workers  by  careful  investigations  on  certain  enzymes 
obtained  from  embryonal  intestine  and  other  embryonal  tissues  could 
not  find  any  marked  difference  between  these  enzymes  and  the  enzymes 
of  the  full  grown  animal.  These  results  speak  against  the  accepted  influence 
of  the  food  and  of  the  processes  depending  upon  the  taking  up  of  food, 
upon  the  formation  of  enzymes.  Recently  the  investigations  of  Lon- 
don 7  and  his  collaborators  upon  the  influence  of  the  food  upon  the 
digestion  juices  have  shown  that  the  amount  of  juice  secreted  is  dependent 
upon  the  constitution  of  the  food  but  not  the  ferment  content  of  the 
same.  The  observations  of  Cohnheim  8  also  speak  against  the  view 
that  the  kind  and  quantity  of  enzymes  secreted  in  the  intestinal  tract 
accommodate  themselves  to  the  digestion,  as  he  found  that  the  organism 
secretes  as  much  fluid  (gastric  juice,  pancreatic  juice  and  bile)  when 
already  digested  food  is  introduced  into  the  stomach  as  when  undigested 
food  is  introduced.  Arrhenius  9  has  calculated  from  London's  figures, 
that  the  total  amount  of  digestive  juice  secreted  was  proportional  to  the 
quantity  of  food-stuffs.  From  experiments  which  Euler  and  his 
collaborators  have  made  upon  the  formation  of  inverting  enzymes  he 
concludes  that  we  have  inverting  enzymes  whose  formation  is  specific 
by  getting  accustomed  to  the  substrate,  while  the  formation  of  others 
is  in  no  wise  thus  influenced.10 

In  this  connection  we  will  call  attention  to  the  appearance  of  enzy- 
motic  substances  in  the  blood  after  the  subcutaneous  or  intravenous 
(parenteral)  introduction  of  certain  food-stuffs.  Weinland  first  showed 
that  the  parenteral  introduction  of  cane-sugar  caused  the  appearance 
in  the  serum  of  a  cane-sugar  splitting  enzyme.11  Abderhalden  and 
Kapfberger  12  have  substantiated  and  developed  these  observations. 
Bodies  having  a  similar  action  also  appear  after  the  injection  of  milk 


I  Journ.  of  Biol.  Chem.,  4,  501  (1908). 
2Compt.  rend.  soc.  biol.,  58,  701  (1905). 

3  Journ.  of  Physiol.,  34,  93  (1906). 

4  Bioch.  Zeitschr.,  9,  1  (1908). 

5  Pfliiger's  Arch.,  127,  443  (1909). 

6  Amer.  Journ.  of  Physiol.,  20,  81,  97  (1907);  21,  64,  69,  85,  95  (1908). 

7  Zeitschr.  f.  physiol.  Chem.,  68,  366  (1910). 
8 Ibid.,  84,  419  (1913). 

9  tbid.,  63,  323  (1909),  see  also  London,  ibid.,  65,  189  (1910). 

10  Ibid.,  70,  279;  76,  388;  78,  246;   79,  274;  80,  241  (1912). 

II  Zeitschr.  f.  Biol.,  47,  279  (1905). 

12  Zeitschr.  f.  physiol.  Chem.,  69,  23  (1910). 


54  GENERAL  AND  PHYSICO-CHEMICAL. 

sugar  and  of  starch.  Abderhalden  and  his  co-workers  have  shown 
that  the  parenteral  introduction  of  protein  or  peptone  gives  the  blood 
serum  of  the  animal  the  power  of  splitting  proteins,  which  power  is 
destroyed  on  heating  to  60-65°  C.1  The  introduction  of  very  large  quan- 
tities of  sugar  or  proteins  per  os  (over  feeding)  has  the  same  effect  as 
the  parenteral  introduction.  Abderhalden  considers  the  active  sub- 
stances thus  obtained  as  enzymes.  The  question  is  still  undecided  whether 
the  substances  introduced  bring  about  a  formation  of  the  enzymes  or 
whether  they  only  transport  the  already  formed  enzymes  to  the  blood. 

Heat  Production.  The  question  whether  in  the  hydrolytic  processes 
with  the  aid  of  enzymes  heat  is  given  off  or  taken  up  has  been  attacked 
in  two  different  ways.  Grafe  2  could  not  find  either  any  setting  free  or 
taking  up  of  heat  in  the  digestion  of  protein  in  a  Rubner  calorimeter. 
On  the  other  hand  Hari  3  by  determining  the  calorific  values  of  albumin 
before  and  after  digestion  came  to  about  the  same  results.  If  we  exclude 
the  work  developed  in  the  process  then  it  follows  that  the  energy  supply 
of  the  organism  is  not  perceptibly  changed  by  the  hydrolytic  cleavages 
of  the  protein.  The  chief  source  of  energy  is  to  be  sought  in  the  oxida- 
tion processes  that  follow  the  cleavages. 

Modes  of  Action  of  the  Enzymes.  The  enzjrmes  do  not  suffer  any 
appreciable  change  during  the  reaction  they  perform,  and  insignificant 
amounts  of  the  enzyme  are  able  to  decompose  relatively  enormous  amounts 
of  the  substrate.  For  example,  1  part  of  saccharase  can  invert  100,000 
parts  of  cane-sugar  (O'Sullivan  and  Thompson)4  and  1  part  of  rennin 
can  decompose  more  than  400,000  parts  of  casein  (Hammarsten)  5. 
For  these  reasons  the  enzymes  have  for  a  long  time  been  considered  as 
catalytic  substances.  Nevertheless  the  enzyme  reactions  always  take 
place  in  heterogeneous  media  where  on  one  hand  the  enzyme  exists  as 
colloid  and  on  the  other  the  substrate  in  many  cases  is  a  colloid  (starch, 
proteins).  As  above  mentioned,  the  enzymotic  decompositions  are 
often  complicated  by  their  taking  place  over  several  intermediary  steps 
to  the  final  product.  As  indicated  by  several  conditions,  the  enzymes 
also,  before  they  act  upon  the  substrate,  combine  therewith  in  some  way 
or  another.  The  fact  that  the  action  of  an  enzyme  is  dependent  upon  the 
stereometric  construction  (page  61)  of  the  substrate  speaks  essentially 
for  this  view.     The  substrate  also  protects  certain  enzymes  against  destruc- 


» Ibid.,  61,  200;  62,  120,  243  (1909);    64,  100,  423,  426,  427;   66,  88;   69,  23  (1910); 
71,  110,  307,  385  (1911).     See  also  77,  250  (1912). 
2  Arch.  f.  Hygiene,  62,  216  (1907). 
•ranger's  Arch,  115,  11  (1906);  121,  459  (1908). 
*  Journ.  ohem.  Soc,  57,  926  (1890). 
6  See  Maly's  Jahresber,  7. 


ENZYMES.  55 

tive  influences  (heat,  alkalies)1;  According  to  this  only  that  part  of  the 
added  enzyme  which  is  combined  with  the  substrate  is  active.  In 
judging  of  the  rapidity  of  enzyme  reactions  the  following  must  he  con- 
sidered: 

1.  The  velocity  with  which  the  enzyme  combines  with  the  substrate. 

2.  The  result  of  the  division,  i.e.,  how  much  of  the  added  enzyme 
is  combined  with  the  substrate. 

3.  The  velocity  of  the  chemical  processes  produced  by  the  enzyme. 

The  velocity  of  the  combination  of  the  enzyme  with  the  substrate  (1)  can  at 
least  in  many  cases  be  ignored  in  consideration  of  the  time  necessary  for  the 
chemical  reaction  (see  page  37).  This  applies  to  those  cases  where  the  chemical 
transformation  in  the  presence  of  an  excess  of  substrate  at  the  beginning  of  the 
processes  remains  the  same  in  each  successive  time  interval.  The  quantity  of 
enzyme  combined  with  the  substrate,  does  not,  in  these  cases,  increase  with  the 
time,  which  would  be  the  case  if  the  time  necessary  for  the  combination  is  not  in 
comparison  with  that  for  the  chemical  reaction.  Equal  decomposition  for  equal 
time  at  the  beginning  of  the  processes  have  been  found  for  the  following  enzymes — 
invertase,2  diastase,3  trypsin  with  casein,4  as  substrate. 

The  second  question,  as  to  the  division  of  the  enzyme  between  different  phases 
we  will  discuss  after  we  have  spoken  of  the  velocity  of  the  real  chemical  reaction 
(page  58)  as  well  as  the  retardation  of  enzyme  action  (page  62). 

In  regard  to  the  chemical  reaction  they  proceed  probably  in  a  different 
manner  according  to  the  kind  of  combination  between  the  substrate  and 
the  enzyme.  In  one  case  we  can  consider  that  the  combination  of  the 
enzyme  with  the  substrate  is  of  such  a  kind  that  both  form  a  homogeneous 
phase  and  that  one  serves  as  solvent  for  the  other  (page  27).  In  this 
case  the  chemical  reaction  produced  by  the  enzyme  takes  place  in  a 
homogeneous  medium.  Secondly,  we  can  consider  the  combination  of 
the  substrate  and  enzyme  as  an  adsorption  combination  (see  page  27) 
in  which  case  the  combination  does  not  form  a  homogeneous  phase  and 
the  reaction  differs  more  or  less  from  one  taking  place  in  a  homogeneous 
system.  Bearing  this  in  mind  it  would  be  interesting  to  investigate 
whether  the  facts  found  for  enzymotic  reactions  correspond  with 
catalytic  reactions  in  homogeneous  media. 

For  these  latter  the  following  laws  (see  page  33)  have  been  found: 

1.  When   the   quantity   of   catalyst    remains   constant,    the   reaction 


1  O'Sullivan  and  Thompson,  Journ.  Chem.  Soc,  57,  926  (1890);  Bayliss  and  Starling, 
Journ.  of  Physiol.,  30,  71  (1903);  Hedin,  ibid.,  30,  173  (1903);  32,  474  (1905);  Taylor, 
Journ.  of  biol.  Chem.,  2,  90  (1906). 

2  O'Sullivan  and  Thompson,  Journ.  Chem.  Soc,  57,  926  (1S90);  Ducleau,  Traite. 
de  microbiologie  II,  137;  Brown,  Trans.  Chem.  Soc,  81,  373  (1902);  Armstrong, 
Proc.  Roy.  Soc,  73,  500  (1904);  Hudson,  Journ.  Amer.  Chem.  Soc,  30,  1160,  1564 
(1908). 

3  Brown  and  Gliddinning,  Proc  Chem.  Soc,  18,  43  (1902). 
•  Hedin.  Journ.  of  Physiol.,  32,  471  (1905). 


56  GENERAL  AND   PHYSICO-CHEMICAL. 

velocity  for  every  moment  is  proportional  to  the  concentration  of  the 
body  decomposed,  which  is  shown  by  the  velocity  coefficient  in  the  same 
experiment  being  constant  at  different  times. 

2.  The  velocity  coefficient  or  the  reaction  velocity  with  constant 
concentration  of  substrate  is  proportional  to  the  quantity  of  catalyst. 

The  first  law  has  been  shown  for  certain  enzymes  in  case  an  excess 
of  enzyme  is  present,  namely  for  saccharase,1  lactase  2  and  trypsin.3  It 
was  found  that  the  decomposition  in  a  certain  time  was  proportional  to 
the  substrate.  In  other  cases  the  determination  of  the  correctness  of 
the  law  was  accomplished  with  difficulty.  A  part  of  the  enzyme  may 
during  an  experiment  be  either  destroyed  or  in  other  ways  (combining 
with  the  product)  be  put  out  of  action;  then  reverse  reactions  may  take 
place  (page  11)  and  finally  in  many  cases  our  analytical  methods  are 
incapable  of  obtaining  comparative  results  for  different  decompositions, 
as  the  reaction  in  many  cases  takes  place  step  by  step,  or  several  reac- 
tions occur  at  the  same  time.4  Only  in  a  few  cases  with  especially 
simple  reactions  have  constant  values  been  found  for  the  velocity 
coefficient  at  the  beginning,  as  long  as  the  quantity  of  reaction  product 
was  small  and  the  active  quantity  of  enzyme  remained  unchanged 
according  to  the  formula  (see  page  ll).5 

t     Xi         C 
k=  —  log 


t     bC-x 

Recently  Hudson  6  has  found  constant  values  for  k  for  the  entire 
process  of  inversion  of  cane-sugar  by  saccharase  in  a  faintly  acid  reac- 
tion. The  reason  for  the  different  results  of  earlier  investigators 7  is 
due,  in  part,  according  to  Hudson,  to  the  fact  that  the  multirotation 
of  the  glucose  formed  was  not  considered  by  these  experimenters  before 
the  extent  of  inversion  was  determined  polariscopically.  In  the  cleavage 
of  salicin  by  emulsin  Hudson  and  Paine  8  obtained  constant  values 
for  k  in  the  entire  process. 

1  Brown,  Proc.  Chem.  Soc,  18,  14  (1902). 

2  Armstrong,  Proc.  Roy.  Soc,  73,  500  (1904). 

*  Hedin,  Journ.  of  Physiol.,  32,  475  (1905). 

*  Hedin,  Zeitschr.  f.  physiol.  Chem.,  57,  468  (1908). 

6Senter,  Zeitschr.  f.  physik.  Chem.,  44,  257  (1903);  Issajew,  ibid.,  42,  102;  44, 
546;  Eider,  Ilofrneister's  Beitrage,  7,  1  (1906);  Dietz,  Zeitschr.  f.  physiol.  Chem., 
52,  301  (1907);  Taylor,  Journ.  of  biol.  Chem.,  2,  93  (1906);  Nicloux,  Compt.  rend, 
soc.  biol.,  56,  840  (1904);  Rona,  Bioch.  Zeitschr.,  33,  413  (1911);  39,  21  (1912);  Euler, 
Zeitschr.  f.  physiol.  Chem.,  51,  213  (1907). 

6  Journ.  Amer.  Chem.  Soc,  30,  1160,  1564  (1908). 

7  Bee  Henri,  Zeitschr.  f.  physik.  Chem.,  39,  194  (1901)  also  A.  J.  Brown,  Trans. 
Chem.  Soc,  81,  373  (1902). 

8  Journ.  Amer.  Chem.  Soc,  31,  1242  (1909). 


ENZYMES.  57 

The  second  law  for  catalytic  reactions  which  we  have  formulated, 
that  with  constant  quantities  <>f  substrate  the  reaction  velocity  is  pro- 
portional to  the  quantity  of  enzyme,  has  been  shown  in  certain  cases 
where  the  substrate  was  in  excess  (practically  constant  quantity)  namely 
with  kephir  lactase,1  trypsin  with  casein  as  substrate.2  In  the  just- 
mentioned  inonomolecular  enzyme  reactions  the  velocity  coefficient  in 
a  few  cases  was  found  proportional  to  the  quantity  of  enzyme  (catalase 
from  blood,3  erepsin  with  glycyl-glyeine  as  substrate,4  pancreatic  lipase5) 
and  in  others  not  (catalase  from  Boletus  scaber,  lipase  from  pig  fat).6 
It  has  been  shown  for  several  enzymotic  reactions  that  with  the  same 
substrate  the  same  decomposition  can  be  obtained  if  the  time  of  action 
varies  in  inverse  proportion  to  the  added  quantity  of  enzyme.  If  p  is 
the  quantity  of  enzyme  and  t  the  time  of  action,  then  the  decomposition 
is  the  same  in  all  tests  where  p.t  is  the  same  figure.  This  rule  has  been 
found  true  for  the  following  enzymes:  saccharase  (O'Sullivan  and 
Thompson  as  well  as  Hudson  7),  pepsin  (Sjoqvist  8),  rennin  (especially 
Fuld9),  peptone-splitting  enzyme  (Vernon10),  fibrin  ferment  of  snake 
poison  (Martin11),  trypsin  (Hedin  12),  pepsin,  rennin,  trypsin,  pyocy- 
aneus  protease  (Madsen13).  On  the  action  of  trypsin  upon  casein  this 
law  has  been  shown  correct  for  different  stages  in  the  reaction.14  This 
indicates  that  the  progress  of  the  entire  reaction  remains  the  same  with 
different  quantities  of  enzyme,  only  that  the  time  for  the  same  decom- 
position is  inversely  as  the  quantity  of  enzyme.  As  clearly  shown  by 
Hedin,  this  indicates  that  the  velocity  coefficient  is  proportional  to  the 
quantity  of  enzyme  which  is  called  for  by  the  second  law.  If  we  start 
with  the  above-mentioned  assumption  that  only  that  enzyme  is  active 
which  is  combined,  then  it  follows  from  the  proportionality  between  the 
velocity  coefficient  and  the  quantity  of  enzyme,  that  always  the  same 
fraction  of  the  enzyme  is  combined  with  the  substrate,  or  that  the  divi- 
sion of  the  enzyme  remains  independent  of  the  quantity. 

Armstrong,  Proc.  Roy.  Soc,  73,  500  (1904). 

2  Hedin,  Journ.  of  Physiol.,  32,  471  (1905). 

3  Senter,  Zeitschr.  f.  physik.  Chem.,  44,  257  (1903). 

4  Euler,  Zeitschr.  f.  physiol.  Chem.,  61,  213  (1907). 

5  Kastle  and  Loevenhart,  Amer.  Chem.  Journ.,  24,  491  (1900). 

6  Euler,  Hofmeister's  Beitriige,  7,  1  (1906). 

7  Trans.  Chem.  Soc,  57,  926,  1890;  Journ.  Amer.  Chem.  Soc,  30,  1160,  1564  (1908). 

8  Skand.  Arch.  f.  Physiol.,  5,  358  (1895). 

9  Hofmesietr's  Beitriige,  2,  169  (1902). 

10  Journ.  of  Physiol.,  30,  334  (1903). 

11  Ibid.,  32,  207  (1905). 

12  Ibid.,  32,  468  (1905);  34,  370  (1906). 

13  Arrhenius,  Immunochemie,  Leipzig,  1907,  46. 

14  Zeitschr.  f.  physiol.  Chem.,  57,  478  (1908). 


58  GENERAL  AND  PHYSICO-CHEMICAL. 

In  determining  the  quantity  of  enzyme  the  so-called  Schutz's  rule  plays 
an  important  part.  In  its  newest  form  this  is,  that  the  decomposition  is  pro- 
portional to  the  square  root  of  the  quantity  of  enzyme  and  the  time,  or  decom- 
position =  kV pt  where  A;  is  a  constant,  p  the  quantity  of  enzyme  and  t  the  time 
of  the  action.  This  was  first  _shown  by  Schutz  *  for  pepsin  and  also, 
in  this  form,  decomposition  =kvp  as  the  time  (t)  was  constant.  The  form 
decomposition  =fcv  pt  was  given  by  Schutz,  and  Huppert.2  According  to 
Pawlow  this  rule  also  applies  to  trypsin  digestion.3  Schutz's  rule  is  good  for 
a  certain  stage  of  digestion  only  and  it  indicates  that  the  extent  of  the  validity 
must  be  very  dependent  upon  the  method  used  for  the  determination  of  the 
decomposition  as  the  different  digestion  products  are  determined  by  different 
methods.  It  must  also  be  remarked  that  within  the  entire  domain  where 
Schutz's  rule  is  applicable  the  same  value  for  pt  must  correspond  to  the  same 
decomposition,  and  necessarily  the  above-discussed  enzyme-time  rule  must  also 
be  valid.  Schutz's  rule  has  also  been  proved  for  the  action  of  gastric  and  pan- 
creatic lipase.4  According  to  Arrhenius  5  the  validity  of  the  rule  can  be  explained 
by  the  assumption  that  the  enzyme  combines  with  the  reaction  products  so  that 
the  active  mass  of  enzyme  changes  in  inverse  proportion  to  the  auantity  of 
reaction  products. 

Reversibility  of  Enzyme  Action  and  Enzymotic  Syntheses.  Many 
catalytic  processes  have  been  shown  to  be  reversible,  i.e.,  the  same 
catalyst  can  influence  the  reaction  in  different  directions  according  to 
the  concentration  of  the  substances  present.  Thus  far  we  have  only 
spoken  of  enzymotic  cleavages;  according  to  the  above  it  is  to  be  expected 
that  synthetical  processes  can  also  be  produced  by  enzymes. 

The  first  example  of  such  a  reaction  was  given  by  Croft-Hill.6 
He  treated  a  40  per  cent  glucose  solution  with  maltase  at  30°  C.  for  a 
very  long  time  and  concluded  from  the  change  in  rotation  and  reducing 
power  that  some  maltose  was  formed  from  the  glucose.  Emmerling  7 
showed  afterward  that  a  synthesis  of  maltose  did  not  occur,  but  rather 
an  isomeric  carbohydrate,  isomaltose  was  formed,  which  is  not  split  by 
maltase.  According  to  Armstrong  8  emulsin  splits  isomaltose,  but 
not  maltose,  and  therefore  it  can  synthesize  maltose  from  glucose.  A 
similar  reaction  had  previously  been  shown  by  E.  Fischer  and  Arm- 
strong,9 that  kefir-lactase  produced  isolactose  and  not  lactose  from 
galactose  and  glucose.  According  to  Cremer  l0  yeast-press  juice  has 
the  power  of  forming  glycogen  from  glucose  or  fructose. 

1  Zeitschr.  f.  physiol.  Chem.,  9,  577  (1885). 
2Pfliiger's  Arch.,  80,  470  (1900). 
3  Arbeit  der  Verdauungsdriisen,  Wiesbaden,  1898,  33. 

•  Stade,  Hofrneister's  Beitrage,  3,  318  (1903);  Engel,  ibid.,  7,  77  (1906),  see  Fromme, 
ibid.,  7,  77,  (1906). 

5  Immunochemie,     1907,  43. 

6  Journ.  of  chem.  Soc,  73,  634   (1S98). 

>  Ber.  d.  d.  chem.  Gesellsch.,  34,  600  and  2207  (1901). 
8Proc.  Roy.  Soc.  (ser.  B),  76,  592,  (1905) 
'  Her.  d.  d.  chem.  Gesellsch.,  35,  3151,  (1902). 
'"Ibid.,  32,  2062  (1899). 


ENZYMES.  59 

A.  Danilewski  first  made  the  observation  that  concentrated  solutions 
of  peptic  cleavage  products  of  protein  substances  separates  an  insol- 
uble substance  under  the  influence  of  rennin.  This  phenomenon  has 
since  been  observed  by  various  investigators  and  the  precipitate  has 
been  called  plastein  by  Sawjalow  '  and  coagalose  by  Lawrow.2  The  same 
phenomenon  is  also  obtained  by  other  proteolytic  enzymes.3  The  plas- 
tein* are  considered  by  various  investigators  as  synthetically  formed 
protein.  The  best  proof  for  this  view  has  been  given  by  Henriques 
and  Gjalback.  They  show  with  the  formol  titration  (see  Chapter  II) 
that  the  nitrogen  titratable  by  formol  diminishes  in  the  reaction  and 
they  also  found  that  the  nitrogen  precipitatable  by  tannic  acid  was 
increased  in  the  plastein  formation.  In  a  later  work  these  authors 
find  that  peptic  cleavage  products  from  proteins  show  a  plastein  for- 
mation when  under  the  influence  of  pepsin-hydrochloric  acid  in  con- 
centrated solution  while  in  dilute  solution  the  cleavage  goes  further  and 
they  conclude  from  this  that  the  process  is  reversible.  Even  protein 
which  has  been  partly  split  by  acid  or  alkali  shows  a  plastein  formation 
with  pepsin-hydrochloric  acid.4 

The  behavior  of  amygdalin  and  its  cleavage  products  with  enzymes 
requires  special  mention.  The  cleavage  takes  place  step  by  step  as 
follows : 

C:oH2-NOi1+H20  =  Cr,H5.CH(OCr)H1105).CN+CGH12OG.   .     (1) 

Amygdalin  Mandelic  acid  nitrileglucoside  Glucose 

C6H5.CH(OC6H„05).CX  +  H20  =  Cr,H5.CH™+C6H1206.  .      (2) 

Uri 

Mandelic  acid  nitrileglucoside  Mandelic  acid  nitrile        Glucose 

CoH5CH^  =  CY,H5C<(°+KCX (3) 

Mandelic  acid  nitrile       Bcnzaldehyde      Hydrocyanic  acid 

The  entire  process  with  the  formation  of  the  end  products  sugar, 
benzaldehyde  and  hydrocyanic  acid  takes  place  under  the  influence  of 
emulsin  from  almonds.  The  first  part  of  the  process  can  be  especially 
brought  about  by  the  influence  of  yeast  (Fischer)5  and  the  second  and 
third  parts  under  the  influence  of  prunase  from  the  leaves  of  Pruneae.6    Of 

1  Zeitschr.  f.  physiol.  Chem.,  54, ,119  (1907). 

2  Ibid.,  .51,  1:  53,  1  (1907);  56,  343  (1908);  60,  520  (1909). 

3Kurajeff,  Hofmeister's  Beitriige,  4,  476  (1904);   Numbers,  ibil.,  4,  543  (1904). 

4  Zeitschr.  f.  physiol.  Chem.,  71,  485  (1911);  81,  439  (1912). 

5  Ber.  d.  d.  chem.  Gesellsch.,  28,  1508  (1896). 

6H.  E.  Armstrong,  E.  F.  Armstrong  and  Horton,  Proc.  Roy.  Soc,  85,  359,  363, 
370  (1912). 


60  GENERAL  AND  PHYSICO-CHEMICAL. 

the  three  above  given  reactions  1  and  3  can  be  reversed  by  enzymes  and 
indeed  1  even  by  using  yeast  (Emmerling)1  and  3  with  emulsin  (Rosen- 
thaler  2) .  In  the  last  instance  the  reaction  is  asymmetric  in  that 
the  d-form  of  the  mandelic  acid  nitrile  is  formed.  The  asymmetric  C 
atom  is  marked  in  the  above  formula.  Subsequently  Rosenthaler  was 
able  to  divide  the  emulsin  into  a  splitting  component  (5-emulsin)  and 
a  synthetical  form  (or-emulsin)3.  In  connection  with  the  views  on  the 
structure  and  mode  of  action  of  enzymes  it  is  of  special  interest  that 
recently  Bredig  and  Fiske  4  have  been  able  to  prepare  the  two  optical 
antipodes  of  mandelic  acid  nitrile  from  benzaldehyde  and  hydrocyanic 
acid  by  means  of  optically  active  catalysts.  By  using  quinine  as 
catalyst  the  dextro-rotatory  nitrile  was  formed  and  by  quinidine  (iso- 
meric with  quinine  but  opposed  in  regard  to  rotation  power)  the  laevo- 
rotatory  nitrile  was  formed.  This  indicates  that  possibly  the  enzymes 
also  have  an  asymmetric  structure.  The  synthetic  formation  of  gluco- 
sides  by  the  aid  of  emulsin  has  also  been  performed  by  van't  Hoff.5 

An  undoubted  synthesis  of  fat  and  other  ester-like  combinations 
of  fatty  acids  is  also  known.  Kastle  and  Loevenhart  6  have  shown 
the  formation  of  ethyl  butyrate  from  ethyl  alcohol  and  butyric  acid 
under  the  influence  of  a  pancreas  enzyme.  In  an  analogous  manner 
Hanriot  7  obtained  monobutyrin  from  butyric  acid  and  glycerin  with 
blood  serum.  Pottevin  8  by  means  of  a  pancreas  enzyme  transformed 
oleic  acid  and  glycerin  into  mono-  and  triolein  as  well  as  oleic  acid  esters 
with  monatomic  alcohols.  The  synthetical  action  of  the  pancreas  has 
been  closely  studied  bjr  Dietz.9 

The  enzyme  used  by  Dietz  was  insoluble  in  water,  and  its  action  was  tested 
with  z'-amyl  alcohol  and  n-butyric  acid  or  the  corresponding  ester.  It  was  shown 
that  the  reaction  took  place  in  the  insoluble  phase  (enzyme).  From  the  formula 
alcohol -f  acid  tester + water,  it  follows  that  when  the  molecular  concentrations 
of  alcohol,  aeid,  ester  and  water  are  designated  Ca,  Cs.  Ce.  Cw,  the  reaction  velocity 

dx 
of  the  ester  formation  for  a  homogeneous  system  is  -j7=k1.CA-Cs—ki.CE.Cw  (see 

dx 
page  32),  which  equation  can  be  simplified  to  -rr  =  ki.Cs—k2.CE  as  the  alcohol 

and  water  were  in  excess  and  their  concentration  considered  as  constant  and 
included  in  the  constants  h  and  k2.    At  equilibrium  we  have  kiCs=k2Cs  or 

1  Ber.  d.  d.  chem.  Gesellsch.,  34,  3810  (1901). 

*  Bioch.  Zeitschr.,  14,  238  (1908). 
» Ibid.,  17,  257  (1909). 

♦Bioch.  Zeitchr,  46,  7  (1912). 

8  Sitzungsber.  preuss.  Akad.  Wiss.,  1909,  s.  1065;   1910  s.  963. 

8  Arner.  Chem.  Journ.,  24,  491  (1900). 

7Compt.  rend.,  132,  212  (1901). 

»nnd.,  136,  1152  (1903),  138,  378  (1903);  Ann.  Inst.  Past.,  20,  901  (1906). 

•  Zeitschr.  f.  physiol.,  Chem.,  52,  279  (1907). 


ENZYMES.  61 

j-=7T  =K  (page  32).     It  follows  that  the  same  equilibrium  is  attained  irrespective 

of  whether  we  start  with  alcohol+acid,  or  ester+H20.  The  equilibrium  is  also 
independent  of  the  antecedents  as  well  as  the  quantity  of  enzyme. 

On  comparing  the  equilibrium  constants  (A')  which  are  obtained  with  dif- 
ferent quantities  of  ester  or  acid,  it  is  shown  that  in  the  above  equation  V(7s 
must  be  introduced  instead  of  Ce  in  order  to  obtain  constant  values  for  A'.  In 
the  saponification  of  the  ester  the  reaction  velocity  is  proportional  to  \^Cs, 
and  not  Ce.  According  to  Dietz  this  is  due  to  the  fact  that  the  system  is  a 
heterogeneous  one,  and  that  only  that  part  of  the  ester  which  is  absorbed  by 
the  solid  phase  (enzyme)  takes  part  in  the  reaction.  The  velocity  constant  of 
the  ester  formation  is  shown  to  be  proportional  to  the  quantity  of  enzyme. 

According  to  what  was  stated  above  (page  35),  the  equilibrium  in  a  reversible 
reaction  must  be  independent  of  the  nature  of  the  catalyst.  This  was  not  the 
case  in  Dietz's  experiments.  With  picric  acid  as  the  catalyst  a  different  equilib- 
rium was  obtained  than  with  the  pancreas  enzyme.  With  the  acid  as  catalyst 
the  equilibrium  was  moved  toward  the  direction  of  the  ester.  While  this  action 
is  not  understood  it  may  perhaps  be  explained  by  the  fact  that  the  system  in  one 
case  was  homogeneous  and  in  the  other  case  heterogeneous. 

Similar  observations  that  the  enzymotic  end-condition  can  be  different  from 
the  stabile  end-condition  of  the  same  system  have  previously  been  made  by 
Tammann,1  but  in  these  cases  generally  so-called  false  equilibrium  existed,  which 
for  example,  by  the  addition  of  more  enzyme  changed,  so  that  the  cleavage  pro- 
ceeds further.  These  false  equilibria  are  generally  caused  by  the  enzyme  being 
destroyed  or  put  out  of  action  in  other  ways. 

Among  the  enzymotic-ester  syntheses  we  must  also  include!  he  or- 
mation  of  carbohydrate  phosphoric  acid  ester  in  fermenting  sugar  solu- 
tions in  the  presence  of  soluble  phosphates,  as  first  observed  by  Harden 
and  Young.2    These  will  be  discussed  in  detail  in  Chapter  III. 

It  is  seen  that  enzymotic  syntheses  are  known.  From  this  it  fol- 
lows that  the  questionable  enzyme  reactions  are  to  be  considered  as 
reversible.  In  certain  cases  another  substance  which  cannot  be  split 
by  the  enzyme  is  formed  while  in  other  cases  the  opposite  direction  of  the 
reaction  can  be  detected  by  means  of  various  constituents  of  the  same 
enzyme  solution. 

Specificity  of  Enzyme  Action.  It  has  been  known  for  a  long  time 
that  a  great  difference  exists  in  regard  to  the  action  of  enzymes  in  the 
sense  that  different  enzymes  act  only  upon  certain  classes  of  bodies  (pro- 
teins, carbohydrates,  fats).  Then  there  also  exist  differences  in  the 
manner  in  which  different  enzymes  of  the  same  group  influence  different 
members  of  the  same  class  (maltase,  lactase,  saccharase).  Finally,  it  is 
possible  for  one  enzyme  to  attack  one  of  two  optical  antipodes  and  the 
other  not  at  all,  or  only  to  a  slight  degree.  That  optical  antipodes 
are  burned  with  unequal  facility  in  the  organism  was  shown  by  E.  Fischer, 
and  that  of  the  numerous  aldohexoses  only  three,  rf-glucose,  d-mannose 

1  Zeitschr.  f.  physiol.  Chem.,  16,  271  (1892). 

2  Proc.  Roy.  Soc.  B,  1908  p.  209. 


62  GENERAL  AND  PHYSICO-CHEMICAL. 

and  d-galactose,  and  of  the  ketohexoses  only  one,  d-fructose  are  fer- 
mentable; and  then  that  the  synthetically  prepared  stereoisomeric 
glucosides  behave  differently  with  the  enzymes.  Thus  of  two  isomeric 
glucosides,  one  methyl-d-glucoside,  the  (a)  was  attacked  by  yeast  and 
the  other  (j3)  only  by  emulsin,  while  the  corresponding  methyW-glucosides 
were  not  split  by  either  of  these  enzymes.  The  corresponding  glucoside 
obtained  from  galactose  behaves  in  a  similar  manner.1  On  the  behavior 
of  amygdalin  to  various  enzymes  see  page  59.  In  connection  with 
these  observations  Fischer  presents  the  theory  that  for  the  action  of 
an  enzyme  a  certain  correspondence  in  stereometric  structure  of  the 
enzyme  and  substrate  must  exist;  the  enzyme  must  fit  the  substrate 
somewhat  like  a  key  fitting  a  lock. 

Then  followed  similar  observations  of  Darin,2  who  found  that  racemic 
mandelic  acid  ester,  on  incomplete  hydrolysis  by  liver  press-juice,  yielded 
a  strongly  dextrorotatory  acid,  while  the  ester  remaining  was  levorotatory. 
The  dextrorotatory  ester  was  more  quickly  hydrolyzed  than  the  levoro- 
tatory ester.  Finally,  we  must  mention  the  investigations  of  Fischer 
and  Abderhalden  3  on  the  cleavage  of  polypeptides  by  pancreas 
press-juice.  From  abundant  material  they  concluded  that  those  polypep- 
tides which  consist  entirely  of  the  optical  forms  of  amino-acids  occurring 
in  nature  are  hydrolyzed  and  the  others  not.  If  in  a  racemic  form  besides 
a  polypeptide  consisting  of  natural  amino-acids,  another  occurs  also, 
then  only  the  first  is  hydrolyzed.  Besides  this,  other  factors  are  also 
of  importance.  Thus  Z-leucyl-glycine  is  not  hydrolyzed,  although  both 
constituents  occur  in  nature.  The  size  of  the  molecule  seems  also  to  be 
of  importance,  as  mono-,  di-  and  triglycyl-glycine  are  not  split,  while 
tetraglycyl-glycine  is.     See  also  Chapter  VIII. 

Retardation  of  Enzyme  Action.  There  are  several  reasons  for  the 
assumption  that  the  hydrolytic  enzymes  are  only  active  after  they  have 
combined  with  the  substrate.  From  this  it  follows  that  those  substances 
which  prevent  the  formation  of  such  combination  may  cause  the  retarda- 
tion of  enzyme  action.  For  this  reason  the  enzyme  action  is  retarded 
by  such  substances  which  adsorb  the  enzyme  (page  49).  Hedin4  has 
made  experiments  on  the  retarding  action  of  charcoal  upon  the  action 
of  trypsin  upon  casein,  and  the  action  of  rennin  upon  milk  and  it  was 
shown  that  the  retardation  was  more  pronounced  if  the  powder  and 
enzyme  were  allowed  to  act  upon  each  other  before  the  substrate  was 
added  than  if  this  was  present  from  the  beginning.     This  fact  indicates 

1  Zeitschr.  f.  physiol.  Chem.,  26,  60  (1898)  (collection  of  Fischer's  works). 
Mourn,  of  Physiol.',  30,  253  (1903);  32,  199  (1905). 
•Zritechr.  f.  physiol.  Chem.,' 46,  52  (1905);  61,  264  (1907). 

•Bioch.  Jpurn.,  1,  484;  2,  81  (1906);  Zeitschr.  f.  physiol.  Chem.,  50,  497  (1907); 
60,  143  (1909).     See  also  Jahnson-Blohm,  ibid.,  82,  178  (1912). 


ENZYMES.  63 

that  the  adsorption  process  is  only  reversible  with  great  difficulty  or 
that  the  enzyme  to  a  certain  extent  is  fastened  to  the  charcoal.  That 
the  substrate  influences  the  formation  of  adsorption  combination  is 
shown  by  the  fact  that  the  substrate  is  also  adsorbed  by  the  charcoal. 
A  small  part  of  the  adsorbed  enzyme  can  indeed  be  subsequently 
displaced  on  the  charcoal  by  other  adsorbable.  substances  and  in  this  way 
become  active  again.  As  various  substrates  are  unequally  adsorbed 
by  charcoal  the  retardation  is,  therefore,  also  different  in  degree.  The 
retardation  of  the  saccharase  action  by  charcoal  is  the  same  as  for  the 
retardation  of  the  trypsin  or  rennin  action  (Eriksson  j). 

The  action  of  several  enzymes  is  retarded  by  normal  serum.  This 
was  first  observed  by  Hammarsten  and  Roden  2  for  the  action  of  rennin. 
Besides  this  certain  constituents  of  the  serum  as  well  as  other  protein 
containing  fluids  have  a  retarding  action  and  in  many  such  cases  the 
order  of  the  addition  of  the  bodies  is  important.  The  retardation  by 
charcoal  corresponds  to  this  retardation  in  several  ways  and  this  has 
led  Hedin  3  to  the  assumption  that  the  retardation  in  both  cases  is  brought 
about  by  a  colloidal  reaction  (adsorption)  between  the  enzyme  and  a 
solid  or  colloid  phase.  The  facts  correspond  to  this  assumption  namely 
that  during  the  action  of  the  retarding  substance  upon  the  enzyme 
the  amount  of  water  present  is  without  importance  for  the  final  result 
of  retardation.  Such  a  retardation  by  normal  serum  or  fluids  con- 
taining protein  has  been  observed  in  the  following  cases:  retardation  of 
trypsin  digestion  of  casein  by  native  seralbumin,4  retardation  of  the  action 
of  rennin  by  neutral  serum  and  by  white  of  egg,5  and  the  action  of  sac- 
charase by  serum.6  Besides  this  Hedin  7  found  a  similar  retardation  by 
seralbumin  upon  the  digestion  of  casein  by  means  of  the  a-protease  of  the 
spleen.  The  retardation  by  normal  serum  or  seralbumin  has  been  shown 
in  the  cases  investigated  not  to  be  a  specific  kind,  i.e.,  a  given  enzyme 
is  retarded  about  to  the  same  extent  regardless  from  what  species  of 
animal  it  was  prepared. 

A  specific  retardation  due  to  kind  have  been  observed  in  the  following 
cases : 

1.  The  antienzyme  obtained  by  immunization  (see  page  66)  retards 
in  those  cases  tested,  only  or  chiefly  the  enzyme  used  in  the  immuniza- 


lIbid.,  72,  313  (1911). 

2  Upsala  lakarefor.  forh.,  22,  546  (1887). 

3  Bioch.  Journ.,  1,  484  (1906);  Zeitschr.  f.  physiol.  Chem.,  60,  364  (1909);   Ergebn. 
d.  Physiol.,  9,  433  (1910). 

*  Journ.  of  Physiol..  32,  390  (1905);   Bioch.  Journ.,  1,  474  (1906). 

5  Zeitschr.  f.  physiol.,  60,  85,  364;  63,  143  (1909). 

6  Ibid.,  72,  313  (1911). 

7  Hammarsten's  Festschr.,  1906. 


64  GENEEAL  AND  PHYSICO-CHEMICAL. 

tion.  Hildebrandt  l  first  produced  an  anti-enzyme  toward  emulsin; 
and  Morgenroth  2  obtained  in  a  similar  manner  an  anti-rennin  in  goats' 
serum;  Bordet and Gengou 3  immunized  against  fibrin  ferment,  Sachs'4 
against  pepsin,  Schutze  as  well  as  Bertarelli  5  against  various 
plant  lipases,  Schutze  6  against  lactase,  Preti  as  well  as  Schutze  and 
Braun  7  against  diastase,  £.  Meyer  8  against  the  proteases  of  bacillus 
prodigiosus  and  bacillus  pyocyaneus. 

2.  The  retarding  body  of  the  rennin  enzyme  which  was  obtained 
by  treating  a  neutral  infusion  of  the  mucous  membrane  with  dilute 
ammonia  and  neutralizing,  has  been  recently  shown  by  Hedin  9  to 
chiefly  retard  the  enzyme  of  the  same  species  (see  Chapter  VIII).  In 
these  cases  the  importance  of  the  order  of  treatment  was  also  evident. 

Most  of  the  retarding  substances  contained  in  the  serum  lose  their 
retarding  power  on  sufficiently  heating  them.  This  also  occurs  in  cer- 
tain cases  by  treatment  with  acid.  Thus  normal  horse  serum  as 
well  as  egg-white  lose  their  ability  to  retard  rennin  by  treatment 
with  very  dilute  hydrochloric  acid  and  for  this  reason  rennin  which 
has  been  inactivated  by  serum  or  egg-white  can  be  set  free  again 
by  the  use  of  hydrochloric  acid  (Hedin)  10.  Native  seralbumin  loses 
its  power  of  attaching  itself  to  trypsin  by  treatment  with  dilute 
acetic  acid. 

Certain  proteins  which  are  digested  with  difficulty  retard  the  diges- 
tion of  more  readily  digestible  ones  without  the  order-phenomenon  being 
observed.  In  such  cases  the  total  digestion  is  probably  diminished 
because  the  more  difficultly  digested  protein  as  substrate  attracts  a 
part  of  the  enzyme.  As  the  order-phenomenon  does  not  exist,  the  enzyme 
is  taken  up  in  a  complete  and  readily  reversible  manner  (enzyme  devia- 
tion Hedin).11  It  is  easily  understood  that  the  retardation  must  be 
less  effective  than  in  those  cases  where  the  enzyme  is  attached  to  the 
retarding  substance.  The  tryptic  digestion  of  casein  in  the  presence 
of  seralbumin,  treated  with  acid,  is  diminished  by  enzyme  deviation  as 
well  as  the  digestion  of  readily  split  proteins  is  retarded  by  egg-white 


1  Virchow's  Arch.,  131,  33  (1893). 

2Centralbl.  f.  Bakt.,  26,  349  (1899);  27,  357  (1900). 

'Ann.  inst.  Past.  15,  129  (1901). 

*  Fortschr.  d.  Med.,  20,  593  (1901). 

6  Deutsch.  med.  Wochenschr.,  1904;  Centralbl.  f.  Bakt.,  40,  231  (1905). 

'Zeitschr.  f.  Hyg.,  48,  457  (1904). 

7Bioch.  Zeitschr.,  4,  6  (1907);  Zeitschr.  exp.  Pathol,  u.  Therap.,  6,  307  (1909). 

»Bioch.  Zeitschr.,  32,  280  (1911). 

•Zeitschr.  f.  physiol.  Chem.,  72,  187;  74,  242;  76,  355  (1911). 

"Zeitschr.  f.  physiol.  Chem.,  60,  85,  364  (1909). 

»  Ibid.,  52,  412  (1907). 


ENZYMES.  65 

which  is  difficult  to  digest  (Delezenne  and  Pozerski,1  Vernon,2 
Qompel  and  Henri,3  Hedin4). 

At  this  time  we  must  also  mention  the  retarding  action  which  the 
proteolytic  primary  cleavage  products  (proteoses,  peptones)  exert  upon 
digestion.  These  products  are  further  split;  a  part  of  the  enzyme  is 
combined  with  the  products  and  in  this  way  prevented  from  dissolv- 
ing new  protein  (Hedin).5  The  retarding  power  of  proteoses  and  pep- 
tones upon  rennin  action  is  probably  similar  to  the  above.6 

Finally,  the  end  products  of  enzymotic  activity  i.e.,  bodies  which 
cannot  be  further  split  by  the  enzyme,  have  also  a  retarding  action  on 
the  enzyme  action.  That  the  inversion  of  cane-sugar  is  retarded  by 
invert  sugar  has  been  claimed  by  many  (Henri,7  A.  J.  Brown,8  Baren- 
drecht,9  Armstrong10),  and  indeed  Barendrecht  claims  that  glucose 
as  well  as  fructose  has  a  retarding  action,  and  that  galactose  has  an 
even  stronger  retarding  action  than  the  direct  cleavage  products  of  cane- 
sugar.  H.  E.  and  E.  F.  Armstonng11  found  that  saccharase,  maltase  and 
lactase  are  retarded  by  just  those  varieties  of  sugar  which  are  produced 
by  their  activity.  The  accumulation  of  the  amylolytic  cleavage  prod- 
ucts have  according  to  Sh.  Lea,12  a  retarding  action  upon  saliva. 

The  retarding  action  of  amino-acids  upon  the  decomposition  of  glycyl- 
Z-tyrosine  by  yeast-press  juice  has  recently  been  studied  by  Abderhalden 
and  Gigon.13  They  found  that  cleavage  of  peptides  is  retarded  by 
those  optically  active  amino-acids  which  occur  in  the  proteins.  This 
result  is  remarkable  in  consideration  of  the  observations  of  Fischer 
and  Abderhalden  that  only  those  polypeptides  were  split  by  pancreatic 
juice  which  are  composed  of  natural  optically  active  amino-acids  (page  62). 

The  retardation  of  the  action  of  papain  by  egg  protein  and  by  serum,  which 
is  prevented  by  heating  or  action  of  hydrochloric  acid,  as  shown  by  the  investiga- 
tions of  Delezenne,  Mouton  and  Pozerski  as  well  as  by  Jonescu  and  Sachs  u 
is  a  peculiar  behavior. 

1  Compt.  rend.  soc.  biol.,  55,  935  (1603). 

2  Journ.  of  Physiol.,  31,  495  (1904). 

3  Compt.  rend.  soc.  biol.,  58,  457  (1906). 

4  Zeitschr.  f.  physiol.  Chem.,  52,  422  (1907). 

5  Zeitschr.  f.  physiol.  Chem.,  52,  422  (1907). 

6  Ibid.,  46,  307. 

7  Zeitschr.  f.  physik.  Chem.,  39,  194  (1901). 

8  Journ.  Chem.  Soc,  81,  382  (1902). 

9  Zeitschr.  f.  physik.  Chem.,  49,  456  (1904). 

10  Proc.  Roy.  Soc.  (ser.  B),  73,  516  (1904). 

11  Ibid.,  79,360  (1907). 

12  Journ.  of  Physiol.,  1911. 

13  Zeitschr.  f.  physiol.  Chem.,  53,  251  (1907). 

14  Delezenne,  Mouton  and  Pozerski,  Compt.  rend.,  142;  Jonescu,  Bioch.  Zeitschr., 
2;   Sachs,  Zeitschr.  f.  physiol.  Chem.,  51,  488  (1907). 


66  GENERAL  AND   PHYSICO-CHEMICAL. 

In  consideration  of  what  has  been  said  (page  58)  about  enzymotic 
syntheses  it  seems  very  possible  in  the  retardation  of  enzymotic  cleavages 
by  means  of  cleavage  products  that  we  are  dealing  with  synthetic 
processes  where  the  cleavage  products  supply  the  material.  This  is  espe- 
cially shown  by  the  above-mentioned  investigations  of  Rosenthaler 
on  emulsin  that  the  retarding  action  of  benzaldehyde  or  of  hydrocyanic 
acid  upon  emulsin  action,  as  shown  by  Tammann,1  is  explainable  by 
syntheses.  Lichwitz  2  considers  the  interaction  of  the  products  as  a 
reversible  paralyzation  of  the  enzyme. 

Appendix :  Antigens  and  Anti-bodies.  In  connection  with  the  retar- 
dation of  enzyme  action  we  can  also  call  attention  to  other  similar  proc- 
esses. Under  the  name  antigen  we  include  those  substances  which, 
when  injected  into  animals,  cause  the  formation  of  bodies  in  the  organ- 
ism with  which  they  can  in  some  way  or  another  react.  The  process 
is  called  immunization  and  the  bodies  formed  are  called  anti-bodies  or 
in  certain  cases  immune  bodies.  General)}''  these  anti-bodies  are  specific 
in  the  sense  that  they  only  react  with  the  corresponding  antigen.  The 
chemical  constitution  of  the  antigen  as  well  as  of  the  anti-body  is  not 
known;  they  belong  perhaps  to  the  colloids,  or  at  least  they  occur  asso- 
ciated with  colloids. 

The  antigens  are  either  substances  soluble  in  water  or  occur  as 
constituents  of  the  cells.  We  will  first  discuss  the  antigens  soluble  in 
water. 

To  this  group  belong,  in  the  first  place,  certain  poisonous  substances 
of  animal  or  plant  origin  (toxins),  for  example,  snake  poisons,  bacterial 
poisons,  ricin  (from  the  seeds  of  Ricinus  communis),  also  enzymes  as  well 
as  certain  proteins  without  special  action.  The  reaction  with  the  anti- 
bodies (which  are  obtained  in  the  blood  serum  of  animals)  manifests 
itself  with  the  poisons  by  the  suppression  of  the  poisonous  action,  with 
the  enzymes  by  retardation  of  the  enzyme  action,  and  with  certain  pro- 
teins by  formation  of  a  precipitate  which  contains  the  antigen  as  well 
as  the  anti-body.     Anti-bodies  of  this  last  type  are  called  precipitins. 

The  longest  known  (due  to  the  epoch-making  investigations  of  v. 
Behring  3)  and  best  studied  are  those  anti-bodies  which  are  produced 
by  toxins  and  which  neutralize  the  action  of  the  toxins  upon  the  animal 
organism  (antitoxins).  According  to  the  older  view  this  takes  place 
by  some  sort  of  an  action  of  the  anti-body  upon  the  cells  sensitive  to  the 
toxins.  After  it  was  shown  that  the  toxins  could  also  be  neutralized 
in  vitro  by  the  anti-bodies,  it  is  now  generally  accepted  that  the  neu- 


lIbirL,  16,  271  (1892). 
-  Ibid.,  78,  128  (1912). 
*  Deutsch.  ix.ed.  Wochenschr.,  1892;  Zeitschr.  f.  Hygiene,  12  (1892). 


ENZYMES.  67 

tralization  is  brought  about  by  some  sort  of  a  combination  between  the 

toxin  and  the  anti-body.      The  views  are  very  contradictory  in  regard 
to  the  nature  of  this  combination  and  the  manner  in  which  it  is  formed. 

The  oldest  theory,  which  has  contributed  much  to  our  knowledge 
of  these  conditions,  is  that  of  P.  Ehrlich,  whom  we  must  thank  for  the 
method  of  measuring  the  quantity  of  toxin  by  injection  into  an  ani- 
mal. The  quantity  of  toxin  which  is  just  sufficient  to  kill  a  guinea-pig 
of  given  weight  in  a  certain  time  is  selected  as  the  unit.  According  to 
so-called  side-chain  theory  of  Ehrlich  l  the  toxins  firstly  have  a  so- 
called  haptophore  group,  by  means  of  which  the  toxin  can  attach  itself 
to  a  certain  cell,  and  secondly,  a  so-called  toxophore  group,  by  which 
the  toxin  exerts  its  poisonous  action.  The  formation  of  anti-body 
after  the  injection  of  the  toxins  Ehrlich  explains  by  the  fact  that  those 
cells  which  are  attacked  by  the  toxins  are  supplied  with  so-called  recep- 
tors, which  just  fit  the  haptophore  group- of  the  toxins;  the  toxins  are 
thus  anchored  on  the  questionable  cells  and  can  then  begin  their  action 
by  aid  of  the  toxophore  group.  By  the  attachment  of  the  receptors,  the 
cells  are  induced  to  produce  new  receptors,  and  indeed,  so  many  recep- 
tors are  produced  that  they  are  thrown  off  and  appear  free  in  the  blood 
plasma.  The  receptors  circulating  in  the  blood  are  the  anti-bodies. 
As  these  are  able  to  combine  with  the  toxins  they  can  protect  against 
the  toxin  those  cells  which  are  supplied  with  the  same  receptor  under 
whose  influence  they  were  found.  The  toxophore  group  of  the  toxins 
can  gradually  be  destroyed  on  keeping.  A  toxin  so  changed  can  be 
continuously  anchored  to  cell-receptors  and  in  this  way  form  anti-bodies, 
but  cannot  produce  any  poisonous  action.  A  toxin  without  toxophore 
groups  is  called  a  toxoid  by  Ehrlich.  It  follows  that  the  toxoids  can 
combine  with  the  anti-bodies. 

According  to  Ehrlich,  on  the  neutralization  of  a  toxin  a  chemical 
combination  takes  place  between  the  toxin  and  the  anti-body,  and  so 
much  of  this  combination  is  formed  that  either  the  toxin  or  the  anti- 
body is  completely  consumed.  Now  the  bacterial  poisons  are  not 
simple  bodies,  but  mixtures  of  several  poisons  of  different  toxicity  and 
different  avidity  toward  the  anti-bodies.  Generally  the  most  poison- 
ous is  first  neutralized,  but  it  also  occurs  that  a  less  poisonous  or  indeed 
a  non-poisonous  body  is  first  combined  with  the  anti-body  (proto-toxoids) 
or  that  non-poisonous  bodies  are  combined  parallel  with  the  true  toxins 
(syntoxoids).  The  less  poisonous  or  non-toxic  bodies  first  combined 
after  the  binding  of  the  true  toxins  are  called  toxons  (also  epitoxoids). 
According  to  the  relative  quantity  and  the  avidity  of  the  different  con- 
stituents of  the  toxic  solution,  the  addition  of  a  certain  quantity  of  anti- 
body can  produce  entirely  different  results. 

1  See  Michaelis,  Die  Bindungsgesetze  von  Toxin  und  Antitoxin,  Berlin,  1905. 


68  GENERAL  AND  PHYSICO-CHEMICAL. 

Arrhenius  opposes  Ehrlich's  theory  that  the  combination  between 
toxin  and  anti-body  is  of  a  chemical  nature,  but  claims,  that  their  for- 
mation does  not  proceed  until  one  of  the  components  has  been  used  up. 
An  equilibrium  is  established  between  the  free  toxin  and  the  free  anti- 
body on  one  side  and  the  combination  of  the  two  on  the  other,  which 
the  law  of  mass  action  requires  according  to  the  formula: 

C     .     C     =     K     .     CN  (page  32). 

toxin     anti-body  toxin  +anti-body 

For  tetanolysin  (a  substance  obtained  from  tetanus  cultures,  which  dissolves 
red-blood  corpuscles)  and  its  anti-body,  as  well  as  for  diphtheria  toxin  and  the 
corresponding  anti-body,  n=2  was  found,  i.e.,  in  the  combination  of  a  molecule 
of  toxin  with  a  molecule  anti-body  two  molecules  toxin-antitoxin  combination 
was  formed. 

The  toxic  action  which  a  mixture  of  toxin  and  anti-body  exerts 
depends  upon  the  quantity  of  toxin  which,  according  to  the  above 
formula,  must  always  remain  free.1  According  to  this  theory  the  toxin 
is  a  unit  poison,  as  Arrhenius2  now  admits  with  Ehrlich,  that  the 
poison  is  gradually  transformed  into  a  non-toxic  or  only  slightly  toxic 
substance  which  has  the  same  ability  to  combine  with  antitoxin  as  the 
toxin  itself. 

Ehrlich's  theory,  as  well  as  that  of  Arrhenius  admits  of  a  chem- 
ical combination  between  the  antigen  and  the  anti-body.  According 
to  Ehrlich  besides  this  the  substrate  (or  the  cells  sensitive  to  the  anti- 
gen) combines  with  the  antigen,  which  is  not  conformable  with  the  theory 
of  Arrhenius. 

The  combination  toxin-anti-body  is  first  gradually  produced,  and 
then  it  is  taken  up  from  all  sides  so  that  the  toxin  is  fastened  to  the 
anti-body  by  a  secondary  process  (exception,  cobra  poison).  The  com- 
bination toxin-antitoxin  is  not  reversible  in  the  ordinary  sense.  This 
is  most  easily  shown  by  the  fact  that  to  a  certain  limit  more  toxin  is 
neutralized  according  to  the  time  allowed  to  elapse  before  the  quantity 
of  toxin  remaining  free  is  determined  by  injection  into  an  animal  or  in 
other  ways.3  In  certain  cases  it  is  possible  to  obtain  the  toxin  again  in 
an  active  form  from  the  toxin-antitoxin  combination,  and  indeed  by 
treatment  with  very  dilute  hydrochloric  acid  (Morgenroth4).  See 
also  page  64  on  the  setting  free  of  rennin  from  its  combination  with 
normal  scrum  and  with  egg-white.     Hedin5  has  also  been  able  to  obtain 

«  Zeitschr.  f.  physik.  Chem.,  44,  7  (1903). 
3  Immunochemie,  Leipzig,  1907,  132. 

3  Martin  and  Cherry,  Proc.  Roy.  Soc,  1898,  420'. 

4  Berl.  klin.  Wochenschr.,  1905,  No.  5;  Festschr.  z.  Eroffnung  d.  pathol.  Instit. 
Berlin,  1906;  Virchow's  Arch.,  190,  371  (1907). 

*  Zeitschr.  f.  physiol.  Chem.,  77,  229  (1912). 


ENZYMES.  69 

the  rennin  again  in  an  active  form,  from  the  combination  of  the  rennin 
with  anti-rennin  obtained  by  immunization  by  treatment  with  hydro- 
chloric acid  and  then  neutralizing. 

Recently  a  third  manner  of  considering  the  toxin-antitoxin  reaction 
has  been  presented  which  is  based  on  the  fact  that  the  reaction  takes 
place  in  a  heterogeneous  system.  According  to  this  the  reaction  is  con- 
sidered as  an  adsorption  process,  and  in  support  of  this  assumption,  sev- 
eral examples  can  be  given  where  finely  divided  solids  or  colloid  sub- 
stances take  up  toxins  or  enzymes,  in  an  irreversible  manner  (Nernst,1 
Biltz,2  Landsteiner3). 

In  reference  to  the  formed  antigens  we  must  call  attention  to  the  fol- 
lowing : 

If  certain  cells,  for  example,  bacteria,  blood-corpuscles,  and  sperma- 
tozoa are  injected  into  animals,  then  anti-bodies  are  formed  which  have 
been  called  immune  bodies  (also  amboceptors  or  sensibilizators) .  By 
themselves  the  immune  bodies  are  inactive,  but  form  with  complements, 
substances  occurring  in  normal  serum,  so-called  cytotoxins,  which  destroy 
the  kind  of  cells  active  in  their  formation.  These  cytotoxins  are  called 
bacteriolysins,  hemolysins,  etc.,  according  to  the  kind  of  cells  used. 
The  immune  bodies  are  specific  in  that  they  together  with  the  com- 
plement only  attack  those  cells  from  which  they  are  formed  and 
they  are  also  stable  against  heat;  the  complements  can  act  together 
with  different  immune  bodies  and  are  very  unstable,  as  they  are  gen- 
erally destroyed  by  heating  to  56°  C.  for  one-half  hour.  Other  anti- 
bodies, produced  under  the  influence  of  injected  cells,  show  their  action 
by  flocking  together  and  agglutinating  the  cells  set  free  in  their  forma- 
tion.    These  anti-bodies  are  called  agglutinins. 

In  regard  to  the  immune  bodies,  Ehrlich  believes  that  they  com- 
bine with  those  cells  under  whose  influence  they  have  been  formed 
and  also  with  the  complements.  They  serve  to  fasten  (amboceptors) 
the  complement,  which  produces  the  real  poisonous  action,  to  the  cells. 
The  immune  bodies  correspond  therefore  to  the  haptophore  groups  of 
the  toxins  and  the  complements  of  the  toxophores.  According  to 
Bordet  the  immune  bodies  act  upon  the  cells  in  the  way  that  the  latter 
are  sensitive  toward  the  complements  (sensibilizators). 

If  a  certain  immune  serum  is  heated  to  56°  then,  according  to  what 
has  been  given,  the  complement  is  destroyed  and  the  serum  now  con- 
tains only  the  amboceptor  of  the  original  cyto-toxin  and  this  amboceptor 
can  be  made  active  again  by  the  addition  of  normal  serum  (complement). 


»  Zeitschr.  f.  Elektrochem.,  10,  379  (1904). 

2Ber.  d.  d.  Chem.  Gesellsch.,  37,  3147  (1904);   Beitr.  z.  exp.  Therapie,  1,  30  (1905). 

3  Zeitschr.  f.  Chem.  u.  Ind.  d.  Koll.,  3,  221  (1907);   Bioch.  Zeitschr.,  15,  33  (1908). 


70  GENERAL  AND  PHYSICO-CHEMICAL. 

If.  therefore,  an  antigen  of  the  corresponding  immune  serum  be  headed 
to  56°  (amboceptor)  and  mixed  with  sufficient  amount  of  normal  serum 
(complement),  then  the  complement  is  bound  up  so  that  when  subsequently 
serum-free  red-blood  corpuscles  and  a  certain  quantity  of  immune  serum, 
obtained  by  immunization  with  these  and  after  losing  its  complement 
by  heating  to  56°,  are  added,  no  solution  of  the  red-blood  corpuscles 
(haemolysis)  takes  place.  If  in  the  first  mixture  either  the  antigen  or  the 
corresponding  amboceptors  are  absent  then  the  complement  is  not 
combined  and  a  haemolysis  occurs  because  the  complement  cannot  unite 
with  the  haemolytic  amboceptors  added.  In  this  manner  it  has  been 
attempted  to  determine  the  presence  of  an  antigen  or  of  amboceptors 
which  fit  with  the  antigen  (method  of  complement  deviation). 

The  protective  substances  formed  by  immunization  can  protect  the 
organism  against  many  fatal  doses  of  the  antigen  and  this  protective 
power  can  be  brought  about  by  the  parenteral  introduction  of  the  immune 
serum  of  another  organism.  The  immunity  is  called  active  when  the 
organism  obtains  the  antigen  and  itself  produces  the  corresponding 
protective  substance.  On  the  contrary  the  immunity  is  called  passive 
if  the  organism  receives  the  anti-body  formed  in  another  living  being 
by  active  immunization. 

During  immunization  under  certain  circumstances  it  is  observed 
that  a  condition  of  super-sensitiveness  toward  the  antigen  exists.  This 
super-sensitiveness  occurs  only  toward  the  antigen  used  and  is  therefore 
specific.  The  same  has  been  observed  in  using  the  soluble  as  well  as  the 
formed  antigens.  This  mysterious  phenomenon  has  been  called  anaphyl- 
axis. 

V.     IONS  AND   SALT  ACTION. 

We  have  previously  mentioned  various  processes  which  depend  upon 
the  influence  of  ions.  To  these  belong  the  precipitation  of  suspension 
colloids  by  electrolytes  as  well  as  different  catalytic  processes.  That 
in  the  last  case  we  are  dealing  with  the  action  of  ions  is  proven  by  the 
fact  that  the  velocity  coefficient  is  proportional  to  the  concentration 
of  a  certain  kind  of  ion.  Nevertheless  it  has  been  shown,  that  the 
velocity  coefficient  in  the  inversion  of  cane-sugar,  by  acid,  is  only  propor- 
tional to  the  H  ions  when  dilute  acids  are  used.  With  greater  concen- 
tration disturbances  occur  which  can  be  ascribed  to  the  action  of  the 
negative  ions  of  the  acids.  The  catalytic  processes  can  be  influenced 
by  salts  in  a  similar  manner  (salt  action). 

The  enzyme  action  has  shown  itself  proportional  to  the  quantity  of  enzyme 
in  certain  cases.     Euler  '  has  attempted  to  show  a  correspondence  between  ion- 

1  Zeitsehr.  f.  physik.  Chem.,  36,  G41  (1901). 


IONS  AND  SALT  ACTION.  71 

action  and  enzyme  action  by  the  assumption  that  the  enzymes  cause  an  increase 
in  those  ions,  which  could  cause  the  reaction  without  the  presence  of  the  enzyme. 
On  the  other  hand  J.  Loeb  1  believes  that  the  enzymes  can  also  be  electrolytically 
dissociated  and  that  their  action  depends  on  the  amount  of  ions.  Tims  pepsin 
is  a  weak  base  which  forms  a  salt  with  the  hydrochloric  acid  added  and  thai  this 
salt  is  more  strongly  dissociated  than  the  base;  for  this  reason  the  action  of  pepsin 
is  increased  by  acid. 

Many  enzymotic  processes  are  influenced  by  the  presence  of  salts 
of  the  alkalies  or  alkaline  earths.  According  to  Bierry,  Giaja  and  Henri 
as  well  as  Preti  2  pancreatic  juice  dialyzed  for  a  long  time  has  no  action 
upon  starch,  but  becomes  active  again  on  adding  NaCl  or  other  salts. 
According  to  Wohlgemuth3  the  diastatic  power  of  saliva  is  increased 
ten-fold  by  the  addition  of  NaCl.  The  anions  are  the  active  part  in  both 
cases  (see  page  52  on  co-enzymes).  The  strong  retarding  action  which 
NaFl  exerts  upon  the  enzymotic  cleavage  of  esters  is  also  to  be  men- 
tioned (Loevenhart  and  Pierce,  Amberg  and  Loevenhart4). 

Other  actions  of  salts  are  also  ascribed  to  ion-action.  To  these 
belong  the  experiments  of  Dresser  5  according  to  which  mercury  salts, 
which  are  relatively  strongly  dissociated,  have  a  poisonous  action  upon 
organic  formations  (yeast,  frog  heart),  while  potassium-mercury  hypo- 
sulphite was  nearly  non-toxic.  As  the  last-mentioned  salt  contains 
very  few  free  Hg  ions  the  posionous  action  of  the  first  salt  is  ascribed  to 
the  ions.  Paul  and  Kronig  6  have  arrived  at  similar  results  by  inves- 
tigating the  poisonous  action  of  mercury  salts  upon  spores.  They  found 
that  KoCy4Hg,  which  hardly  contains  any  Hg  ions,  is  much  less  poison- 
ous than  an  equivalent  solution  of  HgCy2-  The  same  conditions  were 
observed  by  Maillard  7  for  copper  salts. 

This  leads  us  to  the  question  as  to  the  importance  of  water  and  the 
mineral  bodies,  which  are  of  just  as  great  moment  for  the  life  of  the 
cells  and  their  metabolism  as  the  organic  constituents.  In  regard  to 
the  water  this  follows  from  the  fact  that  the  animal  body  consists  of 
about  two-thirds  water.  If  we  also  recall  that  water  is  of  the 
greatest  importance  for  the  normal  physical  condition  of  the  tissues, 
that  the  solution  of  numerous  bodies  and  the  dissociation  of  chemical 
compounds,  that  all  flow  of  juices,  all  exchange  of  material,  all  supply 
of  food,  all  growth  or  destruction  and  all  removal  of  destructive  prod- 


1  Bioch.  Zeitschr.,  19,  534  (1909). 

2Compt.  rend.  soc.  biol.,  60,  479  (1906);    62,  432,  (1907);    Bioch.   Zeitschr.,  4,  1 
(1907);  40,  357  (1912). 

3  Bioch.  Zeitschr.,  9,  1  (1908). 

4  Journ.  of  Biol.  Chem.,  2,  397  (1907);  4,  149  (1908). 
6  Arch.  exp.  Pathol,  u.  Pharm.,  32,  456  (1893). 

6  Zeitschr.  f.  physik.  Chem.,  31,  411  (1896). 
7Compt.  rend.  soc.  biol.,  50,  1210  (1898). 


72  GENERAL  AND   PHYSICO-CHEMICAL. 

ucts,  are  connected  with  the  presence  of  water,  and  that  besides  this 
the  water  by  its  evaporation  is  an  important  regulator  of  temperature, 
it  is  evident  that  water  must  be  a  necessity  of  life. 

The  mineral  substances  found  habitually  in  the  cells  of  higher  plants 
and  of  animals  are  potassium,  sodium,  calcium,  magnesium,  iron,  phos- 
phoric acid,  sulphuric  acid,  chlorine,  and  perhaps  also  iodine  (Justus).1 
Besides,  in  certain  cells  or  organs  we  also  find  manganese,  lithium,  barium, 
silicium,  fluorine,  bromine,  and  arsenic. 

We  are  chiefly  indebted  to  Liebig  for  showing  that  the  mineral 
bodies  are  as  important  for  the  normal  constitution  of  the  organs  and 
tissues,  as  well  as  for  the  normal  performance  of  the  processes  of  life, 
as  the  organic  constituents  of  the  body.  The  importance  of  the  mineral 
constituents  is  evident  from  the  fact  that  we  know  no  animal  tissue 
and  no  animal  fluid  which  is  free  from  mineral  bodies,  and  also  from 
the  fact  that  certain  tissues  or  tissue  elements  contain  chiefly  certain 
mineral  bodies  and  not  others.  In  regard  to  the  alkali  compounds  this 
division  is,  in  general,  as  follows:  The  sodium  compounds  occur  chiefly 
in  the  fluids,  while  the  potassium  compounds  occur  especially  in  the 
form-elements.  Corresponding  to  this,  the  cells  contain  chiefly  potas- 
sium as  phosphate,  while  they  are  less  rich  in  sodium  and  chlorine  com- 
pounds. The  fundamental  experiments  of  Forster2  have  shown  us  that 
inorganic  salts,  as  constituents  of  the  food  are  necessary  for  the  animal 
organism. 

We  have  already  called  attention  to  the  importance  for  every  organ- 
ism of  the  salts  for  the  production  of  a  rather  constant  osmotic  pressure. 
That  the  importance  of  the  salts  is  not  limited  to  the  maintenance 
of  the  osmotic  pressure  follows  from  the  fact  that  different  salt  solutions 
of  the  same  osmotic  pressure  are  not  of  the  same  value  for  the  main- 
tenance of  the  functional  powers  on  extirpated  organs.  Since  S. 
Ringer3  showed  that  various  organic  structures  retained  their  best 
functional  activity  in  a  solution  which  contained  NaCl,  CaCl?  and 
KC1  at  the  same  time,  various  investigators  have  given  the  most  suit- 
able composition  of  such  solutions.  For  the  transfusion  fluid  for  the 
mammalian   heart    Locke4  suggests  the   following   composition;     NaCl 

1  Justus,  Virchow's  Arch.,  170,  176  and  190.  In  regard  to  arsenic  see  the  works 
of  Gautier,  Compt.  rend.,  129,  130,  131,  139;  Bertrand.  ibid.,  134;  Segale,  Zeitschr. 
f.  physiol.  Chem.,  42;  Kunkel,  ibid.,  44.  In  regard  to  the  barium  see  Schultze  and 
Thierf elder,  Sitzungsber.  d.  Gesellsch.  naturforsch.  P'reunde,  1905,  No.  1,  and  in 
regard  to  lithium  see  Hermann,  Pfluger's  Arch.,  109;  and  in  regard  to  manganese  see 
Bradley,  Journ.  of  Biol.  Chem.,  3. 

2  Zeitschr.  f.  Biol.,  9,  297  (1873);  12,  464  (1877). 

'Journ.  of  Physiol.,  6,  154,  361  (1885);  7,118(1886);  16,  1,  17,  23  (1895);  18, 
425  (1896). 

*  Centralbl.  f.  Physiol.,  14,  672  (1900). 


IONS   AND   SALT  ACTION.  73 

0.9-1  per  cent,  CaCl2  0.02-0.024  per  cent,  KC1  0.02-0.042  per  cent, 
XaHCO:j  0.01-0.03  per  cent.  Each  of  the  salts  NaCl,  CaCl2  and  KC1 
individually  has  a  poisonous  action  upon  the  organ  but  this  action  is 
counteracted  by  the  presence  of  the  two  other  salts  (antagonistic  salt 
action). 

This  neutralizing  action  of  salts  has  been  studied  during  recent 
years  especially  by  J.  Loeb  and  his  collaborators.  As  general  results 
it  has  been  found  that  the  most  favorable  quantity  relations  of  the 
three  salts  NaCl,  KC1  and  CaCk  for  the  maintenance  of  life  is  the  same 
as  exists  in  blood.  Especially  interesting  are  the  experiments  with  the 
Fundulus  heteroclitus,  a  genus  of  killifish.  This  fish,  it  is  remarkable,  can 
also  live  in  distilled  water  and  is  therefore  within  wide  limits,  not  depend- 
ent upon  the  osmotic  pressure  of  the  surrounding  medium.  For  this 
reason  it  is  specially  suited  for  the  study  of  the  poisonous  action  of  salts 
or  mixture  of  salts.  KC1  in  concentrations  in  which  it  exists  in  sea  water 
acts  as  a  poison  upon  these  fishes,  if  it  is  alone  in  solution.  The  same 
is  true  for  NaCl.  On  the  contrary  these  fishes  live  for  an  indefinite 
time  in  a  pure  CaCl2  solution  in  a  concentration  similar  to  sea  water. 
One  mol.  KC1  can  be  very  nearly  de-toxicated  by  17  mol.  NaCl  or  by  8^  mol. 
Na2S04.  \  mol.  K2SO4  is  just  as  poisonous  as  1  mol.  KC1.  The  toxicity 
of  the  potassium  salts  is  therefore  dependent  upon  the  K  ions  and  the 
de-toxicating  substance  on  the  Na  ion.  CaCl2  de-toxicates  a  KC1  solu- 
tion even  when  ^  mol.  CaCb  to  1  mol.  KC1  is  present.  SrCl2  shows 
almost  as  great  a  de-toxicating  action  as  CaCk-  NaCl  in  concentra- 
tions, in  which  it  occurs  in  sea-water  can  only  be  incompletely  de-toxi- 
cated by  KC1;  only  by  the  addition  of  CaClo  can  the  complete  de- 
toxication  be  brought  about.  The  poisonous  action  of  acids  upon 
Fundulus  can  be  arrested  by  neutral  salts.1  Fundulus  can  accommodate 
themselves  to  a  rise  in  temperature;  a  rise  in  temperature  can  be  more 
easily  endured  when  the  concentration  of  the  surrounding  medium  is  raised 
at  the  same  time  (Loeb  and  Wasteneys).  Can  fishes  also  accommodate 
themselves  to  an  abnormal  concentration  of  the  surroundings  as  long 
as  the  rise  in  concentration  takes  place  gradually?  In  both  cases  the 
accommodation,  according  to  Loeb,2  depends  upon  a  slow  proceeding 
process,  possibly  a  tanning  of  the  surface  of  the  animal. 

The  fertilized  eggs  of  the  Fundulus  develop,  according  to  Loeb,  just 
as  well  in  water  free  from  salt  as  in  sea-water.  If  the  fertilized  eggs  are 
placed  in  a  NaCl  solution  of  the  same  osmotic  pressure  as  the  sea-water 


^ioch.  Zeichr.,  31,  450;  32,  155,  308;  33,  480-  489  (1911);  39,  167;  43,  181 
(1912). 

2  Loeb  and  Wasteneys,  Journ.  of  exp.  Zool.,  12,  543  (1913)  and  Loeb,  Bioch. 
Zeitschr.,  53,  391  (1913). 


74  GENERAL  AND  PHYSICO-CHEMICAL. 

they  die;  the  toxicity  of  the  NaCl  solution  can  be  arrested  by  small 
quantities  of  almost  any  salt  with  polyvalent  cations.  Not  only  the  salts 
of  the  alkaline  earths,  but  also  those  of  the  heavy  metals  (for  instance 
zinc  sulphate  or  lead  acetate)  can  neutralize  the  toxicity  of  the  NaCl  in 
proper  concentration.1  The  eggs  can  develop  in  solutions  which  kill  the 
completed  fish. 

The  antagonistic  action  of  salts  upon  organic  structures  depends, 
according  to  Loeb,  upon  the  fact  that  the  salts  mixed  in  proper  propor- 
tions causes  a  "  tanning  "  of  the  protoplasmic  surface  of  the  cells  whereby 
the  cells  become  impermeable  for  certain  destructive  substances  to  which 
the  salts  also  belong.  The  fertilized  eggs  of  Fundulus  can.  be  tanned 
by  NaCl + a  heavy  metal  but  not  the  completed  fish.2  Many  observa- 
tions indicate  that  the  egg  is  more  permeable  after  fertilization  than 
before.3 

Appendix 21  Determination  of  the  Reaction  of  a  Solution.  The 
reaction  of  the  solution,  in  which  a  chemical  reaction  takes  place,  plays 
an  important  role  in  many  cases.  As  the  acid  or  alkaline  reaction  of  a 
solution  depends  upon  the  amount  of  H  or  OH  ions  it  is  often  of  import- 
ance to  be  able  to  determine  the  concentration  of  these  ions  in  solution. 
These  cannot  be  determined  by  titration  with  alkali  or  acid  in  the  pres- 
ence of  organic  salts.  In  this  titration  the  existing  equilibrium  in  the 
solution  is  disturbed  and  therefore  also  other  decompositions  occur 
besides  the  neutralization  of  H  or  OH  ions.  The  quantity  of  alkali  or 
acid  used  does  not  therefore  correspond  to  the  original  concentration 
of  H  or  OH  ions. 

According  to  the  law  of  mass  action  there  exists,  between  the  H  and 
OH  ions  formed  by  the  dissociation  of  the  water  on  the  one  hand  and 
the  concentration  of  the  non-dissociated  molecules  on  the  other,  the 
following  equation 

Ch-Coh  =  Ki-Ch>o 

where  CH,  Coh  represents  the  concentration  of  the  H  and  OH  ions, 
<  ii  o  the  non-dissociated  water  molecules  and  Ki  a  constant.  As  Cr2o 
can  only  be  considered  as  constant  in  certain  dilute  solutions  we  have 
Ch-Coh  =  K)  where  K  is  called  the  dissociation  constant  of  the  water. 
As  K  is  a  constant  it  follows  that  the  figures  for  Cr-  and  Coh  can  be 
calculated,  if  the  other  is  known.  As  it  is  more  convenient  to  determine 
Ch  than  C'oh>  therefore  Cr  is  also  ordinarily  determined  for  solutions 

1  PfliiKer's  Arch.,  88,  68  (1901). 
^Science,  84,  653  (1911). 

3  Lillie,  Amer.  Journ.  of  Physiol.,  27,  289  (1911);  McClendon,  ibid.,  27,  240; 
Science,  32,  122,  317;   Lyon  and  Shackell,  ibid.,  32,  249  (1910). 


IONS  AND  SALT  ACTION.  75 

with  alkaline  reaction.  Complete  investigations  on  this  subject  have 
been  carried  out  by  Sorensen.1  He  found  the  value  10  ~14,14  for  K 
at  18°  C.  Ch  is  determined  in  either  of  two  ways.  The  best  method, 
the  electromotive,  is  based  upon  the  electromotive  force  of  gas  chains, 
as  developed  by  Nernst;2  namely,  if  platinum  foil  covered  with 
platinum  black  is  introduced  into  a  watery  solution  and  this  saturated 
with  hydrogen,  then  a  difference  of  electrical  potential  is  produced  between 
the  platinum  and  the  solution  and  this  potential  is  theoretically  propor- 
tional to  the  concentration  of  the  hydrogen  ions  in  the  solution.  We 
cannot  give  any  further  detail  as  to  this  theory  or  to  the  performance 
of  the  measurement  of  the  difference  in  potential.3  If  the  concentration 
of  the  hydrogen  ions  Ch  is  expressed  in  gram  ions  per  liter  by  the  figure 
10~p,  then  according  to  the  suggestion  of  Sorenson  the  name  hydrogen 
ion  exponent  and  the  symbol  Pr-  is  used  f°r  the  numerical  value  of  the 
exponents  of  this  potence.  The  relationship  between  pH  and  the  electro- 
motive force  -k  at  the  contact  between  the  platinum  and  the  solution 
can  be  expressed  graphically  by  a  straight  line;  hence  it  follows  that  if 
r  is  known  then  /  H  can  be  very  easily  found  (the  exponential  line). 

The  other  method  used  by  Sorensen  4  for  the  determination  of  CH 
is  a  colorimetric  method  and  depends  on  the  use  of  indicators.  After 
much  investigation  20  indicators  are  recommended,  of  which  certain  ones 
require  strictly  fixed  methods  of  use.  As  soon  as  more  than  a  qual- 
itative approximation  is  required  then  the  shade  of  color  produced  by 
the  indicator  must  be  compared  with  a  shade  of  color  produced  by  the 
same  indicator  in  a  solution  of  known  concentration  of  H  ions.  Such 
standard  solutions  which  allow  of  a  variation  in  the  concentration  of  the 
H  ions  at  one's  pleasure  have  been  given  by  Sorensen,  and  the  original 
article  gives  a  table  of  curves  from  the  corresponding  value  for  pH  which 
can  be  read  off,  when  the  composition  of  a  standard  solution  is  known.  The 
figure  j  H  for  the  standard  solutions  is  determined  by  aid  of  the  electro- 
motive method.  Standard  solutions  are  selected  so  that  they  serve  as 
natural  protectors  against  too  sudden  changes  in  pn  (so  called  buffer)5. 

As  above  stated  the  dissociation  constant  according  to  Sorensen 
for  water  is  10-1414  at  18°  C.  or  CH-  Coh  =  10~1414.  In  neutral  reac- 
tion CH  =  C0H  and  therefore  CH  =  10~7-07  or  pn  =  7.07.  Smaller  values 
for  ;>h  correspond  to  acid  and  greater  values  to  alkaline  reaction. 

Hasselbach  6  has  suggested  a  modification  of  Sorensen's  method 

1  Bioch.  Zeitschr.,  21,  131  (1909)  also  Ergebn.  d.  Physiol.  Vol.  11. 

2  Zeitschr.  f.  physik.  Chem.,  4,  129  (1889). 

3  In  regard  to  the  determination  see  the  work  of  Sorensen  cited  on  page  74. 

4  Sorensen,  Enzymstudien,  Bioch.  Zeitschr.,  21,  253. 

5  Ibid.,  167. 

6  Bioch.  Zeitschr.,  30,  317  (1910). 


76  GENERAL  AND  PHYSICO-CHEMICAL. 

for  the  electrometric  determination  of  reaction  in  fluids  containing 
carbon  dioxide.  By  the  aid  of  this  method  Hasselbach  and  Lunds- 
gaard  l  have  made  determinations  of  the  reaction  of  the  blood.  From 
these  it  follows  that  at  a  temperature  of  38.5°  C.  where  the  value  for 
pH  =  6.78  corresponds  to  the  neutral  reaction  the  figure  obtained  for  pn 
with  defibrinated  ox-blood  was  7.36  showing  therefore  a  slight  alkaline 
reaction.  The  influence  of  the  respiratory  variation  in  the  CO2  tension 
upon  the  H  ion  concentration  of  the  blood  is  of  a  measurable  size. 
The  total  blood  has  a  greater  H  ion  concentration  than  the  serum  at 
equal  CO2  tension  but  less  than  the  blood  corpuscles.  For  human  blood 
saturated  with  CO2  under  40  mm.  tension  at  38°  C.  Lundsgaaed  2 
found  pH  =  7.19.  Michaelis  and  Davidoff3  found  the  average  values 
of  normal  venous  blood  for  pH  =  7.35  at  37.5°  C. 

1  Biochem.  Zeitschr.,  38,  77  (1911). 

2  Ibid.,  41,  2641(1912). 

3  Ibid.,  46,  131  (1912).. 


CHAPTER  II. 
THE  PROTEIN  SUBSTANCES. 

The  chief  mass  of  the  organic  constituents  of  animal  tissues  consists 
of  amorphous  nitrogenized,  very  complex  bodies  of  high  molecular  weight. 
These  bodies,  which  are  either  proteins  in  a  special  sense  or  bodies  nearly 
related  thereto,  take  first  rank  among  the  organic  constituents  of  the 
animal  body  on  account  of  their  great  abundance.  For  this  reason  they 
are  classed  together  in  a  special  group  which  has  received  the  name 
protein  group  (from  irpayrtvw,  I  am  the  first,  or  take  the  first  place). 
The  bodies  belonging  to  these  several  groups  are  called  protein  sub- 
stances, although  in  a  few  cases  the  protein  bodies  in  a  special  sense  are 
designated  by  the  same  name. 

The  several  protein  substa?ices  x  contain  carbon,  hydrogen,  nitrogen,  and 
oxygen.  The  majority  contain  also  sulphur,  a  few  phosphorus,  and  a 
few  also  iron.  Copper,  chlorine,  iodine,  and  bromine  have  been  found 
in  some  few  cases.  On  heating  the  protein  substances  they  gradually 
decompose,  producing  a  strong  odor  of  burned  horn  or  wool.  At  the  same 
time  they  produce  inflammable  gases,  water,  carbon  dioxide,  ammonia, 
and  nitrogenized  bases,  besides  many  other  substances,  and  leave  a  large 
quantity  of  carbon.  On  deep  hydrolytic  cleavage  they  yield  abundance 
of  a-monamino-acids  of  various  kinds  as  decomposition  products. 

The  nitrogen  occurs  in  the  protein  bodies  in  various  forms,  and  this 
is  also  revealed  in  the  division  of  the  nitrogen  among  the  cleavage  prod- 
ucts. On  boiling  with  dilute  mineral  acids  we  obtain  (1)  so-called  amide 
nitrogen,  which  is  readily  split  off  as  ammonia;  (2)  a  guanidine  residue 
which  is  combined  with  diaminovaleric  acid  as  arginine,  and  which 
has  also  been  called  the  urea-forming  group;  (3)  basic  nitrogen  or  diamino- 
acid  nitrogen,  or  hexone  bases  nitrogen,  which  is  precipitated  by  phos- 
photungstic  acid  as  basic  products  (to  which  also  the  guanidine  residue 
of  arginine  belongs);    (4)  monamino-acid  nitrogen;    and  (5)  the  nitrogen 

1  See  "  Eiweisskorper,"  Ladenburg's  Handworterbuch  der  Chemie,  3,  534-589, 
which  gives  a  complete  summary  of  the  literature  of  protein  substances  up  to  1885. 
The  more  recent  literature  may  be  found  in  O.  Cohnheim,  Chemie  der  Eiweisskorper, 
Braunschweig,  1911.  See  also  Oppenheimer's  Handbuch  der  Biochem.  der  Menschen. 
und  der  Tiere,  1908. 

77 


78  THE  PROTEIN  SUBSTANCES. 

in  variable  amounts  which   appears  as  humus-like  melanoidins,   which 
seem  to  be  of  only  secondary  formation  as  products  of  elaboration. 

The  quantitative  division  of  the  total  nitrogen  between  the  above 
five  groups  is  different  in  the  various  protein  substances,  and  more- 
over cannot  be  given  with  certainty,  because  of  the  above-mentioned 
melanoidin  formation  and  the  errors  in  the  methods  used.1  The  follow- 
ing gives  at  least  an  approximate  idea  of  this  division.2  The  loosely 
combined  so-called  amide  nitrogen  seems  to  be  entirely  absent  in  the 
protamines.  In  the  gelatins  we  find  1-2  per  cent,  and  5-10  per  cent 
in  other  animal  protein  substances,3  in  certain  plant  proteins,  the 
prolamines  (see  page  106),  13-25  per  cent  of  the  total  nitrogen  is  amide 
nitrogen.  The  guanidine  nitrogen  may  amount  in  the  protamines  to 
22-44  per  cent  of  the  total  nitrogen,  in  the  histones  to  12-13  per  cent, 
in  the  gelatins  about  8  per  cent,  and  in  the  other  protein  bodies  about 
2-5  per  cent.  As  basic  nitrogen  precipitable  by  phosphotungstic  acid 
(including  the  guanidine  residue)  we  find  35-88  per  cent  in  the  protamines, 
35-42.5  per  cent  in  the  histones,  15-30  per  cent  in  the  other  animal  pro- 
tein substances.  In  the  prolamines  3-6  per  cent  of  the  total  nitrogen  is 
found  as  products  precipitable  by  phosphotungstic  acid  but  in  plant 
globulin  (globulin  of  the  wheat)  indeed  37  per  cent.  The  chief  quantity 
of  the  nitrogen,  55-76  per  cent,  occurs,  with  the  exception  of  the  pro- 
tamines, as  the  monamino-acid  groups.  The  results  for  the  melanoidin 
nitrogen  vary  so  considerably  that  they  will  not  be  mentioned. 

Recently  D.  v.  Slyke  4  has  perfected  a  method  which  is  based  upon  "the 
deamidation  of  the  amino-acids  by  HN02  (see  below)  and  which  allows  of  a  still 
more  detailed  differentiation  of  the  nitrogen  partition.  In  this  method  the 
nitrogen  of  the  ammonia,  the  melanines,  the  cystine,  arginine,  histidine,  proline  and 
oxyproline  besides  one-half  of  the  tryphtophane  nitrogen  as  well  as  the  nitrogen 
of  the  remaining  amino-acids  can  be  specially  determined. 

From  recent  as  well  as  older  observations  it  follows  as  chief  result 
that  the  nitrogen  in  the  proteins  occurs  in  such  combinations  so  that 
on  hydrolysis  with  acids,  its  chief  amount  splits  off  in  the  form  of  amino- 
acids. 

1  See  the  work  of  Hausmann,  Zeitsehr.  f.  physiol.  Chem.,  27  and  29;  Henderson, 
ibid.,  27;  Kossel  and  Kutscher,  ibid.,  30;  Kutscher,  ibid.,  31,  38;  Hart,  ibid.,  33 
Giimbel,  Hofmeister's  Beitriige,  5;   Rothera,  ibid. 

2  See  the  works  given  in  footnote  1  and  Blum,  Zeitsehr.  f.  physiol.  Chem.,  30 
Kossel,  Her.  d.  d.  chem.  Gesellsch.,  34,  3214;  Hofmeister,  Ergebnisse  der  Physiol. 
Jahrg.  I,  Abt.  1,  759,  which  also  contains  the  literature;  Osborne  and  Harris,  Journ 
Amer.  Chem.  Soc,  25;  and  Giimbel,  I.e. 

3  Skraup  and  v.  Hardt-Stremayr,  Monatsh.  f.  Chem.,  29,  found  lower  results  than 
other  investigators  arid  they  found  also  that  about  two-thirds  of  the  amide  nitrogen 
■was  readily  split  off  and  one-third  slowly. 

4  Ber.  d.  d.  chem.  Gesellsch,  43  and  44  and  Journ.  of  biol.  Chem.,  9,  10  and  12. 


NITROGEN   DISTRIBUTION.  79 

By  the  action  of  nitrous  acid  upon  proteins  at  least  a  partial  deamidation 
takes  place  and  so-called  desamino  proteins  arc  obtained.  The  nitrogen  expelled 
originated  from  the  XII,.  groups  according  to  the  formula  RNHi+HNOj=»ROH+ 

Xj+IIjO.  The  amount  of  such  nitrogen  is  generally  only  small,  1-2  per  cent, 
and  for  this  reason  it  has  been  accepted  thai  such  groups  only  occur  in  small 
amounts  in  the  proteins.  This  is  probably  true  for  a  large  number  of  proteins 
but  not  for  all  and  as  example  of  these  we  will  recall  that  Kossel  and  CAMERON  ' 
have  shown  that  those  protamines  which  contain  no  other  hexone  base  besides 
arginine  although  they  have  NHa  groups  at  the  ends  in  the  guanidine  residue 
HN.CNH.NHj  of  the  numerous  arginine  groups,  do  not  yield  any  nitrogen  on 
Using  v.  Sltke's  method  while  those  protamines  containing  lysine  do.  We  must 
be  very  careful  in  drawing  certain  conclusions  from  the  results  obtained  by  the 
action  of  nitrous  acid  upon  proteins. 

The  nitrous  acid  can  develop  nitrogen  from  the  XTH2  groups  of  the  acid  amides 
as  well  as  from  the  XH2  groups  of  the  amino-acids.  On  the  contrary  no  nitrogen 
is  evolved  in  v.  Slyke's  method  from  the  guanidin  groups  and  from  the  peptic! 
combinations  containing  imid  groups  (see  below).  This  is  also  the  reason,  as 
remarked  above,  why  those  protamines  containing  only  arginine  do  not  jdeld 
any  nitrogen  while  those  protamines  which  also  contain  lysine  where  there  exist 
free  XH2  groups  do  give  off  nitrogen.  On  hydrolyzing  these  deamidized  pro- 
tamines and  also  other  deamidized  proteins  we  therefore  do  not  obtain  any  lysin 
as  shown  by  Skraup  and  collaborators  and  by  Levites  for  certain  proteins. 
The  quantity  of  monamino-acid  nitrogen  is  therefore  in  such  cases  found  to  be 
increased. 

According  to  Osborne,  Leavenworth  and  Brautlecht,2  who  worked 
with  plant  proteins,  the  splitting  off  of  XH3  on  the  acid  hydrolysis  of  the  proteins 
was  very  similar  to  the  splitting  off  of  XTH3  from  the  acid  amide  asparagine,  so 
that  the  binding  of  XH2  groups  on  the  carboxyl  groups  seems  very  probable. 
The  quantity  of  NH3  split  off  in  the  hydrolysis  ran  parallel  With  the  amount 
of  asparagine  and  glutamic  acid  present  and  the  quantity  of  XH3,  split  off  by 
hydrolysis  with  alkali  corresponded  nearly  to  the  sum  of  the  ammonia  that  was 
split  off  by  acid  hydrolysis  and  one-half  of  the  arginine  nitrogen.  According  to 
these  investigators  the  XH2  groups  occur  chiefly  as  acid-amide  combinations. 

A  part  of  the  nitrogen  in  the  proteins  occurs  from  the  above,  undoubt- 
edly as  NH2  groups;  the  extent  of  this  part,  which  is  different  in  different 
proteins  cannot  be  positively  given.  The  chief  mass  of  the  nitrogen 
in  the  proteins,  although  other  forms  of  binding  occur,  exists  as  imide- 
like  combinations  of  amino-acids  united  together  and  this  will  be  com- 
pletely developed  in  the  following  pages. 

The  sulphur  occurs  in  the  different  proteins  in  very  different  amounts. 
Certain  of  them,  such  as  the  protamines  and  apparently  also  certain 

1  In  regard  to  the  action  of  nitrous  acid  upon  proteins,  their  deamidation  and  cleav- 
age products  see  C.  Paal,  Ber.  d.  d.  Chem.  Gesellsch.,  29;  H.  Schiff,  ibid.,  1354;  0. 
Loew,  Chemiker  Ztg.,  1896  and  O.  Nasse,  Pfliiger's  Arch.,  6;  Treves  and  Salomone, 
Bioch.  Zeitschr,  7;  Skraup,  Monatsh.  f.  Chem.,  27  and  28,  with  Hoernes,  ibid.,  27, 
with  Kaas,  Annal.  d.  Chem.  u.  Pharm.,  351;  Lampel,  Monatsh.  f.  Chem.,  28;  Traxl, 
ibid.,  2ft;  Levites,  Zeitschr.  f.  physiol.  Chem.,  43,  and  Bioch.  Zeitschr.,  20;  D.  v. 
Slyke,  foot-note  4,  page  78;  Kossel  and  Cameron,  Zeitschr.  f.  physiol.  Chem.,  76;  Kossel 
and  F.  Weiss,  ibid.,  78. 

2  Amer.  Journ.  of  Physiol.,  23. 


80  THE  PROTEIN  SUBSTANCES. 

bacterial  proteids,1  are  free  from  sulphur;  some,  such  as  gelatin  and 
elastin,  are  very  poor  in  sulphur,  while  others,  especially  horn  sub- 
stances, are  relatively  rich  in  sulphur.  On  hydrolytic  cleavage  with 
mineral  acids,  the  sulphur  of  the  protein  substances  is  regularly,  at 
least  in  part,  split  off  as  cystine  (K.  Morner)  or,  with  bodies  poorer  in 
sulphur,  as  cysteine  (Embden)  ,  but  this,  according  to  Morner  and  Patten, 
is  a  secondary  formation.  From  certain  protein  substances  a-thiolactic 
acid  (Suter,  Friedmann,  Frankel),  which  Morner  claims  is  also  pro- 
duced secondarily,  mercaptans  and  sulphureted  hydrogen  (Sieber  and 
Schoubenko,  Rubner),  and  a  body  having  the  odor  of  ethyl  sulphide 
(Drechsel)  have  been  obtained.2 

A  part  of  the  sulphur  separates  as  potassium  or  sodium  sulphide  on 
boiling  with  caustic  potash  or  soda,  and  may  be  detected  by  lead  acetate 
and  quantitatively  determined  (Fleitmann,  Danilewsky,  Kruger, 
Fr.  Schulz,  Osborne,  K.  Morner3).  What  remains  can  be  detected 
only  after  fusing  with  potassium  nitrate  and  sodium  carbonate  and 
testing  for  sulphates.  The  ratio  between  the  sulphur  split  off  by  alkali 
and  that  not  split  off  is  different  in  various  proteins.  No  conclusions 
can  be  drawn  from  this  •  in  regard  to  the  number  of  forms  of  combination 
which  the  sulphur  has  in  the  protein  molecule.  As  shown  by  K.  Morner, 
only  about  three-fourths  of  the  sulphur  in  cystine  can  be  split  off  by 
alkali,  and  the  same  is  true  for  the  cystine-yielding  complex  of  the  pro- 
tein substances.  If  the  quantity  of  lead-blackening  sulphur  in  a  pro- 
tein body  be  multiplied  by  f,  we  obtain  the  quantity  corresponding  to 
the  cystine  sulphur  in  the  body.  By  such  calculation  Morner  found 
in  certain  bodies,  such  as  horn  substance,  seralbumin  and  serglobulin, 
that  the  quantity  of  cystine  sulphur  and  total  sulphur  were  identical, 
and  therefore  we  have  no  reason  for  considering  the  sulphur  in  these 
bodies  as  existing  in  more  than  one  form  of  combination.  In  other 
proteins,  such  as  fibrinogen  and  ovalbumin,  on  the  contrary,  only  one- 
half  or  one-third  of  the  sulphur  appeared  as  cystine  sulphur. 

Just  as  in  the  products  of  acid  hydrolysis  of  proteins  we  know  of 
two  forms  of  oxygen  bondage,  the  hydroxyl  form  OH  and  the  carbonyl 


1  See  Nencki  and  Schaffer,  Journ.  f.  prakt.  Chem.  (N.  F.),  20,  and  M.  Nencki,  Ber. 
d.  d.  chem.  Gesellsch.,  17. 

2  K.  Morner,  Zeitschr.  f.  physiol.  Chem.,  28,  34,  and  42;  Patten,  ibid.,  39;  Embden, 
Urid.,  32;  Suter,  ibid.,  20;  Friedmann,  Hofmeister's  Beitriige,  3;  Sieber  and  Schou- 
benko, Archiv  d.  sciences  biol.  de  St.  Pdtersbourg,  1;  Rubner,  Arch.  f.  Hygiene,  19; 
Drechsel,  Centralbl.  f.  Physiol.,  10,  529;  Frankel,  Sitzungsber.  d.  Wien.  Akad.,  112, 
II  b,  1903. 

*  Fleitmann,  Annal.  dor  Chem.  und  Pharm.,  6fi;  Danilewsky,  Zeitschr.  f.  physiol. 
•Chem.,  7;  Kruger's,  Pflliger's  Archiv,  43;  F.  Schulz,  Zeitschr.  f.  physiol.  Chem.,  25; 
Osborne,  Connecticut  Agric.  Expt.  Station  Report  1900;  Morner,  1.  c. 


HYDROLYSES  OF  PROTEINS.  81 

form  in  CONH;  so  according  to  Treat  B.  Johnson  l  two  analogous  forms 
of  sulphur  bondage  exist  in  the  proteins,  namely  the  mercaptan  form 
SH  as  in  cystine  and  the  form  NH.CH.CS.NH  corresponding  to  the  oxygen 
binding  in  the  polypeptids  (see  page  86).  He  has  in  fact  also  prepared 
thio-polypeptides  from  glycocoll  and  these  were  analogous  to  the  corre- 
sponding glycin  polypeptids  (see  page  88)  and  like  certain  proteins 
gave  HoS  on  acid  hydrolysis. 

The  constitution  of  the  protein  bodies  is  still  unknown,  although 
the  great  advances  made  in  the  last  few  years  have  brought  us  very  much 
closer  to  the  elucidation  of  the  question.  In  studying  the  constitution 
of  the  protein  bodies  they  have  been  broken  up  in  various  ways  into 
simpler  portions,  and  the  methods  used  for  this  purpose  have  been  of 
different  kinds.  In  such  decompositions,  for  which  only  purified  proteins 
are  to  be  used,  first  large  atomic  complexes — proteoses  and  peptones — 
are  obtained  which  still  have  protein  characteristics,  and  these  then 
suffer  further  decomposition  until  finally  we  obtain  simpler,  generally 
crystalline,  or  at  least  well-characterized,  end  products. 

As  to  the  products  obtained  by  hydrolytic  cleavage  with  mineral  acids, 
important  investigations  have  been  carried  out  by  numerous  older  and 
more  recent  experimenters.2  Besides  certain  acids,  which  will  be  men- 
tioned later  and  which  occur  in  few  cases  only,  we  obtain  the  following: 
monamino-acids  such  as  glycocoll,  alanine,  aminovaleric  acid,  leu- 
cine, isoleucine,  serine,  aspartic  and  glutamic  acids,  cysteine  and  its  disul- 
phide  cystine,  phenylalanine,  tyrosine,  pyrollidine — and  oxypyrollidine 
carboxylic  acid,  tryptophane  and  also  the  three  hexone  bases,  histidine, 
arginine  and  lysine,  the  two  latter  being  diamino-acids.  Besides  these 
also  ammonia,  sulphureted  hydrogen,  ethyl  sulphide  and  melanoidins, 
which  latter  seem  to  be  secondary  products,  have  been  obtained. 

On  the  hydrolysis  with  alkalies  we  obtain,  after  a  preliminary  forma- 
tion of  intermediary  steps  which  will  be  discussed  later,  chiefly  the  same 
cleavage  products  as  in  acid  hydrolysis  but  with  the  exception  that  in 
the  alkali  hydrolysis  a  considerable  part  of  the  amino-acids  become 
racemerized  and  therefore  appear  in  optically  inactive  form  while  in  the 
acid  hydrolysis  chiefly  optically  active  acids  are  obtained.  Because 
of  the  action  of  the  alkali  a  part  may  suffer  further  decomposition  which 
loads  to  the  formation  of  simpler  cleavage  products  and  ammonia. 

On  fusing  proteins  with  caustic  alkali,  ammonia,  methyl  mercaptan  and  other 
volatile  products  are  evolved  and  other  products  are  produced  such  as  leucine, 

1  Journ.  of  biol.  Chem.,  9. 

2  In  regard  to  the  literature  see  O.  Cohnheim,  Chemie  der  Eiweisskorper,  Braun- 
schweig, 1911,  and  F.  Hofmeister,  Ergebnisse  der  Physiologie,  Jahrg.  I,  Abt.  1,  759, 
1902;  E.  Fischer,  Untersuchungen  uber  Aminosauren,  Polypeptide  und  Proteine  (1899- 
1906),  Berlin,  1906.     See  also  special  references. 


82  THE  PKOTEIN  SUBSTANCES. 

from  which  then  volatile  fatty  acids  such  as  acetic  acid,  valeric  acid  and  also 
butyric  acid  are  formed,  also  tyrosine  from  which  latter  phenol  is  formed  and 
indol  and  skatol. 

Most  proteins  are  split  by  proteolytic  enzymes  in  the  same  manner 
as  on  hydrolysis  with  acids  or  alkalies,  but  more  or  less  completely  depend- 
ent upon  the  kind  of  enzymes.  In  the  first  place  proteoses  and  peptones 
(see  below)  are  formed,  then  also  polypeptids  and  amino-acids  of 
various  kinds,  in  certain  cases  also  oxyphenylethylamine,  diamines,  and 
a  little  ammonia  and  other  bodies. 

A  great  many  substances  are  produced  in  the  putrefaction  of  pro- 
teins. First  the  same  bodies  as  are  formed  in  the  decomposition  by 
means  of  proteolytic  enzymes  are  produced,  and  then  a  further  decom- 
position occurs  with  the  formation  besides  ammonia,  carbon  dioxide 
and  hydrogen,  of  a  large  number  of  bodies  belonging  in  part  to  the  aliphatic 
and  in  part  to  the  aromatic  and  heterocyclic  series. 

'To  the  aliphatic  series  belong  volatile  fatty  acids  and  as  shown 
by  Neuberg  l  and  collaborators  not  only  fatty  acids  of  the  normal  chain 
but  also  with  branched  chains,  also  optically  active  acids,  also  succinic 
acid,  methane,  methyl  mercaptan  and  others.  To  this  series  belongs 
also  the  two  putrefaction  bases  cadaverine  and  putrescine,  produced  from 
the  diamino  acids,  and  also  the  so-called  ptomaines  or  cadaver  alkaloids 
which  may  originate,  at  least  in  part,  from  other  tissue  constituents 
and  not  from  proteins. 

The  putrefactive  products  of  the  aromatic  and  heterocyclic  series 
originate  from  the  corresponding  amino-acids.  From  tyrosine  the 
aromatic  oxy-acids  such  as  p-oxyphenyl-propionic  acid,  the  p-cresol, 
phenol  and  oxyphenylethylamine  are  formed.  The  phenylalanine  is  the 
mother  substance  of  the  phenylpropionic  acid,  the  phenylacetic  acid  and 
the  phcnylethylamine.  Indolpropionic  acid,  indolacetic  acid,  skatol 
and  indol  originate  from  the  tryptophane  (indolaminopropionic  acid) ;  the 
imidazolpropionic  acid  and  imidazolethylamine  originate  from  the  histi- 
dine.2 

By  the  moderate  action  of  chlorine,  bromine,  or  iodine  upon  proteins, 
these  halogens  enter  into  more  or  less  firm  combination  with  the  proteins 
and  according  to  the  method  of  procedure  we  can  prepare  derivatives 
having  different  but  constant  amounts  of  halogens.  The  proteins  are 
so  changed  that  they  do  not  split  off  sulphur  on  treatment  with  alkali, 
nor  do  they  respond  to  Millon's  or  Adamkiewicz-Hopkins  reaction. 
Side  processes,  oxidations  and  cleavages  may  also  take  place  here.  The 
most  striking  fart  seems  to  be  a  substitution  of  hydrogen  by  iodine  in  the 

1  Bioch.  Zeitsohr.,  37,  where  the  earlier  works  of  Neuberg  are  cited. 
:  A<  kermann,  Zeitschr.  f.  physiol.  Chem.,  (55. 


OXIDATION    OF  PROTEINS.  83 

aromatic  nucleus  of  tyrosine  and  also  perhaps  in  the  indol  nucleus  of 
tryptophane  and  the  imidazol  nucleus  of  histidine.1  Halogen  proteins 
occur,  as  will  be  shown  later,  in  the  animal  kingdom,  especially  in  the 
albuminoid  group  and  indeed  iodized  tyrosine  (3-5  di-iodotyro-incj 
has  been  isolated. 

By  the  oxidation  of  protein  by  means  of  potassium  permanganate,  Maly 
obtained  an  acid,  ojcyprotosulphonic  acid,  C  51.21,  H  6.89,  X  14.59  8  1.77,  O  23.21 
per  cent,  which  is  not  a  cleavage  product,  but  an  oxidation  product  in  which 
the  group  SH  is  changed  into  SO;. OH.  This  acid  does  not  give  the  proper  color 
reaction  with  Millon's  reagent,  yields  no  tyrosine  or  indol,  but  gives  benzene 
on  fusing  with  alkali.  On  continued  oxidation  Maly  obtained  another  acid, 
peroxyproteic  acid,  which  gives  the  biuret  reaction,  but  is  not  precipitated  by 
most  protein  precipitants.  The  oxyprotein  obtained  by  Schulz  on  the  oxida- 
tion of  protein  by  hydrogen  peroxide  is  closely  related  to  oxyprotosulphonic 
acid  in  composition  and  general  characteristics,  but  contains  lead-blackening 
sulphur  and  gives  Millon's  reaction.  The  oxyprotein  is  claimed  to  be  a  pure 
oxidation  product,  while  in  the  production  of  oxyprotosulphonic  acid  Schulz 
claims  that  a  cleavage  takes  place.  According  to  Buraczewski  and  Krauze 
the  oxyprotosulphonic  acid  is  a  mixture  of  several  substances.  According  to 
the  investigations  of  v.  Furth  -  there  exist  at  least  three  different  peroxyproteic 
acids  (from  casein)  which  differ  from  each  other  by  a  different  division  of  the 
nitrogen  in  the  molecule.  On  treatment  with  baryta-water  we  find  that  they 
split  off  basic  complexes  and  oxalic-acid  groups,  and  new  bodies,  the  dcsamino- 
proteic  acids,  which  give  the  biuret  reaction,  are  produced.  These  later  acids,  which 
on  hydrolysis  give  benzoic  acid  but  no  diamino-acids,  may  be  further  oxidized, 
which  is  not  true  of  the  peroxyproteic  acids,  and  yield  a  new  group  of  acids,  the 
kyroproteic  acids,  which  give  the  biuret  reaction,  hold  about  one-half  of  their 
nitrogen  (11.08  per  cent  total  nitrogen)  in  acid-aniide-like  combination,  but 
yield  neither  basic  products  nor  benzoic  acid. 

On  the  oxidation  of  gelatin  or  protein  with  permanganate  we  also  obtain 
oxaminic  acid,  oxamide,  oxalic  acid,  oxaluric-acid  amide,  succinic  acid,  several 
volatile  fatty  acids,  and  guanidine,  which  was  first  shown  by  Lossen  as  an  oxida- 
tion product.3 

On  the  oxidation  of  gelatin  by  ferrous  sulphate  and  hydrogen  peroxide 
Blumenthal  and  Xeuberg  have  obtained  acetone  as  a  product,  and  Orgler 
the  same  from  ovalbumin.     The  action  of  ozone  upon  casein  has  been  studied 

1  In  regard  to  the  action  of  halogens  upon  proteins  see  Loew,  Journ.  f.  prakt. 
Chem.  (N.  F.),  31;  Blum,  Munch,  med.  Wochenschr.,  1896;  Blum  and  Vaubel,  Journ. 
f.  prakt.  Chem.  (X.  F.),  57;  Liebrecht,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30;  Hop- 
kins and  Brook,  Journ.  of  Physiol.,  22;  Hopkins  and  Pinkus,  Ber.  d.  deutsch.  chem. 
Gesellsch.,  31;  Hofmeister,  Zeitschr.  f.  physiol.  Chem.,  24;  Kurajeff,  ibid.,  26;  Oswald, 
Hofmeister's  Beitriige,  3;  C.  H.  L.  Schmidt,  Zeitschr.  f.  physiol.  Chem.,  35,  36,  3"; 
Xeuberg,  Biochem.  Zeitschr.,  6:  Pauly  and  Gundermann,  Ber.  d.  d.  chem.  Gesellsch., 
41,  43;    Krzemecki,  Chem.  Centralbl.,  1912;    Pauly,  Zeitschr.  f.  physiol.  Chem.,  "6. 

'  Maly,  Sitzungsber,  d.  k.  Akad.  d.  Wissenseh.,  Wien,  91  and  97.  Also  Monatshefte 
f.  Chem.,  6  and  9.  See  also  Bondzynski  and  Zoja,  Zeitschr.  f.  physiol.  Chem.,  19; 
Bernert,  ibid.,  26;  Schulz,  ibid,,  29;  Buraczewski  and  Krauze,  ibid,  76;  v.  Furth,  Hof- 
meister's  Beitriige,  6. 

3  Lessen,  Annal.  d.  Chem.  u.  Pharm.,  201;  Kutscher,  Zeitschr.  f.  physiol.  Chem., 
32;  Zicksraf,  ibid.,  41;  Seemann,  ibid.,  44;  Kutscher  and  Schenck,  Ber.  d.  d.  chem. 
Gesellsch..  37  and  38. 


84  THE  PROTEIN  SUBSTANCES. 

by  Harries  and  Langheld  j  and  the  action  of  chlorine  by  Habermann  and 
Ehrenfeld  and  Panzer.2 

Nitric  acid  gives  various  yellow  products,  which  turn  reddish-brown  in 
alkaline  solution.  Of  these  we  must  especially  mention  the  so-called  xantho- 
protein,  besides  nitrated  proteoses  and  peptones.  The  xanthoprotein  does  not 
yield  any  tyrosine  on  acid  hydrolysis  and  it  does  not  give  the  Millon  or  the  lead- 
blackening  reactions.  Among  the  cleavage  products  v.  Furth  2  has  obtained  a 
melanoidin  substance,  xanthomelanoidin. 

On  the  nitration  of  the  protamines  (see  below)  Kossel  4  and  co-workers  have 
obtained  nitroprotamines  which  give  nitroarginine  on  hydrolysis  which  shows  that 
the  nitro  groups  have  entered  the  guanidine  groups  of  the  arginine. 

By  the  dry  distillation  of  proteins  we  obtain  a  large  number  of  decomposition 
products  having  a  disagreeable  burned  odor,  and  a  porous  glistening  mass  of  carbon 
containing  nitrogen  is  left  as  a  residue.  The  products  of  distillation  are  partly 
an  alkaline  liquid  which  contains  ammonium  carbonate  and  acetate,  ammonium 
sulphide,  ammonium  cyanide,  an  inflammable  oil,  and  other  bodies,  and  a  brown 
oil  which  contains  hydrocarbons,  nitrogenized  bases  belonging  to  the  aniline  and 
pyridine  series,  and  a  number  of  unknown  substances. 

The  occurrence  of  protein  substances  which  contain  a  carbohydrate 
group  has  been  known  for  a  long  time.  The  nature  of  this  carbohydrate, 
which  can  be  split  off  by  acid  and  which  may  amount  to  as  much  as  35 
per  cent,  has  been  explained  chiefly  by  the  investigations  of  Friedrich 
MfJLLER 5  and  his  students.  They  have  shown  that  it  is  always  an 
amino-sugar,  and  generally  glucosamine  and  perhaps  galactosamine 
as  |an  exception.  That  so-called  true  proteins  also  yield  a  carbohydrate 
on  hydrolytic  cleavage  was  first  shown  by  Pavy,  using  ovalbumin.  The 
continued  investigations  of  Fr.  Muller,  and  others  have  demonstrated 
that  in  these  cases  the  carbohydrate  is  also  glucosamine.  A  carbohy- 
drate complex,  although  sometimes  only  to  a  very  slight  amount,  has 
been  detected  in  other  proteins,  ovoglobulin,  serglobulin,  seralbumin, 
peaglobulin,  albumin  of  the  gramineae,  yolk-proteid,  and  fibrin.  In 
other  proteins,  on  the  contrary,  such,  as  edestin  (of  the  hemp-seed)  and 
casein,  myosin,  pure  fibrinogen,  and  ovovitellin,  carbohydrates  have 
been  sought  for  with  negative  results.  All  proteins  hence  do  not  contain 
a  carbohydrate  group,  and  future  investigators  must  therefore  decide 
whether  the  carbohydrate  groups  belong  positively  to  the  protein  com- 


1  Blumenthal  and  Neuberg,  Deutsch.  med.  Wochenschr.,  1901;  Orgler,  Hofmeister's 
Beitrage,  1;   Harries  and  Langheld,  Zeitschr.  f.  physiol.  Chem.,  51. 

2  Habermann  and  Ehrenfeld,  Zeitschr.  f.  physiol.  Chem.,  32;  Panzer,  ibid.,  33 
and  34. 

3  See  Maly's  Jahresber,  30,  p.  24. 

4  Kossel  and  Kennaway,  Zeitschr.  f.  physiol.  Chem.,  72,  with  E.  Wechsler,  ibid., 
78  and  with  F.  Weiss,  ibid.,  78. 

6  In  regard  to  the  literature  on  this  subject  see  the  work  of  Fr.  Muller,  Zeitschr. 
f.  Biologie,  42,  and  Langstein,  Ergebnisse  der  Physiologie,  Jahrg.  I,  Abt.  1,  63,  Zeitschr. 
f.  physiol.  Chem.,  41,  and  Hofmeister's  Beitrage,  6.  See  also  Abderhalden,  Bergell, 
and  DOrpinghaus,  Zeitschr.  f.  physiol.  Chem.,  41. 


CARBON  NUCLEI.  85 

plex  or  whether  they  are  united  with  the  protein  only  as  impurities.  Sev- 
eral observations1  show  that  in  working  with  crystalline  proteins  a  con- 
tamination with  other  protein  substances  is  unfortunately  not  excluded, 
and  this  must  not  be  lost  sight  of,  especially  as  the  quantity  of  carbohy- 
drates obtained  is  often  very  small.  In  this  connection  we  must  call 
attention  to  the  findings  of  Osborne  and  collal  (orators  that  on  recrystalliz- 
ing  ovalbumin  six  times  they  found  that  the  glucosamine  content  was 
reduced  to  1.23  per  cent  while  other  investigators  give  7-8-15  per  cent. 
Under  these  circumstances  we  are  not  warranted  in  considering  the 
carbohydrate  groups  as  belonging  to  the  carbon  nucleus  produced  on 
the  destruction  of  the  real  protein  complex. 

The  previously  mentioned  methods  used  in  studying  the  structure 
of  the  protein  substances  are  not  of  the  same  value,  but  they  in  part 
substantiate  each  other.  Of  these  we  must  mention  the  hydrolysis 
by  means  of  boiling  dilute  mineral  acids,  or  by  proteolytic  enzymes, 
as  the  best  methods  for  obtaining  the  carbon  nuclei  in  the  protein  mole- 
cule.    The  most  important  of  the  carbon  nuclei  obtained  are  as  follows: 

I.     The  Nuclei  belonging  to  the  Aliphatic  Series. 

A.  Sulphur  free,  but  containing  nitrogen:  1.  A  guanidine  residue  (combined 
with  ornithine  as  arginine).  2.  Monobasic  monami  no-acids:  Glycocoll,  alanine, 
valine  (amino  valeric  acid),  leucine,  and  isoleucine.  3.  Bibasic  monamino- 
acids:  Aspartic  acid  and  glutamic  acid.  4.  Oxymonamino-acids:  serine  oxy- 
aminosuccinic  acid  and  oxyaminosuberic  acid.  5.  Monobasic  diamino-acids: 
Diaminoacetic  acid,  ornithine  (from  arginine)  and  lysine.  6.  Oxydiamino-acids: 
Oxydiaminosuberic  acid,  oxydiaminosebacic  acid,  diaminotrioxydodecanoic  acid, 
caseanic  and  caseinic  acids. 

B.  Sulphurized:  Cysteine  and  its  sulphide  cystine,  thiolactic  acid  (mercaptans, 
and  ethyl  sulphide). 

II.     The  Nuclei  belonging  to  the  Carbocyclic  Series. 
Phenylalanine  and  tyrosine. 

m.     The  Nuclei  belonging  to  the  Heterocyclic  Series. 
Proline,  oxyproline,  tryptophane  and  histidine. 

In  regard  to  these  carbon  nuclei  it  must  be  remarked  that  they  are 
not  all  found  in  every  protein  body  thus  far  investigated,  and  also  that- 
one  and  the  same  cleavage  product,  such,  for  example,  as  glycocoll, 
leucine,  tryosine,  etc.,  is  obtained  in  very  variable  amounts  from  differ- 
ent protein  substances. 

It  is  very  difficult  to  say  to  what  extent  all  the  above-mentioned 
carbon  nuclei   exist   in  the  protein  molecule.     It   is   not   inconceivable 

1  Osborne,  D.  B.  Jones  and  Leavenworth,  Amer.  Journ.  of  Physiol.,  24. 


86  THE   PROTEIN   SUBSTANCES, 

that  in  the  hydrolysis  certain  carbon  nuclei  may  be  secondarily  formed 
from  others.  Even  if  we  admit  the  above,  still  it  is  undoubtedly  true 
that  the  chief  cleavage  products  of  the  protein  substances  are  amino- 
acids.  Emil  Fischer  has  shown  that  the  amino-acids  have  the  property 
of  readily  grouping  together  when  water  is  split  off  and  the  amide  group 
of  one  amino-acid  unites  with  the  carboxyl  group  of  the  other.  In 
accord  with  this  behavior  we  can,  as  Hofmeister  1  and  others  have 
explained,  but  which  was  first  proved  by  the  epoch-making  investiga- 
tions of  Emil  Fischer,  consider  the  proteins  as  chiefly  formed  by  the 
condensation  of  aminoTacids,  where  the  amino-acids  are  united  to  each 
other  by  means  of  imino-groups  according  to  the  following  scheme: 

— NH.CH.CO— NH.CH.CO NH.CH.CO— NH.CH.CO— 

C4H9  CH2.C6H4(OH)  CH2.COOH    C3He.CH2.NH2 

(Leucine)  (Tyrosine)  (Aspartic  acid)  (Lysine) 

Such  chaining  of  animo-acids  is  for  the  synthesis  of  protein-like  bodies 
of  the  very  greatest  importance.  The  older  statements  of  Grimaux, 
Schutzenberger  and  Pickering  on  the  artificial  preparation  of  pro- 
tein-like substances  where  these  investigators  were  able  to  prepare  sub- 
stances, which  in  many  properties  are  similar  to  the  proteins,  from  various 
amino-acids  either  alone  or  mixed  with  other  bodies  such  as  biuret,  alloxan, 
xanthine,  or  ammonia.  Of  special  interest  are  the  investigations  of 
Curtius  and  his  collaborators,  in  which  they  were  able  to  prepare  syn- 
thetically the  so-called  biuret  base  (triglycyl-glycine  ethyl  ester)  and  sub- 
sequently many  other  bodies  which  were  related  to  the  proteins.  The 
most  important  work  on  the  chaining  of  amino-acids  has  been  per- 
formed by  E.  Fischer  2  and  his  pupils  but  especially  by  Abderhalden. 
They  have  prepared  a  large  number  of  complex  bodies  called  polypeptides 
by  Fischer,  which  according  to  whether  they  contain  two  or  more 
amino-acid  groups  united  together,  are  called  di-,  tri-,  tetrapeptides, 
etc.  As  examples  of  polypeptides  we  will  mention — dipeptides:  glycyl- 
tvrosine,   alanylglycine,   leucylglycine,  leucylcystine,  prolylphenylalanine, 

1  "  Ueber  den  Bau  des  Eiweissmolekiils."  Gesellsch.  deutsch.  Naturforscher  und 
Aertze,  Verhandl.  1902,  and  Ergebnisse  der  Physiologie,  Jahrg.  I,  Abt.  1,  759. 

See  Pickering,  King's  College,  London,  Physiol.  Lab.  Collect.  Papers,  1897,  which 
also  cites  Grimaux'e  work;  also  Journ.  of  Physiol.,  18,  and  Proceed.  Roy.  Soc,  60, 
1897;  Schutzenberger,  Compt.  rend.,  100  and  112;  Curtius,  Journ.  f.  prakt.  Chem. 
(N.  li.  26  and  70,  and  Per.  d.  d.  chem.  Gesellsch.,  37;  Fischer  and  collaboral  >rs, 
Untersuchungen  iiber  Aminosauren,  Polypeptide  und  Proteine  (1S99-1906)  Berlin  190G 
and  1'ir.  d.  d.  chern.  Gesellsch.,  39,  40,  41,  42,  und  Annal.  d.  Chem.  u.  Pharm.,  354,. 
:{.")7.  :{<;:{.  :{C>r>.  :{*;*.>,  37.");  see  also  Abderhalden,  Ber.  d.  d.  chem.,  Gesellsch,  40  to  43  and 
Zeitschr.  f.  physiol.  Chem.,  72,  75,  77. 


POLYPEPTIDES.  87 

leueylhistidine:  tripeptides:  di-glycylglycine,  alanylglycyltyrosine,  leu- 
cyltryptophyl.ulutaniic  acid;  tetra peptides:  glycyfglutamyldiglycine,  dileu- 
cylglycylglycine;  pentapeptidee:  tetraglycylglycine  and  leucyltriglycylgly- 
cine;  hexar  and  heptapeptides:  leucyltetraglycylglycine  and  leucylpentagly- 
cylglycine.  The  most  complex  polypeptide  thus  far  prepared  is  an 
octadecapeptide  with  15  glycocoll  and  3  leucine  residues  namely:  Meucyl- 
triglycyl-/-leucyltriglycyl-/-leucyl-octaglycylglycine  = 

XHoCH(C4H9)( X ).[XHCHL»C,( )]3.XHCH(C4H9)CO. 
[NHCH2CO]3.NHCH(C4H9)CO.[NHCH2CO]8.NHCH2COOH. 

with  the  supposition  that  the  amino-acids  are  here  also  combined  together 
in  the  imide  binding. 

The  large  number  of  amino-acids  isolated  from  the  proteins  make 
a  large  number  of  bindings  possible.  The  number  of  possible  combina- 
tions is  still  further  increased  by  the  fact  that  all  the  amino-acids  with 
the  exception  of  glycocoll  contain  at  least  one  asymmetric  carbon  atom, 
and  this  leads  to  the  possible  formation  of  stereochemically  different 
peptides.  Thus  in  order  to  give  a  simple  example,  from  two  optically 
active  amino-acids,  four  different  isomeric  forms  of  clipeptides  may  occur, 
namely  (if  we  designate  the  optical  antipodes  by  d-  and  /-)  dd,  11,  dl  and  Id. 
Of  these  forms  two  can  form  a  racemic  dipeptide,  thus  c/-alanyl-c?-leucine  + 
/-alanyl-Meucine  and  rf-alanyl-Meucine  +  /-alanyl-f/-leucine.  As  the  pro- 
teins are  optically  active  and  on  hydrolysis  yield  chiefly  optically  active 
amino-acids,  those  polypeptides  which  can  be  built  up  from  the  natural 
amino-acids  of  the  proteins  are  of  special  importance  in  the  study  of  the 
constitution  of  the  proteins. 

Most  of  the  artificial  polypeptides  are  constructed  from  monamino- 
mono-carboxylic  acids,  but  polypeptides  have  also  been  prepared  which 
contain  diamino-acids  or  aniino-dicarboxylic  acids,  and  in  this  way  the 
number  of  possible  polypeptides  becomes  still  greater.  With  an  aminodi- 
carboxylic  acid  such  as  aspartic  acid,  other  amino-acids  can  be  bound 
with  one  carboxyl  group  or  with  both,  but  also,  if  we  start  with  aspara- 
gine,  they  can  be  anchored  with  the  amide  group.  If  we  start  from  the 
acid  amides  we  can  also  obtain  a  peptide  which  still  contains  the  CONH2 
group  and  on  total  hydrolysis  yields  NH3,  like  most  proteins.  A  poly- 
peptide of  this  kind  is  the  tripeptide,  glycl-/-asparaginyl-Meucine  prepared 
by  E.  Fischer  and  Koenigs. 

NH2CH2CO.NHCHCO.NHCH(C4H9)COOH 

CH2CONH2 

In  consideration  of  the  form  of  binding  of  the  sulphur  in  the  proteins 
it  is  interesting  to  consider  the  preparation  of  thiopolypeptids  as  performed 


88  THE   PROTEIN   SUBSTANCES. 

by  Treat  B.  Johnson.1  He  has  prepared  the  following:  thioglycylglycin- 
thioamide  NH2CH2CS.NHCH2CSNH2  which  is  analogous  to  glycylgly- 
cinamide,    NH2CH2CO.NHCH2CONH2    and    also    dithiopiperazine 

.CH2.C0V 

hn/  ">nh. 

XCS.CH2 

Polypeptides  of  higher  amino-fatty  acids  such  as  a-aminolauryla- 
lanine,  a-aminolaurylleucine  and  others  have  been  prepared  by  Hop- 
wood  and  Weizmann.2  These  peptids  are  different  from  the  so-called 
lipopeptids  prepared  by  Bondi  and  his  collaborators3  which  are  not 
chains  of  only  amino-acids  but  combinations  between  a  high  fatty  acid, 
such  as  lauric-  or  palmitic  acid  and  an  amino-acid  (glycocoll  or  alanine) 
or  a  dipeptide  (lauryl-alanylglycine) . 

Methylated  polypeptides  such  as  methyl-  and  dimethylleucylglycine  (E.. 
Fischer  and  Gluud)  and  betaindiglycylglycine 

(CH3)2N.  CH2CO.NHCH2CO.NHCH2CO 

(Abderhalden  and  Kautzsch)  are  also  known.4  Amides  of  amino-acids  and 
dipeptides  have  been  prepared  by  Bergell  5  and  his  co-workers. 

The  methods  used  by  E.  Fischer  in  the  synthetical  preparation  of 
polypeptides  are  chiefly  as  follows: 

The  first  dipeptide  prepared  by  him,  glycylglycine,  he  obtained  from  glycocoll 
ethyl  ester  which  in  water  is  transformed  into  a  diketopiperazine,  glycine  anhy- 
dride, according  to  the  following  equation: 

/CH2.COx 
2(XH2CH2CO.O.C2H6)  =  2C2H6OH+ NH<  >NH. 

NCO.CH/ 

By  the  action  of  dilute  alkali  upon  this  anhydride  with  the  taking  up  of  water  the 
glycylglycine  NH2CH2CO.NHCH2COOH  is  formed,  and  according  to  this  prin- 
ciple other  dipeptides  can  also  be  prepared. 

Another  method  which  has  much  greater  application  consists  in  the  anchoring 
of  an  amino-acid  to  a  halogen  of  an  acid  radical,  for  example,  by  the  action  of 
brompropionyl  bromide  or  chloride  upon  glycocoll  according  to  the  following 
equation : 

CH3CHBrCOCl+NH2CH2COOH=HCl+CH3CHBrCO.NHCH2COOH 

1  Journ.  of  biol.  Chem.,  9. 

2  See  Chem.  Centralbl.,  1911. 

3  Biooh.  Zeitschr.,  17  and  23;  see  also  Abderhalden  and  C.  Funk,  Zeitschr.  f.  physiol. 
Chem.,  66. 

4  Fischer  and  Gluud,  Annal.  d.  Chem.  u.  Pharm.,  369;  Abderhalden  and  Kautzsch, 
Zeitschr.  f.  physiol.  Chem.,  72  and  75. 

6  Ibid.,  64,  65  and  67. 


POLYPEPTIDES.  89 

(brompropionyl  glycine).  On  subsequent  treatment  with  ammonia  the  halogen 
(Br)  is  replaced  by  XH2  and  the  dipeptide  alanylglycine 

CH3CHXH2CO.XHCH2COOH+XHJ3r 

is  obtained.  By  the  second  action  of  brompropionylchloride  and  then  treatment 
with  NH3  we  introduce  a  new  alanyl  group  and  the  tripeptide  alanyl-alanyl 
glycine  is  prepared.  By  the  action  of  a  halogen  derivative  of  an  acid  radical 
another  amino-acid  residue  can  be  introduced,  and  the  chain  of  amino  groups 
can  be  thus  extended. 

The  prolongation  of  the  chain  on  the  other  side,  namely,  at  the  carboxyl, 
Fischer  has  accomplished  by  chlorination  of  the  amino-acids  by  special  treatment 
with  phosphorus  pentachloride.  The  carboxyl  is  thus  transformed  into  COO, 
while  the  acid  at  the  same  time  fixes  a  molecule  of  HC1,  for  example  CH3CHXH2HCI 

COC1 
Just  as  in  the  case  of  the  carboxyl  group  of  an  amino-acid,  so  also  can  a  poly- 
peptide or  its  halogen  acyl  combination  be  chlorinated  and  then  combined  with 
a  new  amino-acid,  or  a  new  peptide.  As  an  example,  Fischer,  from  a-brom- 
isocapronyldiglycyl  glycine,  first  prepared  a-bromisocapronyldiglycylgh'cyl  chlo- 
ride, and  then  with  diglycylglycine  he  obtained  the  heptapeptide  leucyl- 
pentaglycylglycine, 

C4H9CH(XH2)CO.(NHCH,CO)5.XHCH2COOH. 

For  the  various  combinations  of  the  optically  active  amino-acids  to  poly- 
peptides it  was  important  to  possess  methods  of  preparation  of  these  amino-acids, 
and  for  this  purpose  Fischer  in  many  cases  used  the  so-called  Walden's  reversion. 
This  consists  in  that  one  optically  active  amino-acid,  for  example  the  /-form,  is 
transformed  into  the  corresponding  halogen  fatty  acid  by  the  action  of  nitrosyl 
bromide,  yielding  the  optical  antipode  the  d-form.  By  the  action  of  ammonia 
the  c/-amino-acid  is  now  obtained  which  in  the  above-mentioned  manner  can  be 
retransformed  into  the  /-form.  Thus  from  (/-leucine  we  first  obtain  /-bromiso- 
caproic  acid  and  then  by  the  action  of  ammonia  /-leucine  and  in  the  preparation 
of  the  polypeptides  the  same  occurs.  Thus,  for  example,  if  by  reversion  (/-leucine 
is  changed  first  into  /-bromisoeapronyl  chloride,  if  this  last  is  combined  with 
/-leucine,  then  we  obtain  the  dipeptide  /-lcucyl-/-leucine.  On  combination  with 
diglycylglycine  the  tetrapeptide  /-leucyl-diglycyl  glycine  is  produced.  Walden's 
reversion  does  not  take  place  with  all  amino-acids;  other  methods  can  also  be 
used  to  obtain  the  optical  antipodes,  such  as  the  preparation  of  the  alkaloidal 
salts  of  the  benzoyl  or  formyl  combinations  of  the  racemic  amino-acids. 

The  /3-naphthalinsulpho  combination  of  the  polypeptides  and  peptones  may 
serve,  as  Fischer,  Abderhalden  and  Funk  l  have  shown,  in  explaining  the 
structure  of  these  bodies.  By  the  action  of  ^-naphthaline  sulphochloride  the 
NHg  groups  existing  at  the  beginning  of  the  chain  in  the  amino-acids  react  there- 
with and  on  subsequent  total  hydrolysis  this  naphthaline-sulpho  combination 
remains  unsplit.  Thus  for  instance  we  can  differentiate  between  glycylalanine 
and  alanylglycine  because  after  hydrolysis  in  the  first  case  we  obtain  naphthalin- 
sulphoglycine  and  alanine  and  in  the  second  naphthalin-sulphoalanine  and 
glycocoll  (glycine).  Tyrosine  may,  depending  upon  whether  the  XH2  as  well 
as  the  OH  groups  are  free  or  not  or  if  only  one  is  available,  yield  di-  or  mononaph- 
thalinsulpho-derivatives  and  in  this  way  we  can  also  draw  conclusions  as  to  the 
structure  of  tyrosine  containing  peptides. 

The  previously  mentioned  deamidation  method  of  van  Slyke  (page  781  where 
oxyacids  are  formed  by  the  action  of  HX02  upon  the  XH,  groups  can  also  give 

1  E.  Fischer  and  Abderhalden,  Ber.  d.  d.  Chem.  Gesellsch.,  40;  Abderhalden  and 
C.  Funk,  Zeitschr.  f.  physiol.  Chem.,  64. 


90  THE  PROTEIN   SUBSTANCES. 

certain  conclusions  as  to  the  structure  of  the  peptides  by  comparing  the  hydro- 
lytic  products  before  and  after  deamidation. 

A  comparison  of  the  artificially  prepared  polypeptides  with  the  pro- 
teins, and  especially  with  the  cleavage  products  of  these  last,  the  so-called 
preoteoses  and  peptones,  is  of  great  interest  in  several  respects,  especially 
in  connection  with  certain  reactions.  For  instance  there  are  several 
polypeptides  which  give  the  biuret  reaction  which  is  characteristic  of 
the  proteins  in  general,  and  also  several  (polypeptides  containing  tyrosine), 
which  give  Millon's  reaction  (see  further  on).  The  above-mentioned 
octadecapeptide  is  precipitated  by  phosphotungstic  acid,  tannin  and 
ammonium  sulphate ;  we  also  know  tri-  and  pentapeptides  containing 
tyrosine,  which  are  very  similar  in  properties  to  the  proteoses. 

The  behavior  of  the  polypeptides  with  proteolytic  enzymes  is  of 
great  interest.  As  this  interesting  question  will  be  thoroughly  treated 
in  other  chapters  (I  and  VIII)  it  is  sufficient  here  to  recall  that  the 
possibility  that  polypeptides  as  well  as  proteins  are  hydrolyzed  by  the 
same  enzymes,  yielding  amino-acids,  is  a  weighty  proof  of  the  prob- 
ability that  in  the  proteins  the  amino-acid  chains  are  of  the  same  kind 
as  in  the  polypeptides. 

A  very  important  support  for  such  a  view  is  found  in  the  occurrence 
of  polypeptides  among  the  cleavage  products  of  proteins,  a  find  which 
to  a  certain  extent  forms  the  reverse  of  the  above-mentioned  syntheses. 
Such  polypeptides  are  chiefly  di-  but  also  tri-  and  tetrapeptides.  They 
have  been  obtained  in  the  hydrolytic  products  of  silk  waste,  silk  fibroin 
and  elastin  (Fischer,  Abderhalden),  gelatin  (Levene,  Wallace  and 
Beatty)  and  of  gliadin  (Osborne  and  Clapp)1.  Of  special  interest  in 
this  connection  are  those  polypeptides  which  like  glycyl-d-alanine, 
d-alanyl-glycine,  glycyl-Z-tyrosine,  Z-prolyl-Z-phenylalanine  and  d-alanyl- 
glyeyl-Z-tyrosine,  are  identical  with  the  corresponding  synthetically  pre- 
pared polypeptides  or  at  least  very  closely  related. 

We  have  therefore  conclusive  reasons  for  the  assumption  that  in  the 
proteins,  peptide  bindings  chiefly  occur,  i.e.,  a  combination  of  the  a- 
amino-acids  by  means  of  the  imide  binding.  It  is  also  possible  that 
other  linking  may  occur,  and  Fischer  has  also  given  expression  to  such 
a  possibility.  Besides  the  above-mentioned  imide  binding  another  kind 
must  also  without  doubt  exist  in  the  proteins,  namely,  the  anchoring 
of  the  urea-forming  group  (the  guanidine  residue)  with  the  ornithin 
(diamino-valeric   acid)    by   the   imide   binding.     This   imide   linking    is 


1  Fischer  and  Abderhalden,  Sitz.  Ber.  d.  d.  Berl.  Akad.  d.  Wissensch,  30,  and  Ber. 
d.  d.  Chem.  Gesellsch.,  89,  40;  Abderhalden,  Zeitschr.  f.  physiol.  Chern.,  02,  03  and  72; 
Levene  and  Wallace,  ibid.,  47,  with  Beatty,  Ber.  d.  d.  Chem.  Gesellsch.,  39,  and  Bioch. 
Zcitsrhr.,  4;    Osborne  and  Clapp,  Amer.  Journ.  of  Physiol.,  18. 


CLASSIFICATION.  91 

not,  like  the  «-amino-acids,  broken  by  trypsin,  but  rather  by  an  enzyme 
arginase,  discovered  by  K08SEL  and  Dakin.1 

If  the  proteins  are  considered  as  consisting  chiefly  of  peptide-like 
complexes  consisting  of  amino-acids  united  and  containing  also  several 
NH2  groups  at  the  ends,  it  is  readily  understood  that  the  proteins  are 
amphoteric  electrolytes,  like  the  amino-acids,  which  form  salts  with  bases 
as  well  as  with  acids  and  undergo  hydrolytic  dissociation.  As  we  also 
accept  the  theory  that  the  protein  molecule  contains  a  large  number  of 
(  (  M  >II  as  well  as  XHL»  groups,  it  follows  that  the  proteins  may  be  poly- 
basic  acids  as  well  as  polvacidic  bases.  The  different  proteins  act  in 
this  regard  somewhat  differently,  thus  the  protamines  are  strongly  basic 
while  casein  behaves  strikingly  acid,  and  others  take  a  certain  mean 
position.  It  is  unfortunately  impossible  to  base  a  classification  of  the 
proteins  upon  this  behavior,  as  well  as  upon  chemical  constitution. 
The  general  properties,  such  as  solubility  and  precipitation  properties, 
are  too  uncertain  to  aid  us,  and  especially  as  in  the  investigations  of 
proteins  we,  as  a  rule,  cannot  decide  whether  we  are  dealing  with  a  pure 
or  with  a  contaminated  substance,  namely,  with  mixtures.  Experience 
has  shown  that  the  solubility  and  precipitation  properties  of  the  proteins 
are  strongly  influenced  by  the  presence  of  other  bodies,  and  under  such 
circumstances  a  proper  classification,  as  demanded  by  science,  is  impos- 
sible. On  the  other  hand,  a  classification  is  important,  and  as  the 
ones  used  up  to  the  present  time"  were  based  upon  the  solubility  and 
precipitation  properties,  we  give  the  following  schematic  summary  of  the 
chief  groups  of  protein  bodies: 


I.     Simple  Proteins. 
A.     True  Albuminous  Bodies  or  Proteids. 


Albumins, 
Globulins 


Seralbumin, 

Lactalbumin,  and  others. 
Fibrinogen, 
Serglobulins,  and  others. 

Phosphoproteins       (Nucleoalbu-  I  Ovovitellin, 

mins) [  Casein,  and  others. 

(Coagulated  proteins.) 

Histones. 

(Protamines?) 


1  Zeitschr.  f.  physiol.  Chem.,  41. 


92  THE  PROTEIN  SUBSTANCES. 

B.    Albuminoids  or  Albumoids. 
Keratins. 
Elastin. 

Collagen  and  glutin. 
Reticulin. 

(Fibroin,  Sericin,  Coilin,  Cornein,  Spongin,  Byssus,  and  others.) 

C.    Cleavage  Products  of  True  Albuminous  Bodies. 

Alkali  and  Acid  Albuminates. 
Proteoses,  Peptones,  Polypeptides. 
(Amino-acids.) 

II.     Compound  Proteins. 

.  \Mucin  substances, 

[  Ichthulin,  and  others. 

Nucleoproteins. 

Chromoproteins 


J  Hcemoglobin, 
|  Hcemocyanin. 

As  there  are  two  classifications  recognized  by  English-speaking  scien- 
tists we  will  give  the  classifications  adopted  by  the  American  Physio- 
logical Society  and  the  American  Society  of  Biological  Chemists  and 
also  the  British  Medical  Association. 

Classification  adopted  by  the  American  Physiological  Society  and 
the  American  Society  of  Biological  Chemists: 

I.    Simple  Proteins. 

A.  Albumins. 

B.  Globulins. 

C.  Glutelins. 

D.  Prolamins  (Alcohol-soluble  proteins). 

E.  Albuminoids. 

F.  Histones. 

G.  Protamines. 


II.    Conjugated  Proteins. 


A.  Nucleoproteins. 

B.  Glycoproteins. 

C.  Phosphoproteins. 

D.  Haemoglobins. 

E.  Lecithoproteins. 


CLASSIFICATION.  93 

HI.     Derived  Proteins. 

1.     Primary  Protein  Derivatives. 

A.  Proteans. 

B.  Metaproteins. 

C.  Coagulated  proteins. 

2.    Secondary  Protein  Derivatives. 

A.  Proteoses. 

B.  Peptones. 

C.  Peptides. 

Classification  of  proteins  adopted  by  the  British  Medical  Association: 

I.     Simple  Proteins. 

1.  Protamines. 

2.  Histones. 

3.  Albumins. 

4.  Globulins. 

5.  Glutelins. 

6.  Alcohol-soluble  proteins. 

7.  Scleroproteins. 

8.  Phosphoproteins. 

n.    Conjugated  Proteins. 

1.  Glucoproteins. 

2.  Nucleoproteins. 

3.  Chromoproteins. 

HI.    Products  of  Protein  Hydrolysis. 

1.  Infraproteins. 

2.  Proteoses. 

3.  Peptones. 

4.  Polypeptides. 

To  this  summary  must  be  added  that  we  often  find  in  the  investiga- 
tions of  animal  fluids  and  tissues  protein  substances  which  do  not  fall 
in  with  the  above  schemes,  or  are  classified  only  with  difficulty.  At  the 
same  time  it  must  be  remarked  that  bodies  will  be  found  which  seem 
to  rank  between  the  different  groups,  hence  it  is  very  difficult  to  sharply 
divide  these  groups. 


94  THE  PROTEIN   SUBSTANCES. 

I.    Simple  Proteins. 

A.    True  Albuminous  Bodies. 

The  albuminous  bodies  are  never-failing  constituents  of  the  animal 
and  vegetable  organisms.  They  are  especially  found  in  the  animal 
body,  where  they  form  the  solid  constituents  of  the  muscles  and  of  the 
blood-serum,  and  they  are  so  generally  distributed  that  there  are  only 
a  few  animal  secretions  and  excretions,  such  as  the  tears,  the  perspira- 
tion, and  perhaps  the  urine,  in  which  they  are  entirely  absent  or  occur 
only  in  traces. 

All  albuminous  bodies  contain  carbon,  hydrogen,  nitrogen,  oxygen 
and  sulphur;1  a  few  contain  also  phosphorus.  Iron  is  generally  found 
in  traces  in  their  ash.  The  elementary  composition  of  the  different 
albuminous  bodies  varies  a  little,  but  the  variations  are  within  relatively 
close  limits.  For  the  better-studied  animal  albuminous  bodies  the 
following  composition  of  the  ash-free  substance  has  been  found: 

C 50.5  —54.6  percent 

H 6.5  —  7.3       " 

N 15.0  —17.6       " 

S 0.5  —  2,2       " 

P 0.42—  0.85     " 

0 21.50—23.50     " 

The  animal  proteids  are  odorless,  tasteless,  and  ordinarily  amorphous. 
The  crystalloid  spherules  (D otter plattchen)  occurring  in  the  eggs  of  certain 
fishes  and  amphibians,  do  not  consist  of  pure  proteids,  but  of  albuminous 
bodies  containing  large  amounts  of  lecithin,  which  seem  to  be  combined 
with  mineral  substances.  Crystalline  proteids2  have  been  prepared 
from  the  seeds  of  various  plants,  and  crystallized  animal  proteids  (see 
seralbumin  and  ovalbumin,  Chapters  V  and  XII)  can  be  readily  pre- 
pared. In  the  dry  condition  the  proteids  appear  as  white  powders, 
or  when  in  thin  layers  as  yellowish,  hard,  transparent  plates.  A  few 
are  soluble  in  water,  others  only  soluble  in  salt  or  faintly  alkaline  or  acid 
solutions,  while  others  are  insoluble  in  these  solvents.  Solutions  of 
proteids  are  optically  active  and  turn  the  plane  of  polarized  light  to  the 
left.     All  proteids  when  burned  leave  an  ash,  and  it  is  therefore  ques- 


1  See  foot-note  1,  p.  80. 

2  See  Maschke,  Journ.  f.  prakt.  Chem.,  74;  Drechsel,  ibid.  (N.  F.),  19;  Griibler, 
ibid.  (N.  F.),  23;  Ritthausen,  ibid.  (N.  F.),  25;  Schmiedeberg,  Zeitschr.  f.  physiol. 
Chem.,  1;  Weyl.,  1;  ibid.,  1. 


PROPERTIES.  95 

tionable  whether  there  exists  any  proteid  body  which  is  soluble  in  water 
without  the  aid  of  mineral  substances.  Nevertheless  it  has  not  been 
thus  far  successfully  proved  that  a  native  proteid  body  can  be  prepared 
perfectly  free  from  mineral  substances  without  changing  its  constitution 
or  its  properties.1 

As  previously  stated,  the  albuminous  bodies  are  amphoteric  elec- 
trolytes, and  are  polyacidie  liases  as  well  as  polybasic  acids.  The  base- 
and  acid-combining  powers  of  various  proteids  have  been  the  subject  of 
numerous  investigations  which  cannot  be  given  in  short.  In  regard  to 
various  methods  used  in  such  investigations  as  well  as  to  the  dissociation 
of  protein  salts  we  refer  especially  to  the  work  of  T.  B.  Robertson.2 

The  proteids  can  be  salted  out  from  their  neutral  solutions  by  neutral 
salt?  (NaCl,  Na2S04,  MgS04,  [NH4]2S04,  and  many  others)  in  sufficient 
concentrations.  By  this  salting  out  the  properties  remain  unchanged 
and  the  process  is  reversible,  as  on  diminishing  the  concentration  of  the 
salt  the  precipitate  redissolves.  The  various  proteids  act  in  an  entirely 
different  manner  toward  the  same  salt,  and  also  for  one  and  the  same 
proteid  the  behavior  toward  different  neutral  salts  is  different,  as  some 
cause  a  precipitate,  while  others  on  the  contrary  do  not  precipitate. 

The  behavior  of  various  proteids  with  one  and  the  same  salt,  such 
as  MgSCU  or  (NEU^SO-i,  is  often  made  use  of  in  the  isolation  of  the 
proteid,  and  special  methods  of  separation  are  based'  upon  fractional 
precipitation.  It  has  been  shown  that  these  methods  may  lead  to  great 
errors,  and  give  good  results  only  under  special  conditions.3 

The  conditions  are  different  from  those  of  salting  out,  when  the  pro- 
teid solution  is  precipitated  by  salts  of  the  heavy  metals.  Here  the 
precipitates  (often  called  metallic  albuminates)  are  not  true  combina- 
tions in  constant  proportions,  but  are  rather  to  be  considered  as  loose 
adsorption  compounds  of  the  proteid  with  the  salt.4  These  reactions 
are  irreversible  in  so  far  that  dilution  with  water  or  removal  of  the  salt 
by  means  of  dialysis  does  not  restore  the  unchanged  proteid.  On  the 
other  hand  the  precipitate,  at  least  in  certain  cases  may  be  redissolved 
in  an  excess  of  the  salt  solution  or  of  the  proteid  solution,  and  in  this 
sense  the  process  is  a  reversible  one. 


1  See  E.  Harnack,  Ber.  d.  d.  chem.  Gesellsch.,  22,  23,  25,  and  31;  Werigo,  Pfliiger's 
Archiv,  48;   Biilow,  ibid.,  58;   Schulz,  Die  Grosse  des  Eiweissmolekuls,  Jena,  1903. 

2Ergeb.  d.  Physiol.  10;  Journ.  of  physical  Chem.,  14,  15,  and  Journ.  of  biol.  Chem., 
9. 

3  See  Cohnheim,  Chemie  der  Eiweisskorper,  3.  Aufl.,  101 1 ;  Pinkus,  Journ.  of  Physiol., 
27;   Pauli,  Hofmeister's  Beitrage,  3,  p.  225;   Halsam,  Journ.  of  Physiol.,  32. 

4  See  Galeotti,  Zeitschr.  f.  physiol.  Chem.,  40,  42,  44,  and  48  and  Bonamartini  and 
Lombardi,  ibid.,  58.     See  also  the  opposed  views  of  Linpirh.  ibid,  74. 


96  THE  PROTEIN  SUBSTANCES. 

The  precipitation  of  proteids  and  also  other  soluble  proteins  by  salts 
stands  in  close  relation  to  their  colloidal  nature,  and  in  this  connec- 
tion we  refer  to  what  has  been  said  in  Chapter  I.  The  proteids  do  not 
as  a  rule  diffuse  through  animal  membranes,  or  only  to  a  very  slight 
extent,  and  hence  have  in  most  cases  a  pronounced  colloidal  nature  in 
Graham's  sense.  They  belong  to  the  hydrophile  colloids;  their  solu- 
tions show  properties  in  common  with  those  of  typical  colloids  and  also 
true  solutions.  Certain  of  them,  especially  the  peptones  and  a  few 
proteoses,  which  will  be  discussed  later,  seem  to  occupy  an  intermediate 
position,  as  their  solutions  are  characterized  by  a  lesser  viscosity  and 
greater  diffusibility  and  filtration  ability,  are  not  readily  precipitable 
by  alcohol  or  coagulable  by  heat,  and  are  only  partially  precipitable 
by  salts. 

The  solutions  (or  suspensions)  of  proteids  in  water,  the  proteid  hydro- 
sols,  are  converted  by  various  means  into  proteid  hydrogels.  Of  these 
means  we  must  specially  mention  the  following:  nocking  out  with  salts, 
precipitation  with  alcohol,  gelatinization  of  a  gelatin  solution  on  cool- 
ing, and  coagulation  by  the  action  of  enzymes  or  heat. 

Those  proteids  which  occur,  according  to  the  common  views,  pre- 
formed in  the  animal  fluids  and  tissues,  and  which  have  been  isolated 
from  these  by  indifferent  chemical  means  without  losing  their  original 
properties,  are  called  native  proteids.  New  modifications  having  other 
properties  can  be  obtained  from  the  native  proteids  by  heating,  by  the 
action  of  various  chemical  reagents  such  as  acids,  alkalies,  alcohol,  and 
others,  as  well  as  by  proteolytic  enzymes.  These  new  proteids  are  called 
modified  ("  denaturierte  ")  proteids,  to  differentiate  them  from  the  native 
proteids. 

The  precipitation  with  alcohol  is  a  reversible  reaction,  as  the  pre- 
cipitate redissolves  on  subsequent  dilution  with  water.  The  proteids 
are  changed  by  the  action  of  alcohol,  some  readily  and  quickly,  others 
with  difficulty  and  very  slowly;  the  proteid  then  becomes  insoluble  in 
water  and  is  modified. 

On  heating  a  solution  of  a  native  proteid  it  is  modified  at  a  different 
temperature  for  each  different  proteid.  With  proper  reaction  and  other 
favorable  conditions,  for  instance  in  the  presence  of  neutral  salts,  most 
proteids  can  in  this  way  be  precipitated  in  a  solid  form  as  coagulated 
proteid.  The  hydrosol  is  converted  into  hydrogel,  but  as' a  modification 
takes  place,  this  process  is  irreversible.  The  temperature  at  which 
coagulation  occurs  is  a  variable  one  for  the  same  protein  under  different 
conditions  of  the  experiment.  The  various  temperatures  at  which 
coagulation  of  different  proteids  occurs  in  neutral  solutions  containing 
salt  have  in  many  cases  given  us  good  means  for  detecting  and  separating 
proteids.     The  views  in  regard  to  the  use  of  these  means  are  somewhat 


PROPERTIES.  97 

4 

divided  '  and  the  same  applies  to  the  question  as  to  manner  of  heat 
coagulation  and  the  conditions  under  which  it  takes  place. 

The  heat  coagulation  of  a  protein  solution  is  dependent  upon  the  hydrogen 
ion  concentration  of  the  solution.  According  to  Michaelis  and  co-workers 
the  optimum  of  the  hydrogen  ion  concentration  falls  in  the  coagulation  of  a 
protein  solution  (precipitation  of  the  modified  protein)  with  the  isoelectric  point 
of  the  solution  and  the  optimum  of  the  flocking  is  not  changed  in  regard  to  the 
hydrogen  ion  concentration  by  changes  in  the  protein  concentration.  According 
to  Sorensen  and  Jurgexsen,2  on  the  contrary  the  optimal  hydrogen  ion  con- 
centration is  the  same  as  contained  in  a  solution  of  the  pure  protein  in  pure 
water  caused  by  the  electrolytic  dissociation  of  this  protein  and  is  therefore 
independent  of  the  protein  concentration.  This  hydrogen  ion  concentration, 
according  to  these  workers  diminishes  during  the  heat  coagulation  which  they 
consider  as  a  proof  of  the  diminution  in  the  protein  concentration  of  the  solution. 

A  modification  can  be  brought  about  also  by  the  action  of  acids, 
alkalies,  or  salts  of  the  heavy  metals,  in  certain  cases  by  water  alone, 
and  also  by  the  action  of  alcohol,  chloroform  (Salkowski),  and  ether, 
by  violent  shaking  (Ramsden3),  etc. 

An  adsorption  of  proteids  by  a  suspension  colloid  such  as  silicic  acid, 
colloidal  ferric  hydroxide  and  kaolin,  can  easily  take  place,  and  indeed  the 
proteid  of  a  solution  can  be  removed  by  the  use  of  colloidal  ferric  hydrox- 
ide or  shaking  with  kaolin  (Rona  and  Michaelis  4) .  That  the  proteids 
can  serve  as  preventives  in  the  precipitation  of  suspension  colloids  has 
been  mentioned  in  Chapter  I.  In  the  same  manner  a  mastic  suspension 
is  protected  from  the  precipitating  action  of  an  electrolyte  by  an  excess  of 
a  proteid  solution,  while  the  reverse  may  be  brought  about,  namely,  a 
proteid  solution  can  be  precipitated  by  a  large  quantity  of  mastic  emulsion 
in  the  presence  of  a  proportionately  small  amount  of  electrolyte.  The 
method  for  the  removal  of  proteid  from  solutions,  as  suggested  by 
Michaelis  and  Rona,5  is  based  upon  this  behavior. 

We  have  already  discussed  in  Chapter  I  the  electric  charge  of  the 
proteins  under  various  conditions  and  the  migration  of  these  in  electric 
fields  of  currents. 

1  See  Halliburton,  Journ.  of  Physiol.,  5  and  11;  Corin  and  Berard,  Bull,  de  1'Acad- 
roy.  de  Belg.,  15;  Haycraft  and  Duggan,  Brit.  Med.  Journ.,  1890,  and  Proc.  Roy. 
Soc.  Edin.,  1889;  Corin  and  Ansiaux,  Bull,  de  l'Acad.  roy.  de  Belg.,  21;  L.  Fredericq, 
Centralbl.  f.  Physiol.,  3;  Haycraft,  ibid.,  4;  Hewlett,  Journ.  of  Physiol.,  13;  Duclaux, 
Annal.  Institut  Pasteur,  7.  In  regard  to  the  relationship  of  the  neutral  salts  to  the 
heat  coagulation  of  albumins  see  also  Starke,  Sitzungsber.  d.  Gesellsch.  f.  Morph.  u. 
Physiol,  in  Munchen,  1897;  Pauli,  Pfliiger's  Arch.,  78. 

2  See  Ergeb.  d.  Physiol.,  12  which  contains  the  pertinent  literature. 

3  See  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  31;  Fr.  Kriiger,  Zeitschr.  f.  Biologie, 
41;  Loew  and  Aso,  Bull.  Coll.  Agric,  Tokio,  4;  Ramsden,  Zeitschr.  f.  physik.  Chem., 
47  and  Arch.  f.  (anat.  u.)  Physiol.,  1894. 

4  Biochem.  Zeitschr.,  5. 

6  Biochem.  Zeitschr.,  2,  3  and  4. 


98  THE  PROTEIN  SUBSTANCES. 

The  determination  of  the  molecular  weight  of  the  proteids  has  been 
attempted  by  various  methods  which  are  more  or  less  uncertain.1  There 
is  no  doubt  that  the  molecular  weight  of  the  proteids  is  very  high,  but 
the  statements  about  the  size  vary  considerably.  For  the  true  proteids 
thus  far  investigated,  values  ranging  from  4000 — 6000 — 10,000  have 
been  found. 

The  general  reactions  for  the  proteids  are  very  numerous,  but  only 
the  most  important  will  be  given  here.  To  facilitate  the  study  of  these, 
they  have  been  divided  into  the  two  following  groups.  It  must  be 
remarked  that  the  precipitation  reactions  are  not  only  applicable  for 
the  soluble  true  proteids  but  also,  more  or  less,  for  other  soluble  proteins 
in  general.  The  color  reactions  are  applicable  to  all  soluble  or  insoluble 
proteins  with  few  exceptions,  which  will  be  mentioned  later. 

Precipitation  Reactions  of  the  Proteid  Bodies. 

1.  Coagulation  Test.  An  alkaline  proteid  solution  does  not  coagulate 
on  boiling,  and  a  neutral  solution  only  partly  and  incompletely;  the  reac- 
tion must  therefore  be  acid  for  coagulation.  The  neutral  liquid  is  first 
boiled  and  then  the  proper  amount  of  acid  added  carefully.  A  fiocculent 
precipitate  is  formed,  and  with  proper  technique  the  filtrate  should  be 
water-clear.  If  dilute  acetic  acid  be  used  for  this  test,  the  liquid  must 
first  be  boiled  and  then  1,  2,  or  3  drops  of  acid  added  to  each  10-15  cc, 
depending  on  the  amount  of  proteid  present,  and  boiled  before  the  addi- 
tion of  each  drop.  If  dilute  nitric  acid  (25  per  cent)  be  used,  then  to 
10-15  cc.  of  the  previously  boiled  liquid  }5-20  drops  of  the  acid  must 
be  added.  If  too  little  nitric  acid  be  addei,  a  soluble  combination  of  the 
acid  and  proteid  is  formed,  which  is  precipitated  by  more  acid.  A  pro- 
teid solution  containing  a  small  amount  of  salts  must  first  be  treated  with 
about  1  per  cent  NaCl,  since  the  heating  test  may  fail,  especially  on  using 
acetic  acid,  in  the  presence  of  only  a  slight  amount  of  proteid. 

2.  Precipitation  by  Alcohol.  The  solution  must  not  be  alkaline, 
but  must  be  either  neutral  or  faintly  acid.  It  must,  at  the  same  time, 
contain  sufficient  quantity  of  neutral  salts. 

3.  Neutral  Salts,  such  as  Na2S(>4  or  NaCl,  when  added  to  saturation 
precipitate  certain  proteids  but  not  others.  Ammonium  sulphate  when 
dissolved  to  saturation  in  the  liquid  is  considered  as  the  general  pre- 
cipitant for  proteids.  In  the  presence  of  free  acetic  or  hydrochloric 
acid  the  above-mentioned  salts,  NaCl  or  Na2S04,  in  sufficient  con- 
centration, are  also  general  precipitants  for  the  proteids. 

4.  Precipitation  by  Metallic  Salts  such  as  copper  sulphate,  ferric 
chloride,   neutral  and  basic  lead  acetate   (in  small  amounts),   mercuric 

1  See  especially  F.  N.  Schulz,  Die  Grosse  des  Eivveissmoleciile,  Jena,  1903. 


COLOR  REACTIONS.  99 

chloride  and  others.     On  this  is  based  the  use  of  proteids  as  antidotes 
in  poisoning  with  metallic  salts. 

5.  Precipitation  by  Mineral  Acids  at  Ordinary  Temperatures.  The 
proteids  are  precipitated  by  the  three  ordinary  mineral  acids  in  proper 
amounts,  hut  not  by  orthophosphoric  acid.  If  nitric  acid  be  placed  in  a 
test-tube  and  the  proteid  solution  be  allowed  to  flow  gently  thereon,  a 
white  opaque  ring  of  precipitated  proteid  will  form  where  the  two  liquids 
meet  (Heller's  albumin  test). 

6.  Precipitation  by  the  so-called  Alkaloid  Reagents.  To  these  belong 
the  precipitation  by  metaphosphoric  acid  and  by  hydroferrocyanic  acid, 
which  is  carried  out  by  the  aid  of  potassium  ferrocyanide  in  a  liquid 
containing  acetic  acid;  precipitation  by  phosphotungstic  acid  or  phos- 
phomolybdic  acid  in  the  presence  of  free  mineral  acids;  precipitation 
by  potassium-mercuric  iodide  or  potassium-bismuth  iodide  in  solutions 
acidified  with  hydrochloric  acid;  precipitation  by  tannic  acid  in  acetic 
acid  solutions.  The  absence  of  neutral  salts  or  the  presence  of  free 
mineral  acids  may  prevent  the  appearance  of  the  precipitate,  but  after 
the  addition  of  a  sufficient  quantity  of  sodium  acetate  the  precipitate 
will  in  both  cases  appear;  precipitation  by  picric  acid  in  solutions  acid- 
ified by  organic  acids.  Proteids  are  also  precipitated  by  trichloracetic 
acid  in  2-5  per  cent  solutions,  by  phenol,  salicyl  sulphonic  acid,  nucleic 
acid,  taurocholic  acid  and  by  chondroitin  sulphuric  acid  in  acid  solutions. 

Color  Reactions  for  Proteid  Bodies. 

1.  MUlon's  Reaction.1  A  solution  of  mercury  in  nitric  acid  contain- 
ing some  nitrous  acid  gives  a  precipitate  with  proteid  solutions  which 
at  the  ordinary  temperature  is  slowly,  but  at  the  boiling-point  more 
quickly,  colored  red;  and  the  solution  may  also  be  colored  a  feeble 
or  bright  red.  Solid  albuminous  bodies,  when  treated  by  this  reagent, 
give  the  same  coloration.  This  reaction  is  due  to  the  tyrosine  and  is 
also  given  by  other  monohydroxyl  benzene  derivatives.  According  to 
O.  Nasse  2  it  is  best  to  use  a  solution  of  mercuric  acetate  which  is  treated 
with  a  few  drops  of  a  1  per  cent  solution  of  potassium  or  sodium  nitrite; 
previous  to  use  a  few  drops  of  acetic  acid  are  added. 

2.  Xanthoproteic  Reaction.  With  strong  nitric  acid  the  albuminous 
bodies  give,  on  heating  to  boiling,  yellow  flakes  or  a  yellow  solution. 


1  The  reagent  is  prepared  in  the  following  way:  1  pt.  mercury  is  dissolved  in  2  pts. 
nitric  acid  (of  sp.gr.  1.42),  first  cold  and  then  warmed.  After  complete  solution  of 
the  mercury  add  1  volume  of  the  solution  to  2  volumes  of  water.  Allow  this  to  stand 
a  few  hours  and  decant  the  supernatant  liquid. 

2  See  O.  Nasse,  Sitzungsb.  d.  Naturforsch.  Gesellsch.  zu  Halle,  1879,  and  Pfluger's 
Arch.,  83;  see  also  Vaubel  and  Blum,  Journ.  f.  prakt.  Chem.  (N.  F.),  57. 


100  THE  PROTEIN   SUBSTANCES. 

After  making  alkaline  with  ammonia  or  alkalies  the  color  becomes  orange- 
yellow,  due  to  the  nitroderivatives  of  the  benzene  and  indol  groups. 

3.  Adomkieivicz's  Reaction.  If  a  little  proteid  is  added  to  a  mixture 
of  1  vol.  concentrated  sulphuric  acid  and  2  vols,  glacial  acetic  acid  a 
reddish-violet  color  is  obtained  slowly  at  ordinary  temperatures,  but 
more  quickly  on  heating.  According  to  Hopkins  and  Cole  x  this  reaction 
takes  place  only  on  using  glacial  acetic  acid  containing  glyoxylic  acid. 
According  to  them  it  is  better  to  use  a  solution  of  glyoxylic  acid,  which 
can  be  readily  prepared  by  adding  sodium  amalgam  to  a  concentrated 
solution  of  oxalic  acid  and  filtering  after  the  discharge  of  the  gas.  A 
dilute  aqueous  solution  of  the  acid  or  some  of  the  solid  acid  is  added  to  the 
proteid  solution  and  sulphuric  acid  allowed  to  flow  down  the  side  of  the 
test-tube,  when  the  reddish-violet  color  will  appear  at  the  point  of  con- 
tact of  the  two  liquids  or  on  shaking  the  mixture.  This  color  reaction, 
which  is  generally  called  the  Adamkiewicz-Hopkins  reaction  depends 
upon  the  tryptophane  and  therefore  gelatin  (which  does  not  contain 
•any  tryptophane)  does  not  give  this  reaction. 

As  further  color  reactions  we  will  mention:  4.  Biuret  Test.  If  a 
proteid  solution  be  first  treated  with  caustic  potash  or  soda  and  if  then 
a  dilute  copper-sulphate  solution  be  added  drop  by  drop,  first  a  reddish 
then  a  reddish-violet,  and  lastly  a  violet-blue,  color  is  obtained.  5.  Pro- 
teids  are  soluble  on  heating  with  concentrated  hydrochloric  acid,  produc- 
ing a  violet  color,  and  when  they  are  previously  boiled  with  alcohol  and 
then  washed  with  ether  (Liebermann  2)  they  give  a  beautiful  blue 
solution.  This  blue  color  is  due,  according  to  Cole,3  to  a  contamination 
of  the  ether  with  glyoxylic  acid,  which  reacts  with  the  tryptophane 
groups  split  off  by  the  hydrochloric  acid.  The  violet  color  obtained 
with  proteins  not  purified  with  ether  is  also  considered  as  a  tryptophane 
reaction  with  the  furfurol  (oxymethylfurfurol)  formed  from  the  hexose 
containing  protein  by  the  action  of  the  concentrated  hydrochloric 
acid.  Reaction  6  with  concentrated  sulphuric  acid  and  sugar  (in  small 
quantities)  is  explained  in  the  same  way.  The  beautiful  red  coloration 
is  connected  with  the  formation  of  furfurol  from  the  sugar.  7.  With 
7>-dimethylaminobenzaldehyde  and  concentrated  sulphuric  acid  thepro- 
teids  give  a  beautiful  reddish-violet  or  deep-violet  coloration  (0.  Neu- 
bai"er  and  E.  Rohde4).  Other  aldehydes  also  give  color  reactions 
by  virtue  of  the  tryptophane  group  in  proteins.  Other  reactions  are  8; 
Arnold's  reaction  5  is  a  purple-violet  coloration  which  the  proteins  give 

1  Proceed.  Roy.  Soc,  08. 

2Centralbl.  f.  d.  ined.  Wissensch.,  1887. 

3  Journ.  of  Physiol.,  30. 

4  Zeitsche.  f.  physiol.  Chem.,  44. 

0  Arnold,  Zeitschr.  f.  physiol.  Chem.,  70. 


PROTEIN  REACTIONS.  101 

with  sodium  nitroprusside  and  ammonia.  This  reaction  is  not  given  by 
all  proteins  and  is  due  to  the  cystine  groups.  9,  Abderhalden  and 
Schmidt's  reaction  with  triketohydrindenhydrate  which  gives  a  blue 
coloration  on  boiling.  The  triketohydrindenhydrate  (also  called  "Nin- 
hydrin ")  reacts  with  all  compounds  which  have  an  amino  group  in 
the  a-position  to  the  carboxyl,  is  according  to  Abderhalden  and 
Hchmidt  l  an  excellent  reagent  for  the  detection  of  dialyzable  amino- 
acids  and  non-biuret  giving  amino-acid  derivatives.  They  have  been 
able  to  detect  by  this  reagent  such  non-biuret  giving  substances  in  the 
dialysate  on  the  dialysis  of  different  animal  fluids.  They  have  also 
determined  the  delicacy  of  this  reagent  with  different  amino-acids. 

The  biuret  reaction  is  not  only  given  by  protein  substances,  but  also  by  many 
other  bodies.  According  to  H.  Schiff  2  this  reaction  occurs  with  those  bodies 
containing  amino  groups,  COXH2,  CSNH«,  C(NH)XH2  or  also  CHaNHj,  united 
either  directly  by  their  carbon  atoms  or  by  means  of  a  third  carbon  or  nitrogen 
atom.  As  examples  of  such  bodies  we  can  mention  several  diamines  or  amino- 
amides,  such  as  oximide,  biuret,  glycinamide,  a-  and  /3-aminobutyramide,  aspartic- 
acid  amide,  etc.,  although  we  are  not  certain  as  to  the  conditions  necessary  for  the 
bringing  about  of  this  reaction.  The  biuret  reaction  alone  is  therefore  no  proof 
as  to  the  protein  nature  of  a  substance — for  example,  urobilin  gives  a  very  similar 
color  reaction — and  a  protein  substance  can  still  retain  its  protein  nature,  as  by 
the  action  of  nitrous  acid  or  by  a  splitting  off  of  ammonia,  although  it  does  not 
give  the  biuret  reaction. 

The  delicacy  of  the  various  reagents  differs  for  the  different  proteids, 
and  on  this  account  it  is  impossible  to  give  the  degree  of  delicacy  for 
each  reaction  for  all  proteids.  Of  the  precipitation  reactions.  Heller's 
test  (if  we  eliminate  the  peptones  and  certain  proteoses)  is  recommended 
in  the  first  place  for  its  delicacy,  though  it  is  not  the  most  delicate  reac- 
tion, and  because  it  can  be  performed  so  easily.  Among  the  precipita- 
tion reactions,  that  with  basic  lead  acetate  (when  carefully  and  exactly 
executed)  and  with  alcohol  and  the  reactions  given  under  6,  are  the  most 
delicate.  The  color  reactions  1  to  4  show  great  delicacy  in  the  order  in 
which  they  are  given.3 

No  proteid  reaction  is  in  itself  characteristic,  and,  therefore,  in  testing 
for  proteids  one  reaction  is  not  sufficient,  but  a  number  of  precipitation 
and  color  reactions  must  be  employed. 

For  the  quantitative  estimation  of  coagulable  proteids  the  determina- 
tion by  boiling  with  acetic  acid  can  be  performed  with  advantage,  for 
by  operating  carefully,  it  gives  exact  results.  Treat  the  proteid  solution 
with  a  1-2  per  cent  common-salt  solution,  or  if  the  solution  contains 
large  amounts  of  proteid  dilute  with  the  proper  quantity  of  the  above 
salt  solution,  and  then  carefully  neutralize  with  acetic  acid.     Now  deter- 

1  Zeitschr.  f.  physiol.  Chem.,  72  and  85. 

2  Ber.  d.  d.  chem.  Gesellsch.,  29  and  30. 

3  In  regard  to  the  precipitation  and  color  reactions  of  proteids  with  aniline  dyes 
zee  Heidenhain,  Pfluger's  Arch.,  90,  96. 


102  THE  PROTEIN  SUBSTANCES. 

mine  the  quantity  of  acetic  acid  necessary-  to  completely  precipitate 
the  proteids  in  small  measured  portions  of  the  neutralized  liquid  which 
have  previously  been  heated  on  the  water-bath,  so  that  the  nitrate  does 
nut  respond  to  Heller't  test.  Now  warm  a  larger  weighed  or  meas- 
ured quantity  of  the  liquid  on  the  water-bath,  and  add  gradually  the 
required  quantity  of  acetic  acid,  with  constant  stirring,  and  continue 
heating  for  some  time.  Filter,  wash  with  water,  extract  with  alcohol 
and  then  with  ether,  dry,  weigh,  incinerate,  and  weigh  again.  With 
proper  work  the  filtrate  should  not  give  Heller's  test.  This  method 
serves  in  most  cases,  and  especially  so  in  cases  where  other  bodies  are 
to  be  quantitatively  estimated  in  the  filtrate. 

In  many  cases  good  results  may  be  obtained  by  precipitating  all  the 
proteid  with  tannic  acid  and  determining  the  nitrogen  in  the  washed 
precipitate  by  means  of  Kjeldahl's  method.  On  multiplying  the  quan- 
tity of  nitrogen  found  by  6.25  we  obtain  the  quantity  of  proteid.  Many 
other  methods  for  the  quantitative  estimation  of  proteins  have  been 
suggested. 

The  removal  of  proteids  from  a  solution  may  in  most  cases  be  per- 
formed by  boiling  with  acetic  acid.  Small  amounts  of  proteid  which 
remain  in  the  filtrates  may  be  separated  by  boiling  with  freshly  pre- 
cipitated lead  carbonate  or  with  ferric  acetate,  as  described  by  Hof- 
meister.1  .  If  the  liquid  cannot  be  boiled,  the  proteid  may  be  precipi- 
tated by  the  very  careful  addition  of  lead  acetate,  or  by  the  addition 
of  alcohol.  If  the  liquid  contains  substances  which  are  precipitated 
by  alcohol,  such  as  glycogen,  then  the  proteid  may  be  removed  by  tri- 
chloracetic acid  as  suggested  by  Obermayer  and  Frankel.2  Recently 
Michaelis  and  Rona  have  suggested  a  method  for  the  removal  of  proteids 
by  using  kaolin,  colloidal  ferric  hydrate  or  a  mastic  emulsion.  The 
principle  of  these  methods  has  already  been  given  on  page  97  and  in 
regard  to  the  practical  execution  of  the  method  we  refer  to  the  works 
there  cited. 

In  the  precipitation  of  proteid  as  well  as  the  quantitative  estimation  by  means 
of  heat,  it  must  be  borne  in  mind,  as  shown  by  Spiro,3  that  several  nitrogenous 
substances,  such  as  piperidine,  pyridine,  urea,  etc.,  disturb  the  coagulation  of  the 
proteids. 

Synopsis  of  the  Most  Important  Properties  of  the  Different  Groups  of 

Albuminous  Bodies. 

As  it  is  not  possible  to  base  the  classification  of  the  different  proteid 
groups  according  to  their  constitution,  we  are  obliged  to  make  use  of 
their  different  solubilities  and  precipitation  properties  in  their  general 
characterization.  As  there  exist  no  sharp  differences  between  the  various 
groups  in  this  regard  it  is  impossible  to  draw  a  sharp  line  between  them. 

Albumins.  These  bodies  are  soluble  in  water  in  neutral  reaction  and 
are  not  precipitated  by  the  addition  of  a  little  acid  or  alkali.     They  are 

1  Zeitschr.  f.  physiol.  Chem.,  2  and  4. 

2  Oberrnayer,  Wien.  med.  Jahrb.,  1888;  Frankel,  Pfluger's  Arch.,  52  and  55. 
1  Zeitschr.  f .  physiol.  Chem.,  30. 


GLOBULINS.  103 

precipitated  by  the  addition  of  large  quantities  of  mineral  acids  or  metallic 
-alts.    Their  solution  in  water  coagulates  on  boiling  in  the  presence  of 

neutral  salts,  but  a  weak  saline  solution  does  not.  If  NaCl  or  MgS<  '4 
is  added  to  saturation  to  a  neutral  solution  in  water  at  the  normal  tem- 
perature or  at  30°  C.  no  precipitate  is  formed;  hut  if  acetic  acid  i>  added 
to  this  saturated  solution  the  albumins  readily  separate.  When  ammonium 
sulphate  is  added  to  one-half  saturation  the  albumin  solutions  are  not 
precipitated  at  ordinary  temperatures.  Of  all  the  native  proteids  the 
albumins  are  the  richest  in  sulphur,  containing  from  1.6  per  cent  to  2.2 
per  cent.  So  far  as  they  have  been  investigated  they  do  not  yield  any 
glycocoll  on  acid  hydrolysis. 

Globulins.  These  substances  are,  as  a  rule,  insoluble  in  water,  but 
dissolve  in  dilute  neutral  salt  solutions.  The  globulins  are  precipitated 
unchanged  from  these  solutions  by  sufficient  dilution  with  water,  and 
on  heating  they  coagulate.  The  globulins  dissolve  in  Avater  on  the  addi- 
tion of  very  little  acid  or  alkali,  and  on  neutralizing  the  solvent  they 
precipitate  again.  The  solution  in  a  minimum  amount  of  alkali  is  pre- 
cipitated by  carbon  dioxide,  but  the  precipitate  may  in  certain  cases  be 
redissolved  by  an  excess  of  the  precipitant.  The  neutral  solutions  of  the 
globulins  containing  salts  are  partly  or  completely  precipitated  on  satura- 
tion with  NaCl  or  MgSC>4  in  substance  at  normal  temperatures,  depending 
upon  the  kind  of  globulin.  The  globulins  are  completely  precipitated 
by  half-saturating  with  ammonium  sulphate.  The  globulins  contain  an 
average  amount  of  sulphur  generally  not  below  1  per  cent.  As  a  differ- 
ence between  the  albumins  and  globulins  the  latter  yield  glycocoll  among 
the  hydrolytic  cleavage  products,  and  according  to  Obermayer  and 
"Willheim  l  they  contain  fewer  NH2  groups  at  the  end  of  the  chain,  as 
determined  by  formol  titration,  as  compared  to  the  total  number  of  N- 
atoms. 

A  sharp  line  cannot  be  drawn  between  the  albumins  and  globulins 
from  their  properties  and  this  is  shown  from  the  researches  of  Moll,2 
which  showr  that  by  the  action  of  dilute  alkalies  and  warmth  upon 
seralbumin  it  attains  the  properties  of  serglobulin.  It  is  evident  that 
we  are  here  dealing  with  a  change  of  the  external  properties  of  the 
albumins  to  a  greater  similarity  to  those  of  the  globulins,  and  not  with 
a  true  transformation  of  the  albumin,  which  is  free  from  glycocoll,  into 
globulin  which  contains  glycocoll.  The  same  follows  from  the  observa- 
tions of  others.3  This  is  an  instructive  example  of  the  subordinate  impor- 
tance the  solubility  and  precipitation  properties  have  in  the  differentia- 
tion of  various  groups  of  proteids. 

1  Bioch.  Zeitschr.,  38. 

2  Moll,  Hofmeister's  Beitrage,  4  and  7;  also  Breinl,  Arch.  f.  exp.  Path.  u.  Pharm.,  65. 
•Obermayer  and  Willheim,  1.  c;  R.  Gibson,  Journ.  of  biol.  Chem.,  12. 


104  THE  PROTEIN  SUBSTANCES. 

It  is  just  as  difficult  to  draw  a  sharp  line  between  the  globulins  and 
albuminates  as  it  is  between  the  globulins  and  albumins.  Several  globu- 
lins are  very  readily  changed  by  the  action  of  very  little  acid,  as  also  by 
standing  under  water  when  in  a  precipitated  condition,  into  albuminates, 
and  then  become  insoluble  in  neutral  salt  solutions.  Osborne,1  who  has 
closely  studied  this  property  in  connection  with  edestin  (from  hemp-seed) , 
considers  the  globulin,  "  globan,"  which  has  been  made  insoluble  in  salt 
solution,  as  an  intermediate  step  in  the  formation  of  the  albuminate  which  is 
produced  by  the  hydrolytic  action  of  the  H  ions  of  water  or  of  the  acid. 

Phosphoproteins  are  a  group  of  phosphorized  proteids  which  occur 
extensively  in  the  animal  and  plant  kingdoms  and  which  include  the 
nucleoalbumins  and  the  little-studied  lecithalbumins. 

Nucleoalburnins.  These  proteids  behave  like  rather  strong  acids, 
are  nearly  insoluble  in  water,  but  dissolve  easily  with  the  aid  of  a  little 
alkali  and,  in  the*  entire  absence  of  phosphatides,  contain  also 
phosphorus.  Certain  of  the  nucleoalbumins,  resemble  the  globulins  by 
their  solubility  and  precipitation  properties.  Others  resemble  the 
albuminates,  but  differ  from  both  of  these  groups  by  containing  phos- 
phorus. The>T  stand  close  to  the  nucleoproteins  by  their  content  of 
phosphorus,  but  differ  from  these  in  not  yielding  any  purine  bases  on 
cleavage.  It  has  not  yet  been  found  possible  to  obtain  from  the  neucleo- 
albumins  any  proteid-free  pseudonucleic  acids  corresponding  to  the 
nucleic  acids,  but  only  acids  rich  in  phosphorus,  which  always  give  the 
proteid  reactions.2  For  this  reason  the  nucleoalbumins  cannot  be  classed 
as  compound  proteins.  In  peptic  digestion  a  proteid  rich  in  phosphorus 
can  be  split  off  from  most  nucleoalbumins,  and  this  has  been  called 
para-  or  pseudonuclein.  The  claim  made  that  the  pseudonuclein  is  a 
combination  of  proteid  with  metaphosphoric  acid  has  been  shown  to  be 
incorrect  by  the  investigations  of  Giertz.3 

'  The  separation  of  pseudonuclein  in  peptic  digestion  is  no  doubt  characteristic 
of  the  nucleoalbumin  group,  but  the  non-appearance  of  the  pseudonuclein  pre- 
cipitate does  not  entirely  exclude  the  presence  of  a  nucleoalbumin.  The  extent 
of  such  a  formation  is  dependent  upon  the  intensity  of  the  pepsin  digestion,  the 
degree  of  acidity,  and  the  relation  between  the  nucleoalbumins  and  the  digestive 
fluids.  The  separation  of  a  pseudonuclein  may,  as  shown  by  Salkowski, 
not  occur  even  in  the  digestion  of  ordinary  casein,  and  Wr6blewski  and  others4 
did  not  obtain  any  pseudonuclein  at  all  in  the  digestion  of  the  casein  from  human 
milk.  The  most  essential  characteristic  of  this  group  of  proteids  is  that  they  con- 
tain phosphorus,  and  that  the  purine  bases  are  absent  in  their  cleavage  products. 

1  Zeitschr.  f.  physiol.  Chem.,  33. 

2 Levene  and  Alsberg,  ibid.,  31;  Salkowski,  ibid.,  32;  Levene,  ibid.,  32;  A.  Reh, 
Hofmeister'e  Beitrage,  11;   Dietrich,  Bioch.  Zeitschr.,  22. 

3  Giertz,  Zeitschr.  f.  physiol.  Chem.,  28. 

4  Salkowski,  Pfliiper's  Arch.,  63;  Wr6blewski,  Beitrage  zur  Kenntnis  des  Frauen- 
kasei'ns,  Inaug.-Diss.,  Bern,  1894. 


LECITHALBUMINS.  105 

The  nucleoallmmins  arc  often  confounded  with  nucleoproteina  and 
also  with  phosphorized  glucoproteins.  From  the  first  class,  they  differ 
by  not  yielding  any  purine  bases  when  boiled  with  acids,  and  from  the 
second  group  by  not  yielding  any  reducing  substance  on  the  same  treat- 
ment. The  best  studied  member  of  this  group  is  the  casein  of  milk, 
which  will  be  discussed,  in  detail  in  chapter  XIII. 

Neuberg  and  Pollak  '  have  artificially  prepared  phosphoproteins  by  the 
action  of  phosphorus  oxychloride  upon  an  alkaline  solution  of  lactalbumin  or  blood 
globulin.  The  product  obtained  from  lactalbumin  was  rather  close  to  casein 
in  regard  to  composition  and  other  properties. 

Lecithalbumins.  In  the  preparation  of  certain  protein  substances, 
products  are  often  obtained  containing  lecithin,  and  this  lecithin  (see 
the  phosphatides,  Chapter  IV)  can  be  removed  only  with  difficulty  or  in- 
completely by  a  mixture  of  alcohol  and  ether.  Ovovitellin  (Chapter  XII) 
is  such  a  protein  body  containing  considerable  lecithin,  and  Hoppe-Seyler 
considers  it  a  combination  of  proteid  and  lecithin.  Similar  substances 
occur  in  fish-eggs.  These  last  lecithalbumins  often  have  the  solubilities 
of  the  globulins  and  are  readily  soluble  in  dilute  salt  solutions.  The 
behavior  of  the  nucleoalbumin  of  the  eggs  of  the  perch  shows  how  easily 
this  solubility  may  be  changed.  This  nucleoalbumin,  which  contains  con- 
siderable amounts  of  lecithin,  is  readily  soluble  in  dilute  NaCl  solution, 
but  at  ordinary  temperatures  it  is  changed  by  0.1  per  cent  HC1  almost 
instantaneously  and  without  splitting  off  lecithin,  so  that  it  becomes  in- 
soluble in  dilute  salt  solutions  (Hammarsten)  .  Liebermann  2  has  obtained 
];roteids  containing  lecithin  as  an  insoluble  residue  on  the  peptic  digestion 
of  the  mucous  membrane  of  the  stomach,  liver,  kidneys,  lungs,  and  spleen. 
He  considers  them  as  combinations  of  proteid  and  lecithin  and  calls  them 
lecithalbumins.     Further  investigation  of  these  bodies  is  desirable. 

Mayer  and  Terroine  '  have  shown  that  from  lecithin  emulsified  in  water  and 
a  dialyzed  solution  of  ovalbumin  or  dialyzed  blood  serum  a  precipitate  can  be 
obtained  which  has  some  similarity  to  the  lecithalbumins,  but  which  in  other 
respects  is  so  strikingly  different  that  we  are  not  justified  in  calling  this  pre- 
cipitate lecithalbumin. 

Nothing  characteristic  has  thus  far  been  found  which  differentiates 
this  group  from  others  in  the  quantity  of  amino-acids  split  off  on  hydrol- 
ysis. The  members  of  this  group  differ  essentially  among  themselves, 
e.g.,  vitellin  yields  glycocoll  while  casein  does  not. 

In  order  to  give  a  review  of  the  three  above-mentioned  groups  of  pro- 
teids  we  give  (page  106)  a  tabulation  of  the  amounts  of  the  amino-acids 

1  Ber.  d.  d.  chem.  Gesellsch.,  43  and  Bioch.  Zeitschr.,  26. 

2  Hoppe-Seyler,  Med.  chem.  Untersuch.,  1868;  also  Zeitschr.  f.  physiol.  Chem.,  13, 
479;  Hammarsten,  Skand.  Arch.  f.  Physiol.,  17;  Liebermann,  Pfluger's  Archiv,  50 
and  54. 

3  Compt.  rend.  soc.  biol.,  62. 


106 


THE  PROTEIN  SUBSTANCES. 


obtained  on  cleavage,  but  we  must  bear  in  mind  that  the  figures,  because 
of  the  difficulty  in  the  quantitative  estimation,  are  not  quite  exact,  but 
must  be  considered  as  minimum  values.  As  a  representative  of  the  glob- 
ulin group  we  give  fibrin,  which  is  a  coagulated  globulin;  and  as  repre- 
sentative of  the  phosphoprotein  group,  besides  casein  also  ovovitellin, 
although  not  quite  pure.  The  results  are  based  on  100  parts  of  the 
substances. 

The  proteins  occurring  in  the  plant  kingdom  either  in  the  seeds  or 
tuberes  belong  chiefly  to  the  globulins,  which  correspond  essentially  in 
properties  to  the  animal  globulins.  Besides  these  many  other  less 
abundant  proteins  occur,  which  like  the  albumins  are  soluble  in  water, 
while  toward  certain  salts  they  behave  like  globulins.  It  is  not  clear 
whether  the  phosphorized  plant  proteids  contain  their  phosphorus  as 
impurities  or  whether  they  are  the  same  as  the  animal  phosphoproteins. 
In  seeds  there  occur  also  proteins,  which  are  not  represented  in  the  animal 
kingdom  and  of  these  we  must  especially  mention  the  prolamines.  They 
are  soluble  in  alcohol  and  besides  this  they  are  characterized  by  not 
yielding  any  lysine  on  hydrolysis. 


Lact- 
albumin.1 

Ser- 
albumin.2 

Ov- 
albumin. 

Ser- 
globulins.2 

Fibrin.5 

Casein.7 

Vitelline* 

Glycocoll 

0.0 

2.5 

0.9 

19.4 

1.0 
10.1 

2.4 

0.85 

4.0 

3.0711 

0.0 

2.7 

20.0 

0.6 
3.1 

7.7 

2.53 

3.1 

2.1 

1.04 

0.0 

2.2 

2.5 

10.7 

2.2 

9.1 

0.33 

5.17 

1.77 

3.56 

1.71 
4.91 
3.76 
1.34 

3.5 

2.2 

18.7 

2.5 

8.5 

1.513 

3.8 

2.5 

2.8 

3.0 

3.6 

1.0 

15.0 

0.8 
2.0 
10.4 
1.173 
2.5 
3.5 
3.6 

3.06 
4.06 

0.00 
1.50 
7.20 
9.35 
1.4310 
0.50 
1.39 
15.55 
0.07 
3.20 
4.50 
6.70 
0.23 
1.50 
2.50 
3.81 
5.95 
1.60 

1.1« 

Alanine 

0.759 

Valine 

2.408 

Leucine 

11. 08 

Isoleucine 

Serine 

Aspartic  acid 

Glutamic  acid 

Cystine 

2.139 
12. 959 

Phenylalanine 

Tyrosine 

2.S8 
3.379 

Proline 

4.189 

Oxvproline 

Tryptophane 

Histidine 

1.909 

7.469 

4.819 

Ammonia 

1.259 

1  Abderhalden  and  H.  Pribram,  Zeitschr.  f .  physiol.  Chem.,  21. 

2  Abderhalden,  Lehrb.  d.  physiol.  Chem.,  1909. 
8  K.  Morner,  Zeitschr.  f.  physiol.  Chem.,  34. 

4  Osborne  and  co-workers,  Amer.  Journ.  of  Physiol,  24. 

6  Abderhalden  and  Voitinovici,  Zeitschr.  f.  physiol.  Chem.,  52. 

*  Kutscher,  Endprodukte  der  Trypsin  Verdauung,  Habit.  Schrift.,  Marburg,  1899. 

7  Osborne  and  Guest,  Journ.  of  biol.  Chem.,  9. 

8  Abderhalden  and  Hunter,  Zeitschr.  f.  physiol.  Chem.,  48. 
'Osborne  and  D.  B.  Jones,  Amer.  Journ.  of  Physiol.,  24. 

10  Levene  and  D.  v.  Slyke,  Journ.  of  biol.  Chem.,  6. 

11  Colorimetric  determinations  by  Fasal,  Bioch.,  Zeitschr,  44. 


COAGULATED  PROTEINS. 


107 


In  the  tabulation  of  the  hydrolytic  products  of  plant  proteins  we 
give  edestin,  of  the  hemp-seed,  and  legumin  of  the  pea  as  examples  of 
globulins.  The  other  three,  hordein  of  barley,  gliadin  of  wheat  and  zein 
from  corn  belong  to  the  prolamine  group. 


Edestin.1 

Legumin.4 

Hordein.8 

Gliadin.9 

Zein.10 

Glycocoll 

3.8 

3.6 

5.62 
20.9 

0.33 

4.5 
18. 743 

0.25 

2.4 

2.1 

1.7 

2.0 

0.3812 

1.1 
11.7 

1.0 

0.38 
2.08 
1.08 
8.0 
0.53 
5.3 
13.8 

3.75 
1.55 
3.22 

2.42 

10.12 

4.29 

1.49 

0.0 
0.43 
0.13 
5.67 

43. 197 

5.03 

1.67 

13.73 

1.28 
2.16 
0.00 

4.87 

0.689 

2.0 

3.34 

6.62 

0.13 

0.58 

43.66 
0.45 
2.35 
1.20 

13.22 

1.00 
0.61 
3.16 
0.00 
•       5.22 

0.0 

Alanine 

9.79 

Valine 

1.88 

Leucine 

19.55 

Serine 

1.02 

Aspartic  acid 

Glutamic  acid 

Cystine    

1.71 

26.17 

Phenylalanine 

Tyrosine 

Proline 

6.55 

3.55-10. i11 

9.04 

Oxyproline 

Histidine 

0.82 

Arginine. .  . ; 

1  55 

Lysine 

0  00 

Ammonia 

3  61 

Coagulated  Proteins.  Proteins  may  be  converted  into  the  coagu- 
lated condition  by  different  means:  by  heating,  by  the  action  of  alcohol, 
especially  in  the  presence  of  neutral  salts,  by  chloroform,  ether,  and 
metallic  salts,  and  by  the  prolonged  shaking  of  their  solutions  and  in 
certain  cases,  as  in  the  conversion  of  fibrinogen  into  fibrin  (Chapter  V), 
by  the  action  of  an  enzyme.  The  nature  of  the  processes  which  take 
place  during  coagulation  is  unknown.  The  coagulated  albuminous 
bodies  are  insoluble  in  water,  in  neutral  salt  solutions,  and  dilute  acids 
or  alkalies,  at  normal  temperature.  They  are  dissolved  and  converted 
into  albuminates  by  the  action  of  dilute  acids  or  alkalies,  especially 
on  heating. 

Coagulated  proteins  also  seem  to  occur  in  animal  tissues.  We  find, 
at  least  in  many  organs  such  as  the  liver  and  other  glands,  proteins 


1  Abderhalden,  Zeitschr.  f.  physiol.  Chem.,  37  and  40. 

2  Levene  and  D.  v.  Slyke,  Journ.  of  biol.  Chem.,  6. 

3  Osborne  and  Biddle  Amer.  Journ.  of  Physiol,  26. 

4  Osborne  and  Clapp,  Journ.  of  biol.  Chem.  3. 
8  Abderhalden  and  Babkin,  ibid.,  47. 

6  Osborne  and  Clapp,  Amer.  Journ.  of  Physiol.,  19. 

7  Osborne  and  Jones,  ibid.,  26. 

8  Osborne  and  Guest,  Journ.  of  biol.  Chem.,  9. 

9  Abderhalden  and  Samuely,  Zeitschr.  f.  physiol.  Chem.,  44. 

10  Osborne,  Jones  and  Clapp,  Amer.  Journ.  of  Physiol.,  26. 

11  Kutscher,  Zeitschr.  f.  physiol.  Chem.,  38. 

12  Fasal,  Bioch.  Zeitschr.,  44. 


10S  THE  PROTEIN  SUBSTANCES. 

which  are  not  soluble  in  water,  dilute  salt  solutions,  or  very  dilute  alkalies, 
and  only  dissolve  after  being  modified  by  strong  alkalies. 

Histones  are  basic  proteins  which  stand  to  a  certain  extent  between 
the  strongly  basic  protamines  (see  below)  and  the  true  proteins.  Their 
content  of  nitrogen  varies  between  16.5  and  19.8  per  cent,  and  in  certain 
instances  is  not  higher  than  in  other  proteins,  especially  vegetable  pro- 
teins. According  to  Kossel  and  Kutscher  and  Lawrow  they  are, 
on  the  contrary,  richer  in  basic  nitrogen,  and  especially  yield  more  arginine 
than  other  proteins.  Kossel  first  isolated  a  peculiar  protein  substance 
from  the  red  corpuscles  of  goose  blood  which  was  precipitated  by  ammonia, 
and  because  of  its  similarity  in  certain  regards  to  the  peptones  (in  the 
old  sense)  he  called  it  histone.  At  the  present  time  a  number  of  very 
different  bodies  are  described  as  histones,  such  as  those  obtained  from 
nucleohistone  (Lilienfeld),  from  haemoglobin  (globin  according  to 
Schulz),  from  mackerel  spermatozoa  (scombron  according  to  Bang), 
from  the  codfish  (gadushistone  according  to  Kossel  and  Kutscher), 
from  the  burbot  (lotahistone,  Ehrstrom),  and  from  the  sea-urchin 
(arbacin,  Mathews),  although  probably  not  all  are  true  histones, 
especially  the  above  mentioned  globin.1 

Sulphur  has  been  found  in  those  histones  in  which  it  has  been  tested 
for,  but  they  do  not,  at  least  not  all,  give  the  lead-blackening  test  with 
alkali  and  lead  acetate.  They  give  the  biuret  test,  but  as  a  rule  only 
a  faint  Millon's  reaction.  The  goose-blood  histone  first  studied  by 
Kossel  gives  the  three  following  reactions:  First,  the  neutral  salt- 
free  solution  does  not  coagulate  on  boiling;  second,  gives  a  precipitate 
with  ammonia  which  is  insoluble  in  an  excess  of  the  precipitant;  third, 
gives  a  precipitate  with  nitric  acid  which  disappears  on  heating  and 
reappears  on  cooling. 

The  different  histones  behave  differently  with  these  three  reactions, 
and  hence  they  are  not  specific.  On  the  other  hand,  all  histones  seem 
to  be  precipitated  from  neutral  solution  by  alkaloid  reagents,  and  they 
also  produce  precipitates  in  protein  solutions.  These  two  reactions  are 
likewise  not  specific  for  the  histones,  as  the  protamines  have  a  similar 
behavior.  The  histones  differ  from  the  protamines  by  having  a  much 
lower  content  of  basic  nitrogen,  and  also  probably  by  always  containing 
sulphur.  True  proteins,  as  Osborne's2  edestan,  also  give  these  two 
reactions;   therefore  it  is  impossible  by  qualitative  tests  alone  to  identify 


1  Kossel  Zeitschr.  f.  physiol.  Chem.,  8,  and  Sitzungbers.  der  Gesellsch.  zur  Beford. 
d.  ges  Naturwiss.  zu  Marburg,  1897;  Kossel  and  Kutscher,  ibid.,  1900,  and  Zeitschr. 
f  physiol.  Chem.,  31;  Lawrow,  ibid.,  28,  and  Ber.  d.  d.  chem.  Gesellsch.,  34;  Lilienfeld, 
Zeitschr.  f.  physiol.  Chem.,  18;  Schulz,  ibid.,  24;  Bang,  ibid.,  27;  Ehrstrom,  ibid.,,. 
32;  Mathews,  ibid,  23. 

2  Zeitschr.  f.  physiol.  Chem.,  33. 


HISTONES  AND   PROTAMINES.  109 

a  substance  as  a  histone  with  positivencss.  The  large  content  of  basic 
nitrogen  and  of  arginine  is  not  a  sure  point  of  difference  between  hist  ones 
and  other  bodies.  Histone  yields  little  more  than  40  per  cent  basic 
nitrogen,  while  a  heteroproteose  yields  about  the  same,  namely,  39 
per  cent.  Histone  yields  14-15.5  per  cent  arginine  (gadushistone), 
and  the  lotahistone  only  12  per  cent.  The  vegetable  proteid  excelsin 
is  just  as  rich  in  arginine,  namely,  14.14  per  cent  (Osborne  and  Clapp1). 
The  characteristics  of  the  histones,  according  to  Kossel,  are  the  above- 
given  reactions  and  the  high  amount  of  hexone  bases,  especially  arginine. 
The  arginine  nitrogen  amounts  to  about  25  per  cent  of  the  total  nitrogen, 
the  lysine  N  =  7-8.5  per  cent  and  the  histidine  N  =  1.8-4.5  per  cent.  No 
proteids,  with  the  exception  of  certain  protamines,  are  known  for  the. 
present,  which  contain  as  much  arginine  and  lysine  as  the  histones.  On 
hydrolytic  cleavage  the  histones,  like  other  proteins,  but  unlike  the  pro- 
tamines, yield  a  large  number  of  monamino-acids.  Abderhalden  and 
Rona  2  obtained  from  thymus  histone  the  following:  leucine  11.8, 
alanine  3.46,  glycocoll  0.50,  proline  1.46,  phenylalanine  2.20,  tyrosine 
5.20,  and  glutamic  acid  0.53  per  cent. 

On  pepsin  digestion  the  histones,  according  to  Kossel  and  Pringle  3 
yield  so-called  histone-peptone,  which  also  contains  25  per  cent  of  the 
total  nitrogen  as  arginine  nitrogen.  This  histone-peptone  differs  from 
the  protamines  in  not  giving  a  precipitate  with  proteid  in  neutral  or 
ammoniacal  solution,  but  is  precipitated  in  neutral  reactions  by  sodium 
picrate.     This  property  is  used  in  its  isolation. 

According  to  Kossel  the  histones  are  probably  intermediate  bodies 
between  the  protamines  and  protein  bodies  on  the  demolition  of  the 
latter,  and  if  this  be  true,  then  it  is  not  to  be  expected  that  a  sharp  dif- 
ferentiation exists  between  histone  and  proteid,  and  for  this  reason  it 
is  hardly  possible  for  the  present  to  give  a  precise  definition  for  the 
histones. 

Protamines.  In  close  relation  to  the  proteins  stands  a  group  of 
substances,  the  protamines,  discovered  by  Miescher,  which  are  desig- 
nated by  Kossel  as  the  simplest  proteins  or  as  the  nucleus  of  the  pro- 
tein bodies.  Thus  far  they  have  been  found  only  in  combination  with 
nucleic  acids  in  fish  spermatozoa,4  and  the  investigations  of  Kossel  and 
Weiss  5  have  shown  that  the  material  from  which  the  protamines  are 

1  Amer.  Journ.  of  Physiol,  19  and  23. 

2  Zeitschr.  f.  physiol.  Chem.,  41. 

3  Ibid.,  49. 

4  Nelson,  Arch.  f.  exp.  Path.  u.  Pharm.,  59,  has  recently  shown  that  the  body 
called  by  him  thymamine  and  prepared  from  the  thymus  glands,  is  a  protamine,  still 
he  has  not  given  sufficient  evidence  of  the  protamine  nature  of  the  substance. 

6  Zeitschr.  f.  physiol.  Chem.,  52. 


110  THE  PROTEIN   SUBSTANCES. 

formed,  at  least  in  the  salmon,  is  the  muscle  proteid.  The  question  has 
been  raised  whether  the  protamines  are  true  proteids  or  not,  and  whether 
it  would  not  be  more  correct  to  consider  them  as  cleavage  products  of 
proteid,  or  as  fractions  thereof.  According  to  the  generally  accepted 
view  we  will  treat  them  as  true  proteids. 

Protamine  was  discovered  by  Miescher  *  in  salmon  spermatozoa. 
Later  Kossel  and  his  pupils  isolated  and  studied  similar  bases  from  the 
spermatozoa  of  herring,  sturgeon,  mackerel,  and  other  fishes.  As  all 
these  bases  are  not  identical,  Kossel  uses  the  name  protamines  to  designate 
the  group,  and  calls  the  individual  protamines  according  to  their  origin 
salmine,  clwpeine,  scombrine,  sturine,  cyprinine,  cyclopterine,  crenilabrine 
etc. 

They  differ  materially  from  the  proteins  by  the  fact  that  they  yield 
chiefly  diamino-acids  (always  abundant  arginine)  as  cleavage  products, 
and  only  a  small  amount  of  monamino-acids.  They  are  strongly  basic 
substances  rich  in  nitrogen  (about  30  per  cent  or  more)  and  have  high 
molecular  weight. 

The  percentage  composition  of  these  bodies  has  not  been  satisfactorily 
determined.  As  probable  formulae  we  have  for  salmine  C32H54N18O4 
(Miescher,  Schmideberg,  Nelson),  or  C30H57H17CV (Kossel  and  Goto), 
for  clupeine  C30H62N14C9,  and  for  sturine  C36H69H19O7  (Kossel)  or 
C34H71N17O9  (Goto),  or  according  to  Malenuck  C27H55H13O7  for 
sturine  from  Accipenser  Guldenstadtii.  On  boiling  with  dilute  mineral 
acids  as  also  by  tryptic  digestion,  the  protamines  first  yield  peptone- 
like substances  called  protones,  from  which  simple  products  (amino-acids) 
are  derived  on  further  cleavage.  All  protamines  yield  arginine,  the  four 
protamines  salmine,  clupeine,  cyclopterine,  and  sturine,  yielding  87.4, 
82.2,  62.5,  and  58.2  per  cent  respectively.  In  the  three  protamines  sal- 
mine, clupeine  and  scombrine  the  arginine  nitrogen,  according  to  Kossel 
and  Pringle  2,  amounts  to  about  89  per  cent  of  the  total  nitrogen. 
Sturine  yields  besides  this  the  two  hexone  bases  lysine,  12  per  cent,  and 
histidine,  12.9  per  cent.     Histidine  has  not  been  found  in  any  other  pro- 


1  In  regard  to  protamines,  see  Miescher,  Histochemische  und  Physiologische 
Arbeiten,  Leipzig,  1897;  Piccard,  Ber.  d.  deutsch.  chem.  Gesellsch.,  7;  Schmiedeberg, 
Arch.  f.  exp.  Path.  u.  Pharm.,  37;  Kossel,  Zeitschr.  f.  physiol.  Chem.,  22  (Ueber  die 
basischen  Stoffe  des  Zellkerns),  25,  165  and  190,  26,  40,  44,  and  69;  and  Sitzungsber. 
der  Gesellsch.  zur  Beford.  der  ges.  Naturwiss.  zu  Marburg,  1S97;  Berl.  klin.  Woch- 
enschr.,  1904;  Kossel  and  Mathews,  Zeitschr.  f.  physiol.  Chem.,  23  and  25;  Kossel 
and  Kutscher,  ibid.,  31;  Goto,  ibid.,  37;  Kurajeff,  ibid.,  32;  Morkowin,  ibid.,  28; 
Kossel  and  Dakin,  ibid.,  40,  41,  and  44;  Malenuck,  ibid.,  57;  Pringle,  ibid.,  49;  Ken- 
naway,  ibid.,  72;  Cameron,  ibid.,  76;  F.  Weiss,  59,  60  and  78;  Nelson,  Arch.  f.  exp. 
Path.  u.  Pharm.,  59. 

2  Zeitschr.  f.  physiol.  Chem.,  53. 


PROTAMINES. 


Ill 


tamine.  The  carp  protamine,  cyprinine,  occurs  in  two  different  modi- 
fications, namely,  a-  and  jS-cyprinine.  The  a-cyprinine  yields  only  little 
arginine,  4.9  per  cent,  but  the  lysine  content  is  pronounced,  28.8  per  cent. 
Of  the  total  nitrogen  30.3  per  cent  exists  as  Lysine.  Kossel  and  Dakin 
have  obtained  from  salmine  the  following  cleavage  products,  namely, 
arginine  87.4,  serine  7.8,  aminovaleric  acid  4.3,  and  a-pyrrolidine-car- 
boxylic  (proline)  acid  11  per  cent,  and  according  to  them  the  salmine 
contains  about  10  mol.  arginine,  2  mol.  serine,  1  mol.  aminovaleric  acid, 
and  2  mol.  proline.  Scombrine  contains  only  arginine,  alanine,  and  pro- 
line. According  to  Kossel,  every  protamine  contains  only  2  or  3  mon- 
amino-acids  (clupeine  contains  4)  and  for  every  2  molecules  of  arginine 
cnly  1  molecule  of  monamino-acid  occurs.  The  above-mentioned  pro- 
tones  (of  the  salmine  group)  are  symmetrically  constituted  diarginides 
with  a  monamino-acid — for  example  diarginylserine,  diarginylproline  etc., 
— and  these  diarginides  are  united  together  forming  the  protamine. 
Thus  according  to  Kossel  in  clupeine  we  can  accept  the  presence  of 
diarginylalanine,  diarginylserine,  diarginylproline,  and  diarginylvaline 
(Kossel  and  Pringle). 

The  following  summary  according  to  Kossel  gives  a  view  of  the 
cleavage  products  of  the  protamines  thus  far  investigated: 


Alanine 

Serine 

Valine 

Leucine 

Arainine 

Lysine 

Histidine.  . . . 

Proline 

Tyrosine .  .  .  . 
Tryptophane 


Scom- 
brine. 


Salmine. 


+ 

0 
0 
0 

4- 
0 
0 

+ 

0 

0 


0 

+ 
4- 

0 

+ 

0 
0 

+ 

0 
0 


Clupeine. 

Sturine. 

Cyelop- 
terine. 

a-Cyp- 

rinine. 

0-Cyp- 
rimne. 

+ 

+ 

? 

? 

? 

+ 

0 

? 

? 

? 

+ 

0 

? 

+ 

4- 

0 

+ 

? 

9 

? 

+ 

+ 

+ 

+ 

+ 

0 

4- 

0 

+ 

4- 

0 

+ 

0 

0 

0 

4- 

0 

? 

? 

? 

0 

0 

4- 

6 

6 

0 

0 

+ 

0 

0 

Creni- 
labrine. 


? 
? 
? 
? 

+ 

0 
? 

4- 

0 


Solutions  of  these  bases  in  water  are  alkaline  and  have  the  property 
of  giving  precipitates  with  ammoniacal  solutions  of  proteins  or  primary 
proteoses,  but  the  researches  of  Hunter  l  show  that  these  precipitates 
are  not  histones,  as  generally  considered.  The  salts  with  mineral  acids 
are  soluble  in  water,  but  insoluble  in  alcohol  and  ether.  They  are  more 
or  less  readily  precipitated  by  neutral  salts  (XaCl).  Among  the  salts 
of  the  protamines,  the  sulphate,  picrate,  and  the  double-platinum  chloride 
are  the  most  important,  and  are  used  in  the  preparation  of  the  protamines. 
The  protamines  are,  like  the  proteins,  levogyrate;  but  by  the  action  of 
alkali  the  rotation  is  reduced  or  made  to  disappear,  which  according 


1  Zeitschr.  f.  physiol.  Chem.  53. 


112  THE  PROTEIN  SUBSTANCES. 

to  Kossel  and  Weiss  l  depends  at  least  in  part,  to  a  racemisation  of  the 
hexone  bases,  especially  arginine  within  the  protamine  molecule.  They 
give  the  biuret  test  beautifully,  but  with  the  exception  of  cyclopterine, 
/3-cyprinine  and  crenilabrine  do  not  give  Millon's  reaction.  The  pro- 
tamine salts  are  precipitated  in  neutral  or  even  faintly  alkaline  solutions 
by  phosphotungstic  acid,  picric  acid,  chromic  acid,  and  alkali  ferrocy- 
anides. 

The  protamines  are  prepared,  according  to  Kossel,  by  extracting 
the  heads  of  the  spermatozoa,  which  have  previously  been  extracted 
with  alcohol  and  ether,  with  dilute  sulphuric  acid  (1-2  per  cent),  filtering, 
and  precipitating  with  4  vols,  of  alcohol.  The  sulphate  may  be  purified 
by  repeated  solution  in  water  and  precipitation  with  alcohol,  and  if 
necessary,  conversion  into  the  picrate.  For  more  details  see  the  works 
of  Kossel  and  Malenuck.  The  double-platinum  salt  is  best  suited 
for  analysis  and  can  be  obtained,  according  to  Goto,  by  precipitating 
the  methyl-alcohol  solution  of  the  protamine  hydrochloride  with  plat- 
inum chloride.  Miescher  also  precipitates  the  base  as  a  double-plat- 
inum salt. 

B.    Albuminoids  or  Albumoids. 

Under  this  name  we  collect  into  a  special  group  all  those  protein 
bodies  which  cannot  be  placed  in  either  of  the  other  groups.  Most  and 
best  studied  of  the  bodies  belonging  to  this  group  are  important  con- 
stituents of  the  animal  skeleton  or  the  cutaneous  structure.  Some  are 
hardened  secretions,  and  all  occur  as  a  rule  in  an  insoluble  state  in  the 
organism,  and  they  are  distinguished  in  most  cases  by  a  pronounced 
resistance  to  reagents  which  dissolve  proteins,  or  to  chemical  reagents 
in  general,  and  it  is  due  to  these  external  properties  that  they  are  put 
in  a  special  group.  From  a  purely  chemical  standpoint  there  is  no 
reason  why  they  should  be  separated  from  the  true  proteids  in  a  special 
group.  Most  of  the  bodies  belonging  to  the  albuminoids  have  been 
given  on  page  92. 

The  Keratins.  Keratin  is  the  chief  constituent  of  the  horny  struc- 
ture of  the  epidermis,  of  hair,  wool,  of  the  nails,  hoofs,  horns,  feathers, 
of  tortoise  shell,  etc.,  etc.  Keratin  is  also  found  as  neurokeratin  (Kuhne) 
in  the  brain  and  nerves.  The  shell  membrane  of  the  hen's  egg  seems 
also  to  consist  of  keratin,  and  according  to  Neumeister  2  the  organic 
matrix  of  the  eggshells  of  various  vertebrate  animals  belongs  in  most 
cases  to  the  keratin  group. 

1  Zeitschr.  f.  physiol.  Chem.,  59,  60,  and  78. 

2  Kuhne  and  Ewald,  Verh.  d.  naturhistor.-med.  Vereins  zu  Heidelberg  (N.  F.);  1; 
also  Kuhne  and  Chittenden,  Zeitschr.  f.  Biologie,  26;    Neumeister,  ibid.,  31. 


KERATINS.  113 

It  seems  that  there  exist  a  number  of  keratins,  and  these  form  a  special 
group  of  bodies.  This  fact,  together  with  the  difficulty  in  isolating  the 
keratin  from  the  tissues  in  a  pure  condition  without  a  partial  decom- 
position, is  sufficient  explanation  for  the  variation  in  the  elementary 
composition  given  below.  As  examples  the  analyses  of  a  few  tissues 
rich  in  keratin  and  of  keratins  are  given:1 

c  h  n  s  o 

Human  hair 43.72  6.34  15  06  4.95       29.93  (Rutherford 

and  Hawk) 

Nail 51.00  6.94  17.51  2.80         21.75  (Mulder) 

Neurokeratin..  .  .  56.11-58.45  7.26-8.02  11.46-14.32  1.63-2.24   (Kuhne) 

Neurokeratin 56.61  7.45  14.17  2.27        (Argiris) 

Horn  (average) .  .        50.86  6.94  3.20        (Horbaczewski 

Tortoise  shell 54.89  6.56  16.77  2.22       19.56  (Mulder) 

Shell  membrane. .        49.78  6.64  16.43  4.25       22.50  (Lindvall) 

Egg  membrane .  .        53.92  7.33  15.08  1.44        (Pregl) 

(Scy  Ilium) 


Mohr2  has  determined  the  quantity  of  sulphur  in  various  keratin 
substances.  Sulphur  is  in  great  part  in  loose  combination,  and  it  is 
removed  principally  by  the  action  of  alkalies  (as  sulphides),  or  indeed  in 
part  by  boiling  with  water.  Combs  of  lead  after  long  usage  become  black, 
and  this  is  due  to  the  action  of  the  sulphur  of  the  hair.  On  heating  keratin 
with  water  in  sealed  tubes  to  a  temperature  of  150°  C.  or  higher,  it  dis- 
solves with  the  elimination  of  sulphureted  hydrogen  or  mercaptan 
(Batjer),  and  the  solution  contains  proteose-like  substances  (Kruken- 
berg)  called  atmidkeratin  and  atmidkeratose  by  Bauer.3  Keratin  is 
dissolved  by  alkalies,  especially  on  warming,  producing  besides  alkali 
sulphides  also  proteose  substances. 

Besides  the  well-known  cleavage  products  such  as  leucine,  tyrosine, 
aspartie  acid,  glutamic  acid,  arginine,  and  lysine,  Fischer  and  Dorping- 
haus4  have  found  glycocoll,  alanine,  valine,  proline,  serine,  phenyla- 
lanine, and  pyrrolidone-carboxylic  acid  (secondary  from  glutamic  acid) 
among  the  cleavage  products  of  horn  substances.  Emmerling  claims 
to  have  found  cystine  as  a  sulphurized  cleavage  product,  but  K.  Morner 


1  Rutherford  and  Hawk,  Journ.  of  biol.  Chem.,  3;  Mulder,  Versuch  einer  allgem. 
physiol.  Chem.,  Braunschweig,  1844-51;  Kiihne,  Zeitschr.  f.  Biologie,  26;  Horbaczew- 
ski, see  Drechsel  in  Ladenburg's  Handworterbuch.  d.  Chem.,  3;  Lindvall,  Mary's 
Jahresbericht,  1881;   Argiris,  Zeitschr.  f.  physiol.  Chem.,  54;   Pregl.,  ibid.,  56. 

2  Zeitschr.  f.  physiol.  Chem.,  20. 

3  Krukenberg,  Untersuch.  iiber  d.  chem.  Bau  d.  Eiweisskorper,  Sitzungsber.  d. 
Janaischen  Gesellsch.  f.  Med.  u.  Naturwissensch.,  1886;  Bauer,  Zeitschr.  f.  physiol. 
Chem.,  35. 

4  Zeitschr.  f.  physiol.  Chem.,  36,  which  contains  also  the  older  literature. 


114  THE  PROTEIN  SUBSTANCES. 

was  the  first  to  prove  positively  the  abundant  occurrence  of  cystine  in 
the  cleavage  products.  Morner  obtained  from  ox  horn,  human  hair, 
and  the  shell-membrane  of  the  hen's  egg  6.8,  13.92,  and  7.62  per  cent 
cystine  calculated  on  the  basis  of  the  dry  substance.  Buchtala  l  obtained 
the  following  amounts  of  cystine  from  the  respective  keratin  forma- 
tions, namely,  12.98-14.53  per  cent  from  human  hair,  5.15  per  cent  from 
nails,  7.98  per  cent  from  horsehair,  3.20  per  cent  from  horse  hoofs,  7.27 
per  cent  from  ox  hair,  5.37  per  cent  from  ox  hoofs,  7.22  from  pig  bristles, 
2.17  per  cent  from  pig  hoofs,  6.30  per  cent  from  goose  feathers,  2.14 
per  cent  from  chicken  spurs,  1.88  per  cent  from  the  epidermis  scales  of 
chicken  feet  and  4.7  per  cent  from  elephant  epidermis.  From  the  amount 
of  sulphur  split  off  by  alkali,  Morner  concludes  that,  at  least  in  ox 
horn  and  human  hair,  all  the  sulphur  exists  as  cystine.  Galimard  2 
was  able  to  get  only  a  qualitative  test  for  cystine  in  the  keratin  of  the 
adder  eggs.  Suter,  Morner,  and  Friedmann  3  have  obtained  ct-thio- 
lactic  acid  as  a  hydrolytic  cleavage  product  of  the  keratin  substances. 
The  last-mentioned  investigator  was  also  able  to  detect  thioglycolic  acid 
in  the  cleavage  products  of  wool. 

The  shell  membrane  of  the  hen's  egg,  and  the  eggshells  of  amphibians 
and  certain  fishes  are,  as  above  mentioned,  ordinarily  classified  as  kera- 
tins. These  bodies  among  themselves,  as  well  as  on  comparison  with 
other  keratins,  show  a  marked  difference  in  properties,  this  being  very 
evident  from  the  tabulation  on  page  115. 

The  large  quantity  of  cystine  in  the  keratins  is  considered  as  espe- 
cially characteristic,  and  they  differ  in  this  regard  from^he  other  proteins. 
The  shell  membrane  of  the  hen's  egg  behaves  like  a  keratin  in  regard  to  the 
large  amount  of  cystine  contained,  but  differs  essentially  by  the  absence 
of  tyrosine.  It  is  remarkable  that  the  egg  membrane  of  the  Selachii, 
which  biologically  is  analogous  with  ovokeratin,  differs  from  the  typical 
keratins  by  the  absence  of  cystine,  while  it  contains,  on  the  contrary, 
large  amounts  of  tyrosine.  The  typical  keratins  differ  among  them- 
selves in  regard  to  composition,  thus  the  keratin  from  the  sheep  hoofs 
contains  2  per  cent  phenylalanine,  while  this  ammo-acid  is  absent  in  the 
keratin  of  hair  and  feathers.  It  is  difficult  to  say  whether  or  not  this 
is  due  to  a  difference  in  the  purity  of  the  bodies  or  not.  The  keratins 
investigated  chemically,  thus  far,  do  not  form  a  sufficient  characteristic 
group. 


1  Morner,  ibid.,  34  and  42;  Emmerlinp:,  Ref.  in  Chemiker  Zeitung,  1894;  Buchtala, 
Zeitschr.  f.  physiol.  Chem.,  52,  fii*,  and  78. 

*Chem.  Centralbl,  II,  1905. 

1  Suter,  Zeitschr.  f.  physiol.  Chem.,  20;  Morner.  ibid.,  42;  Friedmann,  Hofmeister's- 
Beitrage,  2. 


KERATINS. 


115 


from 

Ho ' 

hair.1 

Keratin 
from 
Sheep 

Keratin 
from 

Goose 
Feathers1 

Keratin 
from 
Sheep 
H<  rn  ' 

Shell 

MlMII- 

brane 
of  the 
Ben  - 
egg.6 

Mem- 
brane 

lin  m 
alt  liar •  .• 

Tortoise 

• 

( 'ht  lone 

Glycocoll 

4.7 

1.5 

0.9 

7   1 

0.6 

0.3 

3.7 

:  98s 

0.0 

3.2 

3.4 

O.til3 

4.45* 

1.123 

0.58 

1     10 

2.80 
11.5 

0.1 

2.3 
12.9 

7.3 

2 . 9 

4.4 

2.6 
1.8 

ii  g 
8.0 
0.4 
1.1 
2.3 

0.0 
3.6 

3.5 

0.45 
1 .6 
4.5 

15.3 
1.1 
2.5 

17.2 
7.5 
1.9 
3.6 
3.7 

2.7 

0.2 

3.9 

3  5 

1.1 

7    1 

1.1 
8.1 
7.62' 

0.0 

4  0 

2.6 
3.2 

5.8 

2.3 

7.2 

■; 

3.3 

10.6 
4.4 
1   7 
3.2 
3.7 

l.i  36 

.Alanine               

5  ■•:; 

Leucine    

:;  26 

Serine.                  





Glutamic  acid  10 



(  \>t ine              

5.19 

Phenylalanine 

Tyrosine       

1     (is 

13  59 

Proline 

Histidine  



Argininc 

Lysine 

— 

Bodies  occur  in  the  animal  kingdom  which  form  to  a  certain  extent 
intermediate  substances  between  coagulated  protein  and  keratin.  C. 
Th.  Morner  u  has  detected  such  a  body  (albumoid)  in  the  tracheal  car- 
tilage which  forms  a  net-like  trabecular  tissue.  This  substance  appears 
to  be  related  to  the  keratins  on  account  of  its  solubilities  and  the  quan- 
tity of  the  sulphur  (lead-blackening)  it  contains,  while  according  to  its 
solubility  in  gastric  juice  it  must  stand  close  to  the  proteins.  Another 
substance,  nearly  like  keratin,  is  the  horny  layer  in  the  gizzard  of  birds. 
According  to  J.  Hedenius  this  substance  is  insoluble  in  gastric  or  pan- 
creatic juice,  and  acts  quite  like  keratin.  According  to  K.  B.  Hofmann- 
and  Pregl,12  who  call  this  substance  koilin,  it  does  not  yield  any  cystine 
on  hydrolysis,  or  at  least  not  a  determinable  quantity.  According  to 
others  the  quantity  of  cystine  is  very  small.     Buchtala  13  obtained  only 


1  Abderhalden  and  Wells,  Zeitschr.  f .  physiol.  Chem.,  46. 

2  Buchtala,  ibid.,  52. 
*  Argiris,  ibid.,  54. 

4  Abderhalden  and  Voitinoviei,  ibid.,  52. 

5  Abderhalden  and  Le  Count,  ibid.,  46. 

6  Abderhalden  and  Ebstein,  ibid  ,  48. 

7  Korner,  ibid.,  34  and  42. 

8  Pregl,  ibid.,  56. 

8  Buchtala,  ibid.,  74. 

10  Abderhalden  and  Fuchs,  Zeitschr.  f.  physiol.  Chem.,  57,  have  shown  that  the 
same  variety  of  keratin,  on  ageing  of  the  horn  structure,  becomes  somewhat  poorer 
in  glutamic  acid. 

11  See  Mary's  Jahresber.,  18. 

12  Hedenius,  Skand.  Arch.  f.  Physiol.,  3;   Hofmann  and  Pregl,  Zeitschr.  f.  physiol 
Chem.,  52. 

13  Zeitschr.  f.  physiol.  Chem.,  69. 


11C  THE  PROTEIN  SUBSTANCES. 

a  little  more  than  0.5  per  cent  pure  crystalline  cystine  and  on  account 
of  the  low  cystine  content  as  well  as  for  other  reasons  the  koilin  differs 
from  the  keratins. 

Keratin  is  amorphous  or  takes  the  form  of  the  tissues  from  which 
it  was  prepared.  It  is  insoluble  in  water,  alcohol,  or  ether.  On  heating 
with  water  to  150-200°  C.  it  dissolves.  It  also  dissolves  gradually  in 
caustic  alkalies,  especially  on  heating.  It  is  not  dissolved  by  artificial 
gastric  juice  or  by  trypsin  solutions.  Keratin  gives  the  xanthoproteic 
reaction,  as  well  as  the  reaction  with  Millon's  reagent,  although  the 
latter  is  not  always  typical. 

In  the  preparation  of  keratin  a  finely  divided  horny  structure  is 
treated  first  with  boiling  water,  then  consecutively  with  diluted  acid, 
pepsin-hydrochloric  acid,  and  alkaline  trypsin  solution,  and,  lastly,  with 
water,  alcohol,  and  ether. 

Elastin  occurs  in  the  connective  tissue  of  higher  animals,  sometimes 
in  such  large  quantities  that  it  forms  a  special  tissue.  It  occurs  most 
abundantly  in  the  cervical  ligament  (ligamentum  nuchae). 

Elastin  used  to  be  generally  considered  as  a  sulphur-free  substance. 
According  to  the  investigations  of  Chittenden  and  Hart,  it  is  a  question 
whether  or  not  elastin  contains  sulphur,  as  it  may  have  been  removed  by 
the  action  of  the  alkali  in  its  preparation.  H.  Schwarz  has  been  able 
by  another  method,  to  prepare  an  elastin  containing  sulphur,  from  the 
aorta,  and  this  sulphur  can  be  removed  by  the  action  of  alkalies,  without 
changing  the  properties  of  the  elastin;  and  Zoja,  Hedin,  Bergh, 
and  Richards  and  Gies  }  have  found  that  elastin  contains  sulphur.  The 
most  trustworthy  analyses  of  elastin  from  the  cervical  ligament  (Nos. 
1  and  2)  and  from  the  aorta  (No.  3)  have  given  the  following  results, 
which  compare  well  with  each  other: 

s  o 

....  21.94  (Horbaczewski  2) 

....  21.79  (Chittenden  and  Hart) 

0.38  (H.  Schwarz) 

Zoja  found  0.276  per  cent  sulphur  and  16.96  per  cent  nitrogen  in 
elastin.  Hedin  and  Bergh  found  different  quantities  of  nitrogen  in 
aorta-elastin,  depending  upon  whether  Horbaczewski's  or  Schwarz's 
method  was  used  in  its  preparation.  In  the  first  case  they  found  15.44 
per  cent  nitrogen  and  0.55  per  cent  sulphur,  and  in  the  other  14.67  per 

1  Chittenden  and  Hart,  Zeitechr.  f.  Biologie,'  25;  Schwarz,  Zeitschr.  f.  physiol. 
Chem.,  18;  Zoja,  ibid.,  2'.l;  Bergh,  ibid.,  25;  Hedin,  ibid.;  Richards  and  Gies,  Amer. 
Journ.  of  Physiol.,  7. 

2  Zeitschr.  f.  physiol.  Chem.,  6. 


C 

H 

N 

1. 

54.32 

6.99 

16.75 

2. 

54.24 

7.27 

16.70 

3. 

53.95 

7.03 

16.67 

ELASTIN.  117 

cent  nitrogen  and  0.66  per  cent  sulphur.  Richards  and  Gies  found 
0.14  per  cent  sulphur  and  16.87  per  cent  nitrogen  in  (last in.  The  ques- 
tion whether  elastin  is  a  unit  body  still  remains  open. 

The  quantity  of  hydroh  tic  cleavage  products  are  given  in  the  table 
on  page  125.  It  is  sufficient  to  here  call  attention  to  the  fact  that  no 
aspartic  acid  and  only  very  little  glutamic  acid  have  been  found.  The 
hexone  bases  have  been  obtained,  but  only  in  very  small  amounts,  so 
that  the  basic  nitrogen  represents  only  3.34  per  cent  of  the  total  nitro- 
gen (Richards  and  Gies).  From  an  elastin  proteose,  Wechsler  l 
obtained  1.86  per  cent  arginine,  0.5  per  cent,  histidine  and  2.48  per  cent 
lysine. 

Indol  and  skatol  have  not  been  found  on  the  putrefaction  of  elastin,2 
but  Schwarz,  on  the  contrary,  obtained  indol,  skatol,  benzene,  and 
phenols  on  fusing  aorta-elastin  with  caustic  potash.  On  heating  with 
water  in  closed  vessels,  on  boiling  with  dilute  acids,  or  by  the  action  of 
proteolytic  enzymes,  the  elastin  dissolves  and  splits  into  two  chief  prod- 
ucts, called  by  Horbaczewski  hemielastin  and  elastinpeptone.  Accord- 
ing to  Chittenden  and  Hart,  these  products  correspond  to  two  proteoses 
designated  by  them  protoelastose  and  deuteroelastose.  The  first  is  soluble 
in  cold  water  and  separates  out  on  heating,  and  its  solution  is  precipi- 
tated by  mineral  acid  as  well  as  by  acetic  acid  and  potassium  ferrocyanide. 
The  aqueous  solution  of  the  other  does  not  become  cloudy  on  heating, 
and  is  not  precipitated  by  the  above-mentioned  reagents. 

Pure  elastin  when  dry  is  a  yellowish-white  powder;  in  the  moist 
state  it  appears  like  yellowish-white  threads  or  membranes.  It  is  insol- 
uble in  water,  alcohol,  or  ether,  and  shows  a  resistance  toward  the  action 
of  chemical  reagents.  It  is  not  dissolved  by  strong  caustic  alkalies  at 
the  ordinary  temperature  and  only  slowly  at  the  boiling  temperature. 
It  is  very  slowly  attacked  by  cold  concentrated  sulphuric  acid,  but  it 
is  relatively  easily  dissolved  on  warming  with  strong  nitric  acid.  Elastins 
of  different  origin  act  differently  with  cold  concentrated  hydrochloric 
acid;  for  instance,  elastin  from  the  aorta  dissolves  readily  therein,  while 
elastin  from  the  ligamentum  nucha?,  at  least  from  old  animals,  dissolves 
with  difficulty.  Elastin  is  more  readily  dissolved  by  warm  concen- 
trated hydrochloric  acid.  It  responds  to  the  xanthoproteic  reaction, 
and  to  that  with  Millon's  reagent,  but  not  to  the  Adamkiewicz- 
Hopkins  reaction. 

On  account  of  its  great  resistance  to  chemical  reagents,  elastin  may 
be  prepared  (best  from  the  ligamentum  nuchae)  in  the  fallowing  way: 
First  boil  with  water,  then  with  1  per  cent  caustic  potash,  then  again 

1  Zeitschr.  f.  physiol.  Chem.,  67. 

2  See  Walchli,  Journ.  f.  prSkt.  Chem.  (N.  F.),  17. 


118  THE   PROTEIN  SUBSTANCES. 

with  water,  and  lastly  with  acetic  acid.  The  residue  is  treated  with  cold 
5  per  cent  hydrochloric  acid  for  twenty-four  hours,  carefully  washed 
with  water,  boiled  again  with  water,  and  then  treated  with  alcohol  and 
ether. 

In  regard  to  the  methods  used  by  Scrwarz  and  by  Richards  and  Gies,  which 
are  somewhat  different,  we  refer  to  the  original  publications. 

Collagen,  or  gelatin-forming  substance,  occurs  very  extensively  in 
vertebrates.  The  flesh  of  cephalopods  is  also  said  to  contain  collagen.1 
Collagen  is  the  chief  constituent  of  the  fibrils  of  the  connective  tissue  and 
(as  ossein)  of  the  organic  substances  of  the  bony  structure.  It  also  occurs 
in  the  cartilaginous  tissues  as  chief  constituent;  but  it  is  here  mixed 
with  other  substances,  producing  what  was  formerly  called  chondrigen. 
Collagen  from  different  tissues  has  not  quite  the  same  composition,  and 
probably  there  are  several  varieties  of  collagen. 

By  continued  boiling  with  wrater  (more  easily  in  the  presence  of  a 
little  acid)  collagen  is  converted  into  gelatin.  Hofmeister2  found  that 
gelatin  on  being  heated  to  130°  C.  is  again  transformed  into  collagen; 
and  this  last  may  be  considered  as  the  anhydride  of  gelatin.  Collagen 
and  gelatin  have  about  the  same  composition.3 


Collagen -50 .  75 

Gelatin  (commercial) ....  49.38 

Gelatin  from  tendons..  .  .  50.11 

Gelatin  from  ligaments. .  .  50.49 

Fish  glue  (isinglass) 48.69 

Gelatins  of  different  origin  show  a  somewhat  variable  composition, 
which  seems  to  indicate  the  occurrence  of  different  collagens.  It  is  diffi- 
cult to  say  whether  the  variable  content  of  sulphur  is  due  to  a  contami- 
nation with  a  substance  rich  in  sulphur  or  to  a  splitting  off  of  loosely 
combined  sulphur  during  the  purification.  C.  Morner4  has  prepared 
a  typical  gelatin  containing  only  0.2  per  cent  of  sulphur  by  a  method 
which  eliminated  any  possible  changes  due  to  reagents. 

Sadikoff  5  has  prepared  gelatins  by  various  methods  from  tendons  and 
from  cartilage.  Those  from  tendons,  some  of  which  were  prepared  after  pre- 
vious tryptic  digestion,  some  after  treatment  with  0.25  per  cent  caustic  potash, 
and  some  after  treatment  with  sodium  hydroxide  and  then  carbonate,  showed 


6.47 

17.86 

24.92           (Hofmeister) 

6.80 

17.97 

0.70    25.13     (Chittenden) 

6.56 

17.81 

0.26     25.26     (van  Name) 

6.71 

17.90 

0 .  57     24 .  33     (Richards  and  Gies) 

6.76 

17.68 

—        —        (Faust) 

1  Hoppe-Seyler,  Physiol.  Chem.,  p.  97. 

2  Zeitschr.  f.  physiol.  Cheni.,  2. 

a  Hofmeister,  1.  c;  Chittenden  and  Solley,  Journ.  of  Physiol.,  12;  van  Name, 
Journ.  of  Exper.  Med.,  2;  Richards  and  Gies,  Amer.  Journ.  of  Physiol.,  8;  Faust, 
Arch.  f.  exp.  Path.  u.  Pharm.,  41. 

4  Zdtechr.  f.  physiol.  Chem.,  28. 

f  Tbid.,  89  and  41. 


COLLAGENS.  119 

somewhat  differenl  physical  properties  among  each  other,  hut  had  about  the  same 
elementary   composition,  with  0.34  <!.;">:!  per  cent  sulphur.     Sadikoff  stems  to 

think  that  the  gelatins  prepared  up  to  this  time  were  perhaps  not  unit  bodies 
but  were  possibly  mixtures.  The  bodies  prepared  by  Sadikoff  from  cartilage 
he  calls  glutting,  because  they  were  essentially  different  from  the  other  gelatins 
or  glutins.  They  were  poorer  in  carbon  and  nitrogen,  17.17  to  17. s7  per  cent, 
but  somewhat  richer  in  sulphur,  0.53-0.718  per  cent,  than  the  tendon  glutin. 
The  gluteins  differ  also  from  the  glutins  in  thai  on  boiling  with  a  mineral  acid 
they  have  a  faint  reducing  action,  and  also  in  that  they  give  a  color  reaction 
with  phloroglucin-hydrochloric  acid  which  is  probably  due  to  contamination.  The 
glutins  differ  from  the  gluteins  by  a  different  behavior  with  certain  salts. 

The  decomposition  products  of  the  collagens  are  the  sum"  as  those  of 
the  gelatins  ami  will  be  found  in  the  table  on  page  1?5.  ( >f  special 
mention  is  the  fact  that  gelatin  contains  no  tyrosine  and  tryptophane 
but  does  yield  considerable  glycocoll.  This  latter  substance  has,  because 
of  its  sweet  taste,  been  called  gelatin  sugar.  Skraup  l  has  obtained  on 
the  hydrolytic  cleavage  of  gelatin  a  crystalline  acid  having  the  formula 
C12H25N5O10,  which  he  calls  glutinic  acid.  Gelatin  yields  considerable 
basic  nitrogen,  according  to  Hausmann,2  35.83  per  cent  of  the  total 
nitrogen.  It  also  yields  considerable  arginine  (9.3  per  cent),  lysine  5-G 
per  cent,  but  only  little  histidine  (0.4  per  cent).  The  aromatic  group  in 
g  latin  is  therefore,  as  directly  shown  by  Fischer  and  also  by  Spiro,3 
represented  by  phenylalanine. 

Collagen  is  insoluble  in  wrater,  salt  solutions,  and  dilute  acids  and 
alkalies,  but  it  swells  up  in  dilute  acids.  By  continued  boiling  with 
water  it  is  converted  into  gelatin.  Various  collagens  are  converted  into 
gelatin  with  varying  readiness;  the  formation  of  gelatin  occurs  also 
from  difficultly  soluble  collagens  by  continuous  boiling  with  water. 
Collagen  is  dissolved  by  the  gastric  juice  and  also  by  the  pancreatic 
juice  ( trypsin  solution)  when  it  has  previously  been  treated  with  acid 
or  heated  with  water  above  70°  C.4  By  the  action  of  ferrous  sulphate 
corrosive  sublimate,  or  tannic  acid,  collagen  shrinks  greatly.  Collagen 
treated  by  these  bodies  does  not  putrefy,  and  tannic  acid  is  therefore  of 
great  importance  in  the  preparation  of  leather. 

Gelatin  or  glutin  is  colorless,  amorphous,  and  transparent  in  thin 
layers.  It  swells  in  cold  water  without  dissolving.  It  dissolves  in  warm 
water,  forming  a  sticky  liquid,  which  solidifies  on  cooling  when  sufficiently 
concentrated.  As  Pauli  and  Rona  5  have  shown,  various  bodies  may 
have   a   different    influence    upon   the   gelatinization-point    of   a   gelatin 

1  Monatshefte  f.  Chem.,  26. 

2  Zeitsehr.  f.  physiol.  Chem.,  27. 

3  Fischer,  Levene  and  Aders,  Zeitschr.  f.  physiol.  Chem.,  35;  Spiro,  Hofmeister's 
Beitrafre,  1. 

4  Kiihne  and  Ewald,  Verh.  d.  Xaturhist.  Med.  Vereins  in  Heidelberg,  1877,  1. 

5  Hofmeister's  Beitrage,  2. 


120  THE  PROTEIN  SUBSTANCES. 

solution;  thus  certain  substances  such  as  sulphates,  citrates,  acetates, 
and  glycerin  may  accelerate,  while  the  chlorides,  chlorates,  bromides, 
alcohol,  and  urea  retard,  this  power. 

Gelatin  solutions  are  not  precipitated  on  boiling,  or  by  mineral 
acids,  acetic  acid,  alum,  basic  lead  acetate,  or  metallic  salts  in  general.  A 
gelatin  solution  acidified  wjth  acetic  acid  may  be  precipitated  by  potas- 
sium ferrocyanide  on  carefully  adding  the  reagent.  Gelatin  solutions 
are  precipitated  by  tannic  acid  in  the  presence  of  salt,  and  according  to 
Trunkel  l  completely  if  the  gelatin  and  tannic  acid  are  in»  the  propor- 
tion 1 : 0.7.  According  to  him  the  precipitation  is  not  due  to  a  chemical 
combination  but  to  an  adsorption  phenomenon.  Solutions  of  gelatin 
in  water  are  also  precipitated  by  acetic  acid  and  common  salt  in  sub- 
stance; mercuric  chloride  in  the  presence  of  HC1  and  NaCl;  by  meta- 
phosphoric  acid  and  phosphomolybdic  acid  in  the  presence  of  acid; 
and  lastly  also  by  alcohol,  especially  when  neutral  salts  are  present. 
Gelatin  solutions  do  not  diffuse.  Gelatin  gives  the  biuret  reaction, 
but  not  Adamkiewicz-Hopkins  reaction.  It  gives  Millon's  reaction 
and  the  xanthoproteic  reaction  so  faintly  that  they  probably  occur  from 
impurities  consisting  of  proteids.  According  to  C.  Morner,  pure  gelatin 
gives  a  beautiful  Millon's  reaction,  if  not  too  much  reagent  is  added. 
In  the  other  case  no  reaction  or  only  a  faint  one  is  obtained. 

By  continued  boiling  with  water  gelatin  is  converted  into  a  non- 
gelatinizing  modification  called  /3-glutin  by  Nasse.  According  to  Nasse 
and  Kruger  the  specific  rotatory  power  is  hereby  reduced  from  — 167.5° 
to  about— 136°.2  According  to  Trunkel,  who  has  especially  studied 
the  rotation  behavior  of  gelatin,  the  rotation  of  /3-glutin  is  less  than  the 
ordinary  a-glutin.  On  prolonged  boiling  with  water,  especially  in  the 
presence  of  dilute  acids,  also  in  the  gastric  or  tryptic  digestion,  the  gelatin 
is  transformed  into  gelatin  proteoses,  so-called  gelatoses  and  gelatin 
peptones,  which  diffuse  more  or  less  readily. 

According  to  Hofmeister  two  new  substances,  semiglutin  and  hemicollin, 
are  formed.  The  former  is  insoluble  in  alcohol  of  70-80  per  cent  and  is  precipitated 
by  platinum  chloride.  The  latter,  which  is  not  precipitated  by  platinum  chloride, 
is  soluble  in  alcohol.  Chittenden  and  Solley  3  have  obtained  in  the  peptic 
and  tryptic  digestion  a  proto-  and  a  deutero-gelatose,  besides  a  true  peptone.  The 
elementary  composition  of  these  gelatoses  does  not  essentially  differ  from  that  of 
the  gelatin. 

Paal  *  has  prepared  gelatin-peptone  hydrochlorides  from  gelatin  by  the 
action  of  dilute  hydrochloric  acid.     These  salts  are  partly  soluble  in  ethyl  and 

1  Bioch.  Zeitschr.,  26. 

2  Nasse  and  Krtiger,  Mary's  Jahresber.,  19,  p.  29.  In  regard  to  the  rotation  of 
/3-glutin.  see  Framm,  Printer's  Arch.,  68;    Trunkel,  1.  c. 

3  Hofmeister,  1.  c;  Chittenden  and  Solley,  1.  c. 
1  Her.  d.  deutsch.  chern.  Gesellsch.,  25. 


RETICUUN.  121 

Diethyl  alcohol,  and  partly  insoluble  1  herein.  The  peptones  obtained  from 
these  salts  contain  less  carbon  and  more  hydrogen  than  the  gelatin  from  which 
they  originated,  showing  that  hydration  has  taken  place.  The  molecular  weight 
of  the  gelatin  peptone  as  determined  by  PaAL,  by  RaOULT's  cryoseopic  method, 
was  200  to  352,  while  that  for  gelatin  was  878  to  960.  The  gelatin  peptones 
isolated  by  Siegfried  and  his  pupils  which  will  be  discussed  below,  are  of  great 
interest. 

Collagen  (contaminated  with  mucoid)  may  be  obtained  from  bones  by 
extracting  them  with  hydrochloric  acid  (which  dissolves  the  earthy 
phosphates)  and  then  carefully  washing  the  acid  out  with  water.  It 
may  be  obtained  from  tendons  by  extracting  with  lime-water  or  dilute 
alkali  (which  dissolve  the  proteids  and  mucin),  and  then  thoroughly 
washing  with  water.  Gelatin  is  obtained  by  boiling  collagen  with  water. 
The  finest  commercial  gelatin  always  contains  a  little  proteid,  which 
may  be  removed  by  allowing  the  finely  divided  gelatin  to  swell  up  in 
water  and  thoroughly  extracting  with  large  quantities  of  fresh  water. 
Then  dissolve  in  warm  water  and  precipitate  with  alcohol. 

Collagen  may  also  be  purified  from  proteids,  as  suggested  by  van 
Name,  by  digesting  with  an  alkaline  trypsin  solution  or  by  extracting 
the  gelatin  for  many  days  with  1-5  p.  m.  caustic  potash,  as  suggested 
by  C.  Morner.  The  typical  properties  of  gelatin  are  not  changed  by 
this. 

Chondrin  or  cartilage  gelatin  is  only  a  mixture  of  gelatin  with  the  specific 
constituents  of  the  cartilage  and  their  transformation  products. 

Reticulin.  The  reticular  tissues  of  the  lymphatic  glands  contain  a 
variety  of  fibers  which  have  also  been  found,  by  Mall  in  the  spleen,  intes- 
tinal mucosa,  liver,  kidneys,  and  lungs.  These  fibers  consist  of  a  special 
substance,  reticulin,  investigated  by  Siegfried.1 

Reticulin  has  the  following  composition:  C  52.88;  H  6.97;  N  15.63; 
S  1.88;  P  0.34;  ash  2.27  per  cent.  The  phosphorus  occurs  in  organic 
combination.  It  yields  no  tyrosine  on  cleavage  with  hydrochloric  acid. 
It  yields,  on  the  contrary,  sulphureted  hydrogen,  ammonia,  lysine, 
arginine,  and  valine.  On  continued  boiling  with  water,  or  more  readily 
with  dilute  alkalies,  reticulin  is  converted  into  a  body  which  is  precipitated 
by  acetic  acid,  and  at  the  same  time  phosphorus  is  split  off. 

Reticulin  is  insoluble  in  water,  alcohol,  ether,  lime-water,  sodium 
carbonate,  and  dilute  mineral  acids.  It  is  dissolved,  after  several  weeks, 
on  standing  with  caustic  soda  at  the  ordinary  temperature.  Pepsin- 
hydrochloric  acid  or  trypsin  does  not  dissolve  it.  Reticulin  responds 
to  the  biuret,  xanthoproteic,  and  Adamkiewicz-Hopkins  reactions, 
but  not  to  Millon's  reagent. 


1  Mall,  Abhandl.  d.  math.-phys.  Klasse  d.  Kgl.  sachs.  Gesellsch.  d.  Wiss.,  1891; 
Siegfried,  Ueber  die  chem.  Eigensch.  der  retikulirten  Gewebe,  Habil.-Schrift,  Leipzig, 
1892. 


122 


THE  PBOTEIN  SUBSTANCES. 


According  to  Tebb  reticulin  is  only  a  somewhat  changed,  impure  collagen 
but  this  is  disputed  by  Siegfried.  » 

It  may  be  prepared  as  follows,  according  to  Siegfried:  Digest  intes- 
tinal mucosa  with  trypsin  and  alkali.  Wash  the  residue,  extract  with 
ether,  and  digest  again  with  trypsin  and  then  treat  with  alcohol  and  ether. 
On  careful  boiling  with  water  the  collagen  present  either  as  contamina- 
tion or  as  a  combination,  with  recticulin  is  removed.  The  thoroughly 
boiled  residue  consists  of  reticulin. 

Ichthylepidin  is  an  organic  compound,  so-called  by  C.  Morner,2  which  occurs 
with  collagen  in  fish-scales  and  forms  about  one-fifth  of  their  organic  substance. 
This  compound,  with  15.9  per  cent  nitrogen  and  1.1  per  cent  sulphur,  stands  on 
account  of  its  properties  rather  close  to  elastin.  .  It  is  insoluble  in  cold  and  hot 
water,  as  well  as  in  dilute  acids  and  alkalies  at  the  ordinary  temperature.  On 
boiling  with  these  it  dissolves.  Pepsin-hydrochloric  acid,  as  well  as  an  alkaline 
trypsin  solution,  also  dissolves  it.  It  responds  beautifully  to  Millon's  reagent, 
the  xanthoproteic  reaction,  and  the  biuret  test.  At  least  a  part  of  the  sulphur 
is  split  off  «by  the  action  of  alkali.  Ichthylepidin  stands  very  close  to  elastin 
in  regard  to  its  solubilities;  but  it  differs  essentially  in  composition  as  it  is  markedly 
poorer  in  glycocoll,  but  much  richer  in  proline  and  glutamic  acid  (Abderhalden 
and  Voitinovici  3). 


As  skeletins,  Krukenberg  4  has  designated  a  number  of  nitrogenized 
substances  which  form  the  skeletal  tissue  of  various  classes  of  inverte- 
brates. These  substances  are  chitin,  spongin,  conchiolin,  byssus,  cornein, 
and  crude  silk  {fibroin  and  sericin).  Of  these,  chitin  does  not  belong  to 
the  protein  substances,  and  silk  is  hardly  to  be  classed  as  a  skeletin. 
Only  those  so-called  skeletins  will  be  discussed  that  actually  belong  to 
the  protein  group,  and  chitin  will  be  discussed  in  another  chapter. 

The  elementary  composition  of  certain  of  the  bodies  belonging  to 
this  group  is  as  follows :  5 


C             H  N 

Conchiolin  (from  the  shells  of  pinna) ..  .  52.70  G.54  1G.G0 

Spongin 46.50  G.30  16.20 

48.75  6.35  16.40 

Cornein 48.96  5.90  16.81 

Fibroin 48.23  6.27  18.31 

"       48.30  6.50  19.20 

Sericin 44.32  6.18  18.30 

44.50  0.32  17.14 


s 

85 
50 


(Wetzel) 

(Crookewitt) 

(Posselt) 

(Krukenberg) 

(Cramer) 

(Vignon) 

(Cramer) 

(Bondi) 


1  Tebb,  Journ.  of  Physiol.,  27;  Siegfried,  ibid.,  28. 

2  Zeitschr.  f.  physiol.  Chem.,  24  and  37.     See  also  Green  and  Tower,  ibid.,  35. 
'  Zeitschr.  f.  physiol.  Chem.,  52,  p.  368. 

4  Grundziige  einer  vergl.  Physiol,  d.  thier.  Geriistsubst.  Heidelberg,  1885. 

■•  Krukenberg,  Her.  d.  d.  chem.  Gesellsch.,  17  and  IS,  and  Zeitschr.  f.  Biologie,  22; 
Croockewitt,  Annal.  d.  Chem.  u.  Pharrn.,  48;  Posselt,  ibid.,  45;  Cramer,  Journ.  f. 
prakt.  Chem.,  96;  Vignon,  Compt.  rend.,  115;  Wetzel,  Zeitschr.  f.  physiol.  Chem.,  29 
and  CentralU.  f.  Physiol.,  13,  113;  Bondi,  Zeitschr.  f.  physiol.  Chem.,  34. 


CORNEIN.  123 

Spongin  forms  the  chief  muss  of  the  ordinary  sponge.  It  dissolves  with 
difficulty  in  concentrated  mineral  acids  but  dissolves  with  readiness  in  caustic 
alkalies.  It  does  not  give  the  MlLLON  reaction  or  An aukiewicz's.  It  gives 
no  gelatin.  On  hydrolysis  spongin  yields  considerable  glycocoll  13.9  per  cent, 
glutamic  acid  l.S.l  per  cent,  leucine  7.f>  per  cent,  proline  6.3  per  cent,  lysine 
3-4   per   cent,    and    arginine   5  6    per   cent.1     Tyrosine   and    phenylalanine    could 

not  be  detected.    After  Hundeshagen   bad  shown   the  occurrence   of  iodine 

and  bromine  in  organic  combination  in  different  sponges  and  designated  the  albu- 
moid  containing  iodine,  iodospongin,  IIaknack  -  later  isolated  from  the  ordinary 
sponge,  by  cleavage  with  mineral  acids,  an  iodospongin  which  contained  about 
II  per  cent  iodine  and  4.5  per  cent  sulphur.  Strauss3  has  obtained  sponginoses 
of  various  kinds  from  spongin  by  dilute  acids.  The  heterosponginose  contained 
the  greater  part  of  the  iodine  and  sulphur,  while  the  deuterosponginose  contained 
the  carbohydrate  groups.  Iodospongin  is  considered  as  a  derivative  of  the 
heterosponginose.  Conchiolin  is  found  in  the  shells  of  mussels  and  snails  and 
also  in  the  eggshells  of  these  animals.  It  yields,  according  to  Wetzel,4  glycocoll, 
leucine,  and  abundance  of  tyrosine.  The  quantity  of  diamino-nitrogen  amounts 
to  8.7  per  cent  and  the  amide  nitrogen  3.47  per  cent  (from  the  shell  of  pinna). 
The  Byssus  contains  a  substance,  closely  related  to  conchiolin,  which  is  soluble 
with  difficulty.  According  to  Abderhalden  5  it  yields  considerable  glycocoll 
and  tyrosine  and  also  alanine,  aspartic  acid  and  very  large  amounts  of  proline. 

Cornein  is  the  name  given  to  the  substance  of  the  axial  system  of 
certain  Anthozoa.  The  substance  occurring  in  the  groups  of  Gorgonia 
and  Antipathes  has  been  called  gorgonin  by  C.  Morner  6  and  differs  from 
the  pen  not  id  in  of  the  Pennatulidea?  by  the  latter  being  readily  soluble 
in  pepsin-hydrochloric  acid.  The  cleavage  products  have  not  been  care- 
fully studied;  one  of  the  crystalline  products,  called  cornicrijstalline  by 
Krikenberg,  is  nothing  but  iodine  crystals,  as  shown  by  Morner. 
After  Drechsel  7  found  nearly  8  per  cent  iodine  in  the  dry  substance  of 
the  axial  system  of  the  Gorgonia  Cavolini,  C.  Morner  showed  that  in 
the  Anthozoa  in  general  the  organic  skeletal  substance  contains  halogens 
in  organic  combination.  Iodine  was  found  in  all  varieties,  and  indeed 
in  amounts  from  traces  up  to  7  per  cent.  Eromine  wras  found,  with  the 
exception  of  two  Antipathes,  in  amounts  of  0.25  to  4  per  cent,  while 
chlorine,  which  was  never  absent,  occurred  as  a  few  tenths  per  cent. 
The  halogens  occur  in  the  organic  skeletal  substance  as  gorgonin  and 
pcnnatulin. 

Drechsel  obtained  leucine,  tyrosine,  lysine,  ammonia  and  an  iodized 
amino-acid,  iodogorgonic  acid,  as  cleavage  products  of  gorgonin.     This  last 

'Abderhalden  and  Strauss,  Zeitschr.  f.  physiol.  Chem.,  48;  Kossel  and  Kutscher, 
end.,  :$!,  20.-). 

2  Zeitschr.  f.  physiol.  Chem.,  24;  Hundeshagen,  Maly's  Jahresber.,  25,  394;  see 
also  L.  Scott,  Biochem.  Zeitschr.,  1. 

'  Biochem.  Centralbl.,  3. 

4  Zeitschr.  f.  physiol.  Chem.  29,  and  Centralbl.  f.  Physiol.,  13,  113. 

5  Zeitschr.  f.  physiol.  Chem.,  55. 

6  Zeitschr.  f.  physiol.  Chem.,  51  and  55. 

7  Zeitschr.  f .  Biol.,  33. 


124  THE  PROTEIN  SUBSTANCES. 

is  identical  with  3-5  di-iodo-tyrosine,  HOI2C6H2.CH2.CHNH2COOH, 
synthetically  prepared  by  Wheeler  and  Jamieson.1  On  acid  cleavage 
of  gorgonin,  Henze  2  obtained  the  three  hexone  bases,  abundant  tyrosine 
and 'very  little  leucine.  On  cleavage  with  barium  hydroxide  he  obtained 
only  lysine,  besides  tyrosine  and  glycocoll  in  larger  amounts. 

Fibroin  and  sericin  are  the  two  chief  constituents  of  raw  silk.  By 
the  action  of  boiling  water  the  sericin  (silk  gelatin)  dissolves  and  can  be 
obtained  by  a  method  suggested  by  Bondi,3  while  the  more  difficultly 
soluble  fibroin  remains  undissolved  in  the  shape  of  the  original  fiber. 
The  sericin,  whose  sufficiently  concentrated  hot  solution  gelatinizes  on 
cooling,  is  precipitated  by  mineral  acids,  several  metallic  salts,  and  by 
acetic  acid  and  potassium  ferrocyanide.  The  spider  silk  investigated 
by  Fischer4  yielded  fibroin  but  not  sericin. 

Abderhalden  and  his  collaborators5  have  investigated  a  great 
number  of  varieties  of  silk  and  found  sericin  in  varying  amounts  (15  to 
28  per  cent).  The  composition  of  the  various  kinds  of  silk  is  char- 
acterized, especially,  by  a  varying  amount  of  glycocoll  and  in  this  regard 
we  can  differentiate  between  two  chief  groups.  The  one  group  is,  like 
the  Italian  silk,  very  rich  in  glycocoll  while  the  other  group,  like  the 
Tussah  silk,  contains  a  much  smaller  quantity  of  glycocoll. 

Sericin,  whose  proper  concentrated  warm  solution  gelatinizes  on 
cooling,  is  precipitated  by  mineral  acids  and  several  metallic  salts  and 
by  acetic  acid  and  potassium  ferrocyanide.  In  regard  to  the  products 
of  hydrolysis  it  differs  very  essentially  from  fibroin  by  being  much  poorer 
in  glycocoll,  alanine  and  tyrosine. 

Fibroin  is  soluble  in  concentrated  acids  and  alkalies  and  reprecipitable 
(in  a  modified  form)  on  neutralization.  It  gives  the  biuret  test  and 
Millon's  and  Adamkiewicz-Hopkiih's  reactions,  the  last  but  faintly. 
Fibroin  has  an  especially  great  interest  because  of  the  hydrolyses  per- 
formed by  Fischer  and  his  co-workers,  and  especially  by  the  finding  of 
the  previously  mentioned  polypeptides  by  these  workers.  Of  the  cleavage 
products  which  characterize  fibroin  we  must  mention  the  large  amount 
of  glycocoll,  alanine  and  tyrosine,  and  the  very  small  amounts  of  hexone 
bases,  besides  the  almost  complete  absence  of  monamino-dicarboxylic 
acids.  The  quantity  of  the  hydrolytic  cleavage  products  of  the  three 
silk  substances,  in  so  far  as  they  have  been  investigated,  are  given  in  the 
following  table,  which  also  includes  the  results  for  elastin,  gelatin,  and 


1  Wheeler  and  Jamieson,  Amer.  Chem.  Journ.,  £3;  Wheeler,  ibid.,  38. 

2  Henze,  Zeitechr.  f.  physio).  Chem.,  38  and  51. 
'  Zeitschr.  f.  physiol  Chem.,  34. 

*  Ibid.,  58. 

8  See  Zeitschr.  f.  physiol.  Chem.,  59,  61,  62,  64,  71,  74,  80. 


ALBUMINATES 


125 


koilin.     The  fibroin  A  came  from  ordinary  silk;   fibroin  B  and  the  sericin 

originated  from  Indian  Tussah  .silk. 


Glycocoll 

Alanine 

Valine 

Leucine 

Serine 

Aspartic  acid . 
Glutamic  acid. 

Cystine 

Phenylalanine 

Tyrosine 

Proline 

Oxyproline.  .  . 

Histidine 

Arginine 

Lysine 


Elastin.1 

Gelatin." 

Koilin.' 

Fibroin   A1 

Fibroin  B* 

Sericin.7 

25.75 

19.25 

1.2 

36.0 

9  5 

1  5 

6.6 
1.0 

21.1 

3.0 

5.8 

21.0 

24  o 

9 .  S 

9.23 

13.2 

15 

1.5 

4.8 

— 

0.4 

— 

1.6 

2  0 

5.4 

— 

1.23 

2.3 

— 

•_'  s 

0.8 

16. 83 

5.2 

— 

10 

1.8 

— 

— 

0.710 

— 

— 

— 

3.9 

1.03 

2  3 

1.5 

0.6 

0.3 

0.34 

— 

5.4 

10.5 

9.2 

10 

1.7 

7.7 
6.4 
0.4 

5.5 

— 

1.0 

3.0 



0.035 





— . 

0.3 

9.3 

3.60s 

1.0 

— 

— 

— 

5.6 

1.645 

■ — ■ 

— 

— 

35.13 

23 . 4 

1.76 
11  70 


S    20 

3.68 


5.249 


C.    Cleavage  Products  of  Simple  Proteins. 

On  the  hydrolysis  of  proteins  by  the  aid  of  acids,  alkalies  or  by 
enzymes,  cleavage  products  are  obtained  which  represent  various  inter- 
mediary steps  between  the  native  proteins  on  one  side  and  the  simple 
cleavage  products,  the  amino-acids,  on  the  other  side.  Among  these 
products  we  have  for  a  long  time  known  two  chief  groups  which  still 
retain,  to  a  high  degree,  their  protein  character,  namely,  the  albuminates 
and  the  proteoses  (and  peptones). 

1.    Albuminates. 

Alkali  and  Acid  Albuminates.  The  native  proteins  are  modified 
by  the  action  of  sufficiently  strong  acids  or  alkalies.  By  the  action  of 
alkalies  all  native  albuminous  bodies  are  converted,  with  the  elimina- 
tion of  nitrogen,  or  by  the  action  of  stronger  alkali,  with  the  extraction 
of  sulphur  also,  into  a  new  modification,  called  alkali  albuminate.  If 
caustic  alkali  in  substance  or  in  strong  solution  be  allowed  to  act  on  a 


1  Cited  from  Abderhalden's  Lehrbuch  d.  physiol.  Chem.,  1909. 

2  Cohnheim,  Chemie  d.  Eiweisskorper  3  d.  Aufl. 
z  Skraup  and  Biehler,  Monatsh.  f.  Chem.,  30. 

4  K.  B.  Hoffmann  and  Pergl.  Zeitschr.  f.  physiol.  Chem.,  52. 
6  v.  Knaffl-Lenz,  ibid.,  52. 

6  Abderhalden  and  Spack,  ibid.,  62. 

7  Strauch,  ibid.,  71. 
8E.  Fischer,  ibid.,  53. 

8  Calculated  as  arginine. 

10  This  figure  is  somewhat  uncertain. 


126  THE  PROTEIN  SUBSTANCES. 

concentrated  protekl  solution,  such  as  blood-serum  or  egg-albumin,  the 
alkali  albuminate  may  be  obtained  as  a  solid  jelly  which  dissolves  in 
water  on  heating,  and  which  is  called  "  Lieberkuhn's  solid  alkali 
albuminate."  By  the  action  of  dilute  caustic  alkali  solutions  on  dilute 
proteid  solutions  we  have  alkali  albuminates  formed  slowly  at  the  ordinary 
temperature,  but  more  rapidly  on  heating.  These  solutions  may  vary 
with  the  nature  of  the  proteid  acted  upon,  and  also  with  the  intensity 
of  the  action  of  the  alkali,  but  still  they  have  certain  reactions  in  common. 

If  proteid  is  dissolved  in  an  excess  of  concentrated  hydrochloric  acid, 
or  if  we  digest  a  proteid  solution  acidified  with  1-2  p.  m.  hydrochloric 
acid  in  the  thermostat,  or  digest  the  proteid  for  a  short  time  with 
pepsin-hydrochloric  acid,  we  obtain  new  modifications  of  proteid  which 
may  show  somewhat  varying  properties,  but  have  certain  reactions  in 
common.  These  modifications,  which  may  be  obtained  in  a  solid  gelat- 
inous condition  on  sufficient  concentration,  are  called  acid  albuminates 
or  acid  albumins,  and  sometimes  syntonin,  though  we  perfer  to  apply 
the  term  syntonin  to  the  acid  albuminate,  which  is  obtained  by  extract- 
ing muscles  with  hydrochloric  acid  of  1  p.  m. 

The  alkali  and  acid  albuminates  have  the  following  reactions  in 
common:  They  are  almost  insoluble  in  water  and  dilute  common-salt 
solution  (see  page  104),  but  they  dissolve  readily  in  water  on  the  addi- 
tion of  a  very  small  quantity  of  acid  or  alkali.  Such  a  solution  as  nearly 
neutral  as  possible  does  not  coagulate  on  boiling  but  is  precipitated  at 
the  normal  temperature  on  neutralizing  the  solvent  by  an  alkali  or  an 
acid.  A  solution  of  an  alkali  or  acid  albuminate  in  acid  is  easily  pre- 
cipitated on  saturating  with  NaCl,  but  a  solution  in  alkali  is  precipitated 
with  difficulty  or  not  at  all,  according  to  the  amount  of  alkali  it  contains. 
Mineral  acids  in  excess  precipitate  solutions  of  acid  as  well  as  alkali 
albuminates.  The  nearly  neutral  solutions  of  these  bodies  are  also  pre- 
cipitated by  many  metallic  salts. 

Notwithstanding  this  agreement  in  the  reactions,  the  acid  and  alkali 
albuminates  are  essentially  different,  for  by  dissolving  an  alkali  albumi- 
nate in  some  acid  no  acid,  albuminate  solution  is  obtained,  nor  is  an  alkali 
albuminate  formed  on  dissolving  an  acid  albuminate  in  water  by  the 
aid  of  a  little  alkali.  In  the  first  case  we  obtain  a  combination  of  the 
alkali  albuminate  and  the  acid,  soluble  in  water,  and  in  the  other  case  a 
soluble  combination  of  the  acid  albuminate  with  the  alkali  added.  The 
chemical  process  in  the  modification  of  proteids  with  an  acid  is  essentially 
different  from  the  modification  with  an  alkali,  hence  the  products  are 
of  a  different  kind.  The  alkali  albuminates  are  relatively  strong  acids. 
They  may  be  dissolved  in  water  with  the  aid  of  CaC03,  with  the  elimina- 
tion of  ( '<>_>.  which  floes  not  occur  with  typical  acid  albuminates,  and 
they  show  in  opposition  to  the  acid  albuminates  also  other  variations 


PROTEOSES  AND  PEPTONES.  127 

which  stand  in  connection  with  their  strongly  marked  acid  nature.  Dilute 
solutions  of  alkalies  act  more  energetically  on  proteida  than  do  acids 
of  corresponding  concentration.  In  the  first  case  a  part  of  the  nitro- 
gen and  often  also  the  sulphur,  is  split  off,  and  from  this  property  we  may 
obtain  an  alkali  albuminate  by  the  action  of  an  alkali  upon  an  acid  albu- 
minate; but  we  cannot  obtain  an  acid  albuminate  by  the  reverse  reac- 
tion (K.  Mokner  l).  This  does  not  exclude  the  possibility  that,  by 
a  more  severe  acid  treatment,  products  can  be  obtained  which  perhaps 
correspond  to  those  products  obtained  by  a  more  mild  alkali  treatment. 

The  preparation  of  the  albuminates  has  been  given  above.  The 
corresponding  albuminate  obtained  by  the  action  cf  alkalies  or  acids 
upon  a  proteid  solution  may  be  precipitated  by  neutralizing  with  acid 
or  alkali.  The  washed  precipitate  is  dissolved  in  water  by  the  aid  of  a 
little  alkali  or  acid,  and  again  precipitated  by  neutralizing  the  solvent. 
If  this  precipitate,  which  has  been  washed  in  water,  is  treated  with 
alcohol  and  ether,  the  albuminate  will  be  obtained  in  a  pure  form. 

In  the  preparation  of  acid  as  well  as  of  alkali  albuminates,  proteoses  and  the 
closely  related  albuminates  are  formed.  The  "  alkali  albumose  "  obtained  by 
Maajb  2  belongs  to  this  class.  The  lysalbinic  acid  and  protalbinic  acid  obtained 
by  Paal  '  from  ovalbumin  are  likewise  alkali  albuminates.  These  have  been 
can  tally  studied  by  Skraup  and  his  co-workers.4 

Desaminocdbtiminic  acid  is  an  alkali  albuminate  which  Schmiedeberg  5  obtained 
by  the  action  of  such  weak  alkali  that  a  part  of  the  nitrogen  was  evolved  but  the 
quantity  of  sulphur  remained  the  same.  The  proteid  combination  obtained  by 
Blum'  by  the  action  of  formol  on  proteid  and  called  by  him  protogen,  has  similarities 
with  the  alkali  albuminates  in  regard  to  solubilities  and  precipitation,  but  is  not 
identical  therewith. 

2.   Proteoses  and  Peptones. 

Peptones  were  formerly  designated  as  the  final  products  of  the  decom- 
position of  protein  bodies  by  means  of  proteolytic  enzymes  in  so  far  as 
these  final  products  are  still  true  proteins,  while  the  intermediate  prod- 
ucts produced  in  the  peptonization  of  proteins,  in  so  far  as  they  are 
not  substances  similar  to  albuminates,  were  designated  as  proteoses 
(albumoses,  or  propeptones) .  Proteoses  and  peptones  may  also  be 
produced  by  the  hydrolytic  decomposition  of    the  proteins    with  acids 

1  Pfluger's  Arch.,  17. 

2  Zeitschr.  f.  physiol.  Chem.,  30. 

3  Ber.  d.  d.  chem.  Gesellsch.,  35. 

4  Hummelberger,  Lampel  and  Woeber,  Monatsh.  f.  Chem.,  30. 
6  Arch.  f.  exp.  Path.  u.  Pharm.,  39. 

6  Blum,  Zeitschr.  f.  physiol.  Chem.,  22.  The  older  investigations  of  Loew  may 
be  found  iu  Maly's  Jahresber.,  18S8.  On  the  action  of  formaldehyde  see  also  Benedi- 
centi,  Arch.  f.  (Anat.  u.)  Physiol.,  1897;  S.  Schwarz,  Zeitschr,  f.  physiol.  Chem.,  30; 
Bliss  and  Xovy,  Journ.  of  Exper.  Med.,  4. 


128  THE  PROTEIN  SUBSTANCES. 

or  alkalies,  and  by  the  putrefaction  of  the  same.  They  may  also  be 
formed  in  very  small  quantities,  as  by-products,  in  the  investigations 
of  animal  fluids  and  tissues,  and  the  question  as  to  the  extent  to  which 
these  exist  preformed  under  physiological  conditions  requires  very 
careful  investigation. 

Between  the  peptone,  which  represents  the  final  cleavage  product, 
and  the  proteose,  which  stands  closest  to  the  original  protein,  we  have 
undoubtedly  a  series  of  intermediate  products.  Under  such  circum- 
stances it  is  a  difficult  problem  to  try  to  draw  a  sharp  line  between  the 
peptone  and  the  proteose  group,  and  it  is  just  as  difficult  to  define  our 
conception  of  peptones  and  proteoses  in  an  exact  and  satisfactory  manner. 

In  the  past  we  used  to  consider  the  peptones  as  the  end  products 
in  the  hydrolysis,  they  still  being  true  proteins,  but  we  must  call  atten- 
tion to  the  fact  that  since  that  time  we  have  learned  of  polypeptide- 
like  cleavage  products  of  the  proteins,  and  also  that  polypeptides  have 
been  prepared  synthetically.  With  this  in  mind  it  is  not  possible  to 
say  what  we  understand  by  the  conception  true  proteid,  and  also  that 
possibly  there  exists  a  large  number  of  intermediary  steps  between  the 
original  modified  proteid  and  the  simplest  cleavage  products.  There 
is  no  doubt  that  those  bodies  which  have  been  called  proteoses  and 
peptones  are  chiefly  mixtures;  and  the  question  has  been  proposed  by 
Abderhalden  l  whether  it  is  not  best  to  drop  the  conception  of  pro- 
teoses and  to  call  all  products  precipitable  by  ammonium  sulphate,  etc., 
and  previously  described  as  proteoses,  peptones. 

Although  there  is  much  in  favor  cf  such  a  proposition,  still  on  account 
of  the  great  importance  which  the  conception  of  the  proteoses  has  gen- 
erally received,  it  is  probably  too  early  to  drop  the  question  of  proteoses 
entirely  from  a  text-book,  and  we  will  therefore,  as  in  the  past  editions, 
discuss  the  historical  development  of  the  proteoses  and  peptones  in 
the  ordinary  sense. 

The  proteoses  (or  albumoses)  used  to  be  considered  as  those  protein 
bodies  whose  neutral  or  faintly  acid  solutions  do  not  coagulate  on  boil- 
ing and  which,  to  distinguish  them  from  peptones,  were  characterized 
chiefly  by  the  following  properties:  The  watery  solutions  are  precipitated 
at  the  ordinary  temperature  by  nitric  acid,  as  well  as  by  acetic  acid  and 
potassium  ferrocyanide,  and  this  precipitate  has  the  peculiarity  of  dis- 
appearing on  heating  and  reappearing  on  cooling.  If  a  proteose  solu- 
tion is  saturated  with  NaCl  in  substance,  the  proteose  is  partly  pre- 
cipitated in  neutral  solutions,  but  on  the  addition  of  acid  saturated 
with  salt  it  is  more  completely  precipitated.  This  precipitate,  which 
dissolves  on  warming,  is  a  combination  of  the  proteose  with  the  acid. 

1  Oppenheirner's  Handb.  der  Biochern.,  Bd.  1,  1908. 


PROTEOSES  AND  PEPTONES.  129 

We  formerly  designated  as  peptones  those  protein  bodies  which  are 
readily  soluble  in  water  and  which  are  not  coagulated  by  heat,  whose 
solutions  are  precipitated  neither  by  nitric  acid,  nor  by  acetic  acid  and 
potassium  ferrocyanide,  nor  by  NaCl  and  acid. 

The  reactions  and  properties  which  the  proteoses  and  peptones  have 
in  common  were  formerly  considered  as  the  following:  They  all 
give  the  color  reactions  of  .the  proteins,  but  with  the  biuret  test  they  give 
a  more  beautiful  red  color  than  the  ordinary  proteins.  They  are  pre- 
cipitated by  ammoniacal  lead  acetate,  by  mercuric  chloride,  tannic,  phos- 
photungstic,  and  phosphomolybdic  acids,  by  potassium-mercuric  iodide 
and  hydrochloric  acid,  and  also  by  picric  acid.  They  are  precipitated 
but  not  coagulated  by  alcohol,  that  is,  the  precipitate  obtained  is  soluble 
in  water  even  after  being  in  contact  with  alcohol  for  a  long  time.  The 
proteoses  and  peptones  also  have  a  greater  diffusive  power  than  native 
proteins,  and  the  diffusive  power  is  greater  the  nearer  the  questionable 
substance  stands  to  the  final  product,  the  now  so-called  true  peptone. 

These  old  views  have  gradually  undergone  an  essential  change.  After 
Heynsius'  *  observation  that  ammonium  sulphate  was  a  general  pre- 
cipitant for  proteins,  and  for  peptones  in  the  old  sense,  Kuhne  and  his 
pupils  2  proposed  this  salt  as  a  means  of  separating  proteoses  and  pep- 
tones. Those  products  of  digestion  which  separate  on  saturating  their 
solution  with  ammonium  sulphate,  or  can  indeed  be  salted  out  at  all, 
are  considered,  by  Kuhne  and  also  by  most  of  the  modern  investigators, 
as  proteoses,  while  those  which  remain  in  solution  are  called  peptones 
or  true  peptones.  These  true  peptones  are  formed  in  relatively  large 
amounts  in  pancreatic  digestion,  while  in  pepsin  digestion  they  are  formed 
only  in  small  quantities  or  after  prolonged  digestion. 

According  to  Schutzenberger  and  Kuhne3  the  proteins  yielded 
two  chief  groups  of  new  protein  bodies  wdien  decomposed  by  dilute 
mineral  acids  or  writh  proteolytic  enzymes;  of  these  the  anti  group  shows 
a  greater  resistance  to  further  action  of  the  acid  and  enzyme  than  the 
other  namely,  the  hemi  group.  These  two  groups  are,  according  to 
KtiHNE,  united  in  the  different  proteoses,  even  though  in  various  relative 
amounts,  and  each  proteose  contains  the  anti  as  well  as  the  hemi  group. 
The  same  is  true  for  the  peptone  obtained  in  pepsin  digestion,  hence  he 
calls  it  amphopeptone.     In  tryptic  digestion  a  cleavage  of  the  ampho- 

1  Pfluger's  Archiv,  34. 

2  See  Kuhne,  Yerhandl.  d.  naturhistor.  Vereins  zu  Heidelberg  (N.  F.),  3;  J.  Wenz, 
Zeitschr.  f.  Biologie,  22;  Kuhne  and  Chittenden,  Zeitschr.  f.  Biologie,  22;  R.  Neu- 
meister,  ibid.,  23;  Kuhne,  ibid.,  29. 

3  Schutzenberger,  Bull,  de  la  Soc.  chimique  de  Paris,  23;  Kuhne,  Yerhandl.  d. 
naturhist.  Vereins  zu  Heidelberg  (N.  F.),  1,  and  Kuhne  and  Chittenden,  Zeitschr.  f. 
Biologie,  19.     See  also  Paal,  Ber.  d.  deutsch.  chem.  Gesellsch.,  27. 


130  THE  PROTEIN  SUBSTANCES. 

peptone  takes  place  into  aniipeptone  and  hemipcptone.  Of  these  two 
peptones  the  hemipeptone  is  further  split  into  amino-acids  and  other 
bodies  while  the  antipeptone  is  not  attacked.  By  the  sufficiently 
energetic  action  of  trypsin  only  one  peptone  remains  to  the  last — the  so- 
called  antipeptone. 

Kuhne  and  his  pupils,  who  have  conducted  extensive  investiga- 
tions on  the  proteoses  and  peptones,  classify  the  various  proteoses  accord- 
ing to  their  different  solubilities  and  precipitation  properties.  In  the 
pepsin  digestion  of  fibrin  l  they  obtained  the  following  proteoses :  (a) 
Heteroproteose,  insoluble  in  water  but  soluble  in  dilute  salt  solution; 
(6)  Protoproleose,  soluble  in  salt  solution  and  water.  These  two  pro- 
teoses are  precipitated  by  NaCl  in  neutral  solutions,  but  not  completely. 
Heteroproteose  may,  by  being  in  contact  with  water  for  a  long  time 
or  by  drying,  be  converted  into  a  modification,  called  (c)  Dysproteose, 
which  is  insoluble  in  dilute  salt  solutions,  (d)  Deuteroproteose  is  a  pro- 
teose which  is  soluble  in  water  and  dilute  salt  solution  and  which  is 
incompletely  precipitated  from  acid  solution  by  saturating  with  NaCl, 
and  is  not  precipitated  from  neutral  solutions. 

The  proteoses  obtained  from  different  protein  bodies  do  not  seem  to  be 
identical,  but  differ  in  their  behavior  to  precipitants.  Special  names  have  been 
given  to  these  various  proteoses  according  to  the  mother-protein,  namely,  albu- 
moses,  globidoses,  vitelloses,  caseoses,  myosinoses,  elastoses,  etc.  These  various 
proteoses  are  further  distinguished,  as  proto-,  hetero-,  and  deuterocaseose,  for 
example.  Chittenden  2  has  suggested  the  common  name  proteoses  for  the  prod- 
ucts formed  intermediary  between  the  proteins  and  peptones  in  the  digestion  of 
animal  and  vegetable  proteins.  We  have  made  use  of  it  in  this  sense  in  pref- 
erence to  the  word  albumose  (which  is  used  in  the  German  and  by  some  other 
writers),  but  which  will  be  used  in  this  book  as  indicating  the  intermediary 
products  in  the  hydrolysis  of  albumins  and  not  as  a  general  term.  Certain 
proteoses  have  also  been  obtained  in  a  crystalline  state  (Schrotter)  . 

Neumeister3  designates  as  atmidalbumose  that  body  which  is  obtained  by 
the  action  of  superheated  steam  on  fibrin.  At  the  same  time  he  also  obtained  a 
substance  called  atmidalbumin,  which  stands  between  the  albuminates  and  the 
proteoses. 

Of  the  soluble  proteoses  Neumeister  designates  the  protoproteose 
and  heteroproteose  as  primary  proteoses,  while  the  deuteroproteoses, 
which  are  closely  allied  to  the  peptones,  he  calls  secondary  proteoses.  As 
essential   differences   between  the  primary  and   secondary  proteoses  he 

'See  Kuhne  and  Chittenden,  Zeitschr.  f.  Biologie,  20. 

*  Kuhne  ami  Chittenden,  Zeitschr.  f.  Biologie,  22  and  26;  Neumeister,  ibid.,  23; 
Chittenden  and  Hartwell,  Journ.  of  Physiol.,  11  and  12;  Chittenden  and  Painter, 
Studies  from  the  Laboratory,  etc.,  Yale  University,  2,  New  Haven,  1887;  (l.ittenden, 
ibid.,  .'5:  Sebelien,  Chem.  Centralblatt,  1890;  Chittenden  and  Goodwin,  Journ.  of 
Physiol..  12. 

3  Zeitschr.  f.  Biologie,  26.  See  also  Chittenden  and  Meara,  Journ.  of  Physiol., 
15,  and  Salkowski,  Zeitschr.  f.  Biologie,  34  and  37. 


PROTEOSES  .WD  PEPTONES.  131 

BUggeste  the  following.1  The  primary  proteoses  are  precipitated  by 
nitric  arid  in  salt-free  solutions,  while  the  secondary  proteoses  arc  pre- 
cipitated only  in  salt  solutions,  and  certain  deuteroproteoses,  such  as 
deuterovitellose  and  deuteramyosinose,  are  precipitated  by  nitric  acid 
only  in  solutions  saturated  with  Na(  '1.  The  primary  proteoses  arc  pre- 
cipitated from  neutral  solutions  by  copper-sulphate  solution  (2:100), 
and  by  NaCl  in  substance,  while  the  secondary  proteoses  are  not.  The 
primary  proteoses  are  completely  precipitated  from  a  solution  saturated 
with  NaCl  by  the  addition  of  acetic  acid  saturated  with  salt,  while  the 
secondary  proteoses  arc  only  partly  precipitated.  The  primary  proteoses 
are  readily  precipitated  by  acetic  acid  and  potassium  ferrocyanide,  while 
the  secondary  are  only  incompletely  precipitated  after  some  time.  The 
primary  proteoses  are  also,  according  to  Pick,2  completely  precipitated 
by  ammonium  sulphate  (added  to  one-half  saturation),  while  the  second- 
ary proteoses  remain  in  solution. 

The  true  peptones,  as  they  were  formerly  considered  to  be,  are  exceed- 
ingly hygroscopic,  and  if  perfectly  dry,  sizzle  like  phosphoric  anhydride 
when  treated  with  a  little  water.  They  are  exceedingly  soluble  in  water, 
diffuse  more  readily  than  the  proteoses,  and  are  not  precipitated  by 
ammonium  sulphate.  In  contradistinction  to  the  proteoses,  the  true 
peptones  are  not  precipitated  by  nitric  acid  (even  in  solutions  saturated 
with  salt),  by  sodium  chloride  and  acetic  acid  saturated  with  salt, 
potassium  ferrocyanide  and  acetic  acid,  picric  acid,  trichloracetic 
acid,  potassium-mercuric  iodide,  and  hydrochloric  acid.  They  are 
precipitated  by  phosphotungstic  acid,  phosphomclybdic  acid,  corrosive 
sublimate  (in  the  absence  cf  neutral  salts),  absolute  alcohol,  and  tannic 
acid,  but  the  precipitate  may  redissolve  on  the  additicn  of  an  excess  of 
the  precipitant.  As  an  important  difference  between  amphopeptone  and 
antipeptone  we  must  also  mention  that  the  former  gives  Millon's 
reaction,  while  the  antipeptone  does  not. 

In  regard  to  the  precipitation  by  alcohol  we  must  call  attention  to  the  observa- 
tions of  Frank  el  that  not  only  are  the  acid  combinations  of  peptone  (Paal) 
soluble  in  alcohol,  but  also  the  free  peptone,  and  Frankel  has  even  suggested  a 
method  of  preparation  based  on  this  behavior.  Schrotter  3  has  also  prepared 
crystalline  proteoses  which  were  soluble  in  hot  alcohol,  especially  methyl  alcohol. 

The  views  on  the  hydrolytic  cleavage  products  of  peptic  and  tryptic 
digestion  which  were  accepted  until  a  few  years  ago  have  recently  been 
considerably  modified  in  several  points. 


1  Xeumeister,  Zeitschr.  f.  Biologic,  24  and  26. 

2  Zeitschr.  f.  physiol.  Chem.,  24. 

3  Frankel,   Zur  Kenntnis  der  Zerfallsprodukte  des  Eiweisses  bei  peptischer  und 
tryptiecher  Verdauung,  Wien,  1896;  Schrotter,  Monatshefte  f.  Chem.,  14  and  16. 


132  THE  PROTEIN  SUBSTANCES. 

The  older  view  that  in  peptic  digestion  only  proteoses  and  peptones, 
but  no  simpler  cleavage  products,  are  formed,  has  been  shown  not  to  be 
true.  The  works  of  Zunz,  Pfaundler,  Salaskin,  Lawrow,  Lang- 
stein,1  and  others  have  shown  that  by  very  lengthy  digestion  amino- 
acids  may  in  part  be  formed  and  also  other  products  such  as  oxyphenyl- 
ethylamine,  tetra-  and  pentamethylenediamine.  The  biuret  reaction 
does  not  disappear  and  the  above  mentioned  products  seem  to  be  formed 
only  under  special  conditions.  In  ordinary,  not  too  lengthy  pepsin, 
digestion,  it  is  generally  admitted  that  no  amino  acids  are  formed  but 
only  proteoses  and  peptones. 

In  connection  with  the  above-mentioned  experimental  results  it  must  be 
remarked  that  not  all  the  products  found,  for  example,  the  oxyphenylethylamine 
and  the  diamines,  are  produced  by  the  action  of  pepsin,  but  rather  by  the  action  of 
other  enzymes.  In  certain  cases,  undoubtedly,  impure  pepsin  was  used,  or  indeed 
autodigestion  of  the  stomach  was  carried  on,  and  the  action  of  other  enzymes 
was  not  excluded.  In  other  cases  the  digestion  with  pepsin  and  considerable 
acid  (even  1  per  cent  H2S04)  was  continued  for  a  very  long  time,  indeed  for  an 
entire  year,  without  controlling  the  influence  of  the  acid  alone  upon  the  proteoses. 

Kuhne's  view  that  in  tryptic  digestion  (pancreatic  digestion)  a 
peptone,  so-called  antipeptone,  always  remains  which  cannot  be  further 
split  is  not  strictly  true.  By  sufficiently  long  autodigestion  of  the  pan- 
creas, Kutscher2  was  able  to  obtain,  as  final  products,  a  mixture  of 
digestion  products  which  failed  to  respond  to  the  biuret  test,  and  the  same 
results  have  been  obtained  by  others.  An  antipeptone  in  the  old  sense, 
i.e.,  a  digestion  product  which  is  resistant  to  tryptic  digestion  but  which 
still  gives  the  biuret  test,  is  without  question  not  always  obtained  as  end 
product  in  trypsin  digestion.  On  the  contrary  as  Fischer  and  Abder- 
halden3  have  shown,  polypeptide-like  bodies  are  produced  in  tryp- 
tic digestion  (and  the  same  is  probably  true  also  for  peptic  digestion) 
which  do  not  give  the  biuret  test,  i.e.,  "  abiuret  "  products,  and  which 
are  resistant  to  further  tryptic  digestion  but  yield  amino-acids  on 
hydrolysis  with  acids.  This  behavior  stands  in  close  relation  to  the 
observation  that  in  tryptic  digestion  certain  amino-acids,  such  for  example, 
as  tyrosine,  tryptophane  and  leucine  are  split  off  earlier  and  more  readily 
than  the  others  of  the  protein  molecule. 

Antipeptone,  which  is  only  attacked  with  great  difficulty  by  trypsin 
has  in  fact  been  isolated  by  Siegfried   (see  below)   and  although  the 

'  Zunz,  Zeitschr.  f.  physiol.  Chem.,  28,  and  Hofmeister's  Beitrage,  2;  Pfaundler, 
Zeitschr.  f.  physiol.  Chem.,  30;  Salaskin,  ibid.,  32;  Salaskin  and  Kowalewsky,  ibid., 
38;  Lawrow,  ibid.,  33;  Langstein,  Hofmeister's  Beitrage,  1  and  2. 

2  Zeitschr.  f.  physiol.  Chem.,  25,  26,  28,  and  Die  Endprodukte  der  Trypsinver- 
dauung,  Habilitationsscbxift  Strassburg,  1899. 

3  Zeitschr.  f.  physiol.  Chem.,  39. 


PROTEOSES  AND  PEPTONES.  133 

views  of  Kuhne  are  not  in  all  points  correct  still  the  fact  remains  that 
under  certain  circumstances  I  he  protein  can  be  split  into  fractions,  of  which 
the  hemi  group  is  further  easily  decomposed  by  enzyme  action  while  the 
other,  the  anti  group,  is  very  much  more  resistant  to  such  action.  It 
also  seems  as  if  the  first  group  is  characterized  by  a  larger  content  of 
tyrosine,  tryptophane  and  the  latter  by  its  content  of  glycocoll,  phenyl- 
alanine and  proline. 

By  the  use  of  the  methods  specially  worked  out  by  the  Hofmeister 
school,  of  fractionally  salting  out  with  ammonium  sulphate  or  zinc  sul- 
phate or  also  by  Siegfried's  iron-alum  method,  numerous  attempts  to 
separate  the  various  proteoses  and  peptones  have  been  made.1  Not 
only  have  we  learned  by  these  methods  of  a  larger  number  of  proteoses, 
but  our  older  conception  of  the  products  formed  primarily  has  been 
materially  modified.  Immediately  at  the  commencement  of  diges- 
tion, even  in  peptic  digestion,  a  splitting  of  the  protein  molecule  into 
several  complexes  takes  place.  In  opposition  to  the  view  of  Huppert,2  that 
the  proteoses,  in  pepsin  digestion,  are  always  derived  from  the  primarily 
formed  acid  albuminate,  Pick  and  Zunz  have  shown  that  several  pro- 
teoses, as  well  as  acid  albuminate,  appear  as  primary  products  at  the 
commencement  of  the  digestion.  According  to  Goldschmidt  3  a  splitting 
off  of  proteoses  and  the  formation  of  acid  albuminate  takes  place  simul- 
taneously by  the  action  of  dilute  acids  alone.  Besides  the  proteoses 
we  also  have,  according  to  Zunz  and  Pfaundler,  even  at  the  beginning, 
other  primary  bodies,  which  cannot  be  salted  out  and  which  do  not 
give  the  biuret  reaction,  but  are  in  part  precipitated  by  phosphotungstic 
acid.  These  little-known  products  seem  to  be  intermediate  between 
the  peptones  and  the  amino-acids,  and  they  correspond  probably  to 
the  polypeptide  bodies  obtained  by  Fischer  and  Abderhalden  in  tryptic 
digestion. 

By  fractional  precipitation  of  Witte's  peptone  with  ammonium  sulphate 
Pick  has  obtained  various  chief  fractions  of  proteoses.  The  first  contains  the 
proto-  and  heteroproteoses  whose  precipitation  limit  lies  at  24-42  per  cent  satu- 
ration with  ammonium  sulphate  solution,  i.e.,  the  presence  of  24-42  cc.  of  the 
saturated  ammonium  sulphate  solution  in  100  cc.  of  the  liquid.  Then  follows 
a  fraction  A  at  54-62  per  cent  saturation,  then  a  third  fraction  B.  with  70-95 
per  cent  saturation,  and  finally  fraction  C,  which  precipitates  from  the  saturated 
solution  on  acidification  with  sulphuric  acid  saturated  with  the  salt. 


1  Umber,  Zeitschr.  f.  physiol.  Chem.,  25;  Alexander,  ibid.,  25;  Pfaundler,  ibid., 
30;  Zunz,  ibid.,  28,  and  Hofmeister's  Beitriige,  2;  Pick,  ibid.,  2,  and  Zeitschr.  f.  physiol. 
Chem.,  24  and  28;  Siegfried,  see  footnote  3,  p.  136. 

2  Schiitz  and  Huppert,  Pfliiger's  Arch.,  80. 

*  F.  Goldschmidt,  Ueber  die  Einwirkung  von  Sauren  auf  Eiweisstoffe,  Inaug.- 
Diss.  Strassburg,  1898. 


134  THE  PROTEIN  SUBSTANCES. 

The  hetero-  and  protoproteoses  *are  not,  according  to  our  present 
views,  the  only  primary  proteoses.  In  the  proteose  fraction  obtained 
on  saturating  with  ammonium  sulphate  in  neutral  liquids,  which  should 
contain  secondary  proteoses  only,  primary  proteoses  such  as  the  gluco- 
proteose  (Pick),  which  contains  a  carbohydrate  group  and  the  so-called 
synproteose  (Hofmeister  l)  occur.  It  is  no  longer  sufficient  to  consider 
an  unequal  ability  to  be  salted  out,  as  an  essential  difference  between 
the  primary  and  secondary  proteoses. 

There  is  no  doubt  that  there  exists  a  large  number  of  so-called  pro- 
teoses having  various  precipitation  properties,  and  different  other  prop- 
erties and  new  differences  appear,  while  working  with  them  according  to 
different  methods.  For  example  Rona  and  Michaelis  2  find  that  cer- 
tain proteoses  are  precipitated  by  mastic  emulsion  while  others  are  not. 
Those  that  are  precipitatable  by  mastic,  can  all  be  salted  out,  while  all 
those  that  can  be  salted  out  are  not  all  precipitated  by  mastic.  The 
hetero-  and  protoproteoses  act,  according  to  Zunz  3  like  strong  protec- 
tion colloids  toward  colloidal  gold,  which  is  not  the  case  with  the  others, 
and  also,  according  to  this  worker,  the  so-called  proteoses  are  more  readily 
precipitated  by  chondroitin-sulphuric  acid  and  acetic  acid  than  the  so- 
called  secondary  proteoses.  According  to  Hunter4  only  the  primary 
proteoses  are  precipitated  by  protamines  while  the  secondary  are  not. 
It  is  also  possible  that  numerous  intermediary  members  exist  between 
those  proteoses  which  stand  close  to  the  original  protein  and  those  that 
are  further  removed.  The  difficulties  in  isolation  and  purification  of 
these  different  members  are  so  very  great  that  the  proteoses  thus  far 
isolated  must  not  be  considered  as  chemical  individuals.  Under  these 
circumstances  the  above-mentioned  differentiation  and  classification 
of  the  various  proteoses  is  of  little  value  and  a  more  detailed  discus- 
sion of  the  properties  of  the  various  proteoses  thus  far  isolated  is  with- 
out interest. 

It  would  be  of  great  interest  if  certain  differences  in  the  chemical 
structure  of  the  different  proteoses  could  be  determined  with  certainty. 
Such  differences  are  claimed  to  have  been  found  in  certain  cases.  Thus 
Hart  has  found  that  the  heteroproteose  (from  muscle  syntonin)  was 
considerably  richer  in  arginine  and  poorer  in  histidine  than  the  proto- 
proteose,  and  Pick  has  also  found  marked  differences  between  the  hetero- 
and  protoproteose  from   fibrin.      The  hetero-proteose  yields  very  little 

1  Ueber  Bau  unci  Gruppirung  der  Eiweisskorper,  Ergebnisse  der  Physiol.,  Jahrg.  I, 
Abt.  1 ,  783. 

"-  Biochem.  Zeitsohr.,  3. 

3  Arch,  internat.  d.  Physiol.,  1  and  5,  and  Bull.  Soc.  Scienc.  med.  et  natur.  Brux- 
elles,  (54. 

Mourn,  of  Physiol.,  37. 


PROTEOSES  AND   PEPTONES.  135 

tyrosine  and  indoi  but  abundant  leucine  and  glycocoll,  and  about  39 
per  cent  of  the  total  nitrogen  in  a  basic  form.  The  protoproteose, 
according  to  Pick,  on  the  contrary  yields  considerable  tyrosine  and  indol, 
only  little  leucine  but  no  glycocoll,  and  contains  only  about  25  per  cent 
basic  nitrogen.  Friedmann,  Hart,  and  Levene  have  obtained  very 
similar  results  in  regard  to  the  quantity  of  basic  nitrogen  in  the  two-pro- 
teoses,  although  Leyene  as  well  as  Adler  l  did  not  find  the  same  results 
as  Pick  in  regard  to  the  amounts  of  monamino-acids  in  the  two  proteoses. 
The  work  of  Levene,  v.  Slyke  and  Birchard  2  show,  in  many  important 
points,  a  decided  contradiction  to  the  statements  of  Pick  and  these 
divergent  results  may  possibly  be  explained  by  the  fact  that  they  were 
not  working  with  pure  substances,  but  rather  with  mixtures. 

According  to  Pick  the  heteroproteose  is  also  more  resistant  toward 
tryptic  digestion  than  the  protoproteose,  a  behavior  which  coincides 
with  Kuhne's  view  of  a  resistant  atomic  complex,  an  anti  group,  in  the 
protein  bodies.  Kuhne  and  Chittenden  3  regularly  obtained  on  the 
tryptic  digestion  of  heteroproteose  a  separation  of  so-called  antialbumid, 
a  body  which  is  attacked  with  great  difficulty  in  tryptic  digestion,  but 
which  separates  as  a  jelly-like  mass  and  which  is  richer  in  carbon  (57.5- 
58.09  per  cent),  but  poorer  in  nitrogen  (12.61-13.94  per  cent),  than  the 
original  protein.  The  occurrence  of  such  resistant  complexes  in  diges- 
tion has  also  been  repeatedly  observed. 

This  antialbumid  later  attracted  increased  interest,  because  as 
first  found  by  Danilewsky  and  later  other  investigators  have  shown, 
that  solutions  of  rennin,  gastric  juice,  pancreatic  juice,  and  papain  cause 
a  similar  coagulum  in  not  too  dilute  proteose  solutions.  These  coagula, 
called  plasteines  (coagulum  by  rennin)  by  Sawjalow,  and  coaguloses 
(coagulum  by  papain)  by  Kurajeff,4  are  similar  in  many  respects  to 
antialbumid,  having  a  higher  content  of  carbon  (57-60  per  cent)  and 
nitrogen  (13-14.6  per  cent).  In  other  cases  the  quantity  of  carbon  as 
well  as  nitrogen  is  lower  (Lawrow). 

We  cannot  for  the  present  make  any  positive  statement  as  to  the 
importance  and  mode  of  formation  of  the  coaguloses  or  plasteins.     It 


1  Hart,  Zeitschr.  f.  physiol.  Chem.,  33;  Pick,  ibid.,  28;  Friedmann,  ibid.,  29;  Levene, 
Joum.  of  Biol.  Chem.,  1;  R.  Adler,  Die  Heteroalbumose  und  Protalbumose  des  Fibrins 
Dissert.  Leipzig,  1907. 

2  Joum.  of  Biol.  Chem.,  8  and  10. 

3  Zeitschr.  f.  Biol.,  19  and  20. 

4  The  works  of  Danilewsky  and  Okunew  are  cited  and  reviewed  in  the  following 
Sa\Vjalow,  Pfliiger's  Arch.,  85,  and  Centralbl.  f.  Physiol.,  16;  and  Zeitschr.  f.  physiol. 
Chem.,  54;  Lawrow  and  Salaskin,  Zeitschr.  f.  physiol.  Chem.,  36;  Lawrow,  ibid.,  51, 
53,  56  and  60;  Kurajeff,  Hofmeister's  Beitrage,  1  and  2:  see  also  Sacharow,  Biochem. 
Centralbl.,  1,  233;  Levene  and  v.  Slyke,  Biochem.  Zeitschr.,  13. 


136  THE  PROTEIN  SUBSTANCES. 

is  rather  generally  admitted  that  they  are  formed  by  a  synthesis,  a  view 
which  has  received  support  by  the  investigations  of  V.  Henriques  and 
Gjaldbak.1  According  to  Sawjalow  a  plastein  is  not  formed  from  a 
proteose  alone,  but  always  from  a  mixture  of  proteoses.  Lawrow  claims 
that  they  may  be  produced  from  proteoses  as  well  as  from  polypeptide 
substances,  and  correspondingly  we  must  differentiate  between  the  coag- 
uloses  or  coagulosogens  from  the  proteose  group  coaproteoses,  and  from 
the  polypeptide  group  or  coapeptides.  The  latter  yield  on  hydrolysis 
chiefly  monamino-acids,  while  the  first  yield  also  basic  nitrogenous  prod- 
ucts. Perhaps  the  plasteinogen  investigated  by  Bayer2  which  differs 
essentially  from  the  true  proteid  in  its  elementary  composition  as  well 
as  from  other  coaguloses,  belongs  to  the  coapeptides. 

The  different  behavior  on  saturating  their  solution  with  ammonium 
sulphate  has  been  generally  used,  as  above  remarked,  for  years  to  dif- 
ferentiate between  the  proteoses  and  peptones.  Those  precipitable 
by  this  salt  were  called  proteoses,  and  those  not  were  called  peptones. 
This  method  of  division,  which  never  had  sufficient  support  and  which 
was  perfectly  arbitrary,  cannot  be  considered  at  the  present  time.  We 
know  now,  thanks  to  the  works  of  Emil  Fischer  and  his  co-workers, 
that  there  are  polypeptides  either  prepared  artificially  or  found  among 
the  cleavage  products  of  the  proteins,  which  are  precipitated  by  ammo- 
nium sulphate.  At  the  present  it  is  generally  conceded  that  the  peptones 
in  the  ordinary  sense  are  only  a  mixture  of  different  bodies.  The  chief 
step  in  these  investigations  must  be  the  isolation  from  this  mixture 
of  unit  bodies  with  definite  chemical  characteristics.  Of  such  bodies, 
besides  the  polypeptides  previously  mentioned  and  studied  by  Fischer 
and  others,  we  must  mention  the  products  isolated  by  Siegfried  and 
his  pupils.3 

These  so-called  peptones  are  in  part  peptic-peptones  and  partly 
tryptic-peptones,  and  some  are  prepared  from  proteid  (fibrin)  and  others 
from  gelatin.  The  tryptic  fibrin-peptones  are  antipeptones  in  Kuhne's 
sense  because  they  are  very  resistant  to  the  further  action  of  trypsin. 
They  are  according  to  Neumann  simultaneously  bibasic  acids  and  mono- 
acidic  bases.  They  give  the  biuret  reaction,  but  not  Millon's  reaction; 
they  contain  no  tyrosine  and  yield  on  hydrolysis,  arginine,  lysine,  glutamic 
acid,  and  it  seems  also  aspartic  acid.  A  pepsin-glutin  peptone  isolated 
by  Siegfried  and  Schmitz  yielded  arginine,  lysine,  glutamic  acid,  gly- 

1  Zeitschr.  f.  physiol.  Chem.,  71  and  81. 

2  Hofmeister's  Beitriige,  4;  see  also  L.  Rosenfeld,  ibid.,  9;  J.  Lukomnik,  ibid.,  9 
and  F.  Micheli,  Biochem.  Centralbl.,  6,  p.  562. 

'  The  works  of  Siegfried  and  his  pupils,  Fr.  Muller,  Borkel,  Miihle,  Kriiger,  Scheer- 
rnesser,  Neumann,  H.  Schmitz,  may  be  found  in  Arch.  f.  (Anat.  u.)  Physiol.,  1894  and 
Zeitschr.  f.  physiol.  Chem.,  21,  41,  43,  45,  48,  59,  and  65  and  Pfliiger's  Arch.  136. 


PROTEOSES  AM)  PEPTONES.  137 

cocoll  and  besides  these  also  leucine  and  proline  although  not  in  quan- 
tities that  could  be  determined.  Of  the  total  nitrogen  they  found 
i'9-7  per  cent  arginine,  9.1  per  eent  lysine,  19.2  per  cent  glycocoll,  9.3 
percenl  glutamic  acid  and  12.7  per  cent  proline  and  leucine  together. 
Siegfried  has  given  proof  in  several  ways  as  to  the  purity  and  unity 
of  the  peptones  isolated  by  him. 

In  another  manner,  namely  by  fractional  precipitation  with  metallic  salts, 
especially  with  mercuric-potassium  iodide  and  the  preparation  of  phenyliso- 
cyanate  compounds,  Hofmeister  and  his  pupils  Stookey,  Raper  and  Rogo- 
zinski  i  have  isolated  peptones  or  polypeptide-like  bodies  from  blood  proteid. 
One  of  these,  called  arginine-histidine  peptone,  yielded  arginine  and  histidine  as 
basic  hydrolytic  products  while  another  yielded  chiefly  lysine^as  basic  product 
and  hence  was  called  ly sine-peptone. 

From  glutin-pcptone,  Siegfried,  on  warming  with  hydrochloric 
acid,  obtained  a  base,  C21H39N9O8,  which  can  also  be  directly  obtained 
from  gelatin.  This  he  calls  a  kyrin,  because  it  is  to  be  considered  as  a 
basic  protein  nucleus,  and  he  calls  this  special  one  glutokyrin.  The 
glutokyrin  gives  the  biuret  reaction  and  is  considered  as  a  basic  peptone. 
On  complete  hydrolytic  cleavage  it  yields  arginine,  lysine,  glutamic 
acid,  and  glycocoll.  Of  the  total  nitrogen  two-thirds  belong  to  the 
bases  and  one-third  to  the  amino-acids.  Recently  he  with  0.  Pilz 
on  further  hydrolysis  has  prepared  a  /3-glutokyrin,  which  only  yielded 
arginine,  lysine  and  glutamic  acid.  Similar  basic  nuclei,  protokyrins, 
have  recently  been  obtained  by  Siegfried  2  from  fibrin  and  casein,  using 
the  same  method.  Caseinokyrin  gives  a  non-crystalline  sulphate,  but 
a  crystalline  phosphotungstate.  The  free  caseinokyrin  has  an  alkaline 
reaction,  gives  the  biuret  test,  and  its  composition  corresponds  to  the 
formula  C23H47N9O8.  It  yields  arginine,  lysine,  and  glutamic  acid  on 
cleavage.  The  basic  nitrogen  amounts  to  about  85  per  cent  of  the  total 
nitrogen,   and  caseinokyrin,   behaves  in  this  respect  like  a  protamine. 

Among  the  known  cleavage  products  of  proteins,  arginine  is  the  only 
one  which,  up  to  the  present,  is  never  absent,  and  for  this  reason  Ave 
designate  as  proteins  only  those  atomic  complexes  which  contain,  besides 
chained  monamino-acids,  also  arginine,  or,  more  simply,  show  the  prev- 
iously mentioned  imide  bindings.  Hence  caseinokyrine,  which  yields 
only  arginine,  lysine  and  glutamic  acid,  and  scombrin,  which  yields 
only  arginine,   proline,   and   alanine,   are  the  simplest  known  proteins. 

Scombrin  belongs  to  the  previously  mentioned  group  of  protamines 
which,  according  to  Kossel,3  are  formed  by  a  successive  cleavage  of  the 


1  Hofmeister's  Beitrage,  7,  9,  and  11. 

2  Kgl.  Sachs.  Ges.  d.  Wiss.,  Math.-Phys.    Klasse,    1903,   and   Zeitschr.  f.  phvsiol 
Chem.,  43,  with  Pilz.,  ibid.,  58. 

3  Zeitschr.  f.  phvsiol.  Chem.,  44. 


138  THE  PROTEIN  SUBSTANCES. 

typical  protein.  The  occurrence  of  basic  protokyrins  in  the  hydrolytic 
cleavage  of  genuine  proteins  like  gelatin  has  given  valuable  support  to 
Kossel's  theory  as  to  a  basic  nucleus  in  the  protein  bodies. 

On  account  of  the  cleavage  taking  place  in  digestion,  the  digestive 
products  should  have  a  lower  molecular  weight  than  the  original  protein. 
This  is  really  the  case  as  shown  by  molecular  weight  determinations. 
As  these  determinations  have  been  made  upon  impure  substances  or 
mixtures,  the  results  1  obtained  are  only  of  little  value.  The  same  is 
true  for  the  elementary  analysis  of  the  proteoses  and  peptones.2 

■v  — 

In  the  preparation  and  separation  of  various  proteoses  and  peptones 
all  precipitable  protein  is  always  removed  first  by  neutralization  and 
then  by  boiling.  The  proteoses  may  then  be  separated  from  the  pep- 
tones by  means  of  ammonium  sulphate  according  to  Ivuhne's  method, 
and  divided  into  different  fractions  according  to  the  method  of  Pick 
and  the  Hofmeister  school.  The  separation  and  preparation  of  pure 
hetero-  and  protoproteoses  can  be  best  performed  by  the  method  sug- 
gested by  Pick,  but  this  method,  as  well  as  that  with  ammonium  sulphate, 
gives  good  results  only  when  the  precautions  suggested  by  Haslam  3 
are  carefully  followed.  We  can  here  only  refer  to  the  cited  works  of 
Kuhne  and  co-workers,  of  E.  Zunz  and  especially  those  of  the  Hof- 
meister and  the  Siegfried  schools.  In  regard  to  the  literature  on  the 
detection  of  proteoses  and  peptones  in  animal  fluids  we  refer  to  Chapters 
V  and  XIV. 

If  we  wish  to  detect  the  presence  of  so-called  true  peptone,  by  means 
of  the  biuret  reaction  in  a  solution  saturated  with  ammonium  sulphate, 
we  add  a  slight  excess  of  a  concentrated  solution  of  caustic  soda  and 
cool,  and  then  add  a  two  per  cent  solution  of  copper  sulphate  drop  by 
drop,  after  the  sodium  sulphate  has  separated  out. 

In  the  quantitative  estimation  of  proteoses  and  peptones  we  make 
use  of  the  nitrogen  estimation,  the  biuret  test  (colorimetric),  and  the 
polarization  method.     These  methods  do  not  give  exact  results. 

The  polypeptides  have  had  their  most  important  properties  dis- 
cussed on  pages  85-91,  and  of  the  cleavage  products  of  the  proteins  only 
the  amino-acids  remain  to  be  discussed. 


1  Sabanejew,  Ber.  d.  d.  chem.  Gesellsch.,  26,  385;  Paal,  ibid.,  27,  1827;  Sjoqvist, 
Skand.  Arch.  f.  Physiol.,  5. 

-  Klernentary  analyses  of  proteoses  and  peptones  will  be  found  in  the  works  of 
Kuhne  and  Chittenden  and  their  pupils,  cited  in  footnote  2,  p.  130;  also  by  Herth, 
Zeitschr.  f.  physiol.  Chem.,  1,  and  Monatshefte  f.  Chem.,  5;  Maly,  Pfluger's  Arch. 
9,  20;  Henninger,  Compt.  rend.,  86;  Schrotter,  1.  c,  Paal,  1.  c. 

3  Journ.  of  Physiol.,  32  and  36. 


GLYCOCOLL.  139 


3.     The  Amino-acids.1 

CH2(NH2)     ,  „    ,     , 

Glycocoll  (ami no-acetic  acid),  Cl>H5N(>2  =  /,      __        ,  also  called  gly- 

COOH 

cine   or  gelatin  sugar,  is  found   in    the    muscles   of   the    invertebrates, 

but  has  chief  interest  as  a  hydrolytic  decomposition  product  of  protein 
bodies,  especially  fibroin,  spider-silk  elastin,  gelatin,  and  spongin,  as 
well  as  of  hippuric  acid  and  glycocholic  acid. 

Glycocoll  forms  colorless,  often  large,  hard  rhombic  crystals  or  four- 
sided  prisms.  The  crystals  have  a  sweet  taste  and  dissolve  readily  in 
cold  water  (4.3  parts).  Glycocoll  is  insoluble  in  alcohol  and  ether  and 
dissolves  with  difficulty  in  warm  alcohol.  Like  the  amino-acids  in  gen- 
eral it  combines  with  acids  and  alkalies.  With  the  latter  compounds 
we  must  mention  those  with  copper  and  silver.  Glycocoll  dissolves 
cudHc  hydroxide  in  alkaline  liquids,  but  does  not  reduce  at  boiling  heat. 
A  boiling-hot  solution  of  glycocoll  dissolves  freshly  precipitated  cupric 
hydroxide,  forming  a  blue  solution,  which  in  proper  concentration  deposits 
blue  needles  of  copper-glyeocoll  on  cooling.  The  compound  with  hydro- 
chloric acid  is  readily  soluble  in  water  but  less  soluble  in  alcohol. 

Sorensen  2  finds  that  phosphotungstic  acid  does  not  precipitate 
glycocoll  from  dilute  solutions  but  only  from  concentrated  ones.  By  the 
action  of  gaseous  HC1  upon  glycocoll  in  absolute  alcohol,  beautiful 
crystals  are  obtained  of  the  hydrochloride  of  glycocoll-ethyl  ester,  which 
melts  at  144°  C.  and  from  which  the  glycocoll-ethyl  ester  can  be  obtained 
by  the  method  suggested  by  E.  Fischer3  for  the  separation  of  glycocoll 
from  the  other  amino-acids.  On  shaking  with  benzoyl  chloride  and 
caustic  soda,  hippuric  acid  is  formed,  and  this  is  also  made  use  of  in 
different  ways  in  detecting  and  isolating  glycocoll  (Ch.  Fischer,  Gox- 
nermaxn,  Spiro4).  The  /3-naphthalene-sulpho-glycine  with  a  melt- 
ing-point of  159°,  the  4-nitro-tolulene-2-sulpho-glycine,  melting  at  180°, 
and  the  a-naphthylisocyanate  compound  melting  at  190.5-191.5°  are 
also  of  importance.  On  putrefaction  methane  is  probably  produced 
from  glycocoll. 

Glycocoll  can  be  best  prepared  from  hippuric  acid  by  boiling  it  with 
4  parts  dilute  sulphuric  acid  (1:6)  for  ten  to  twelve  hours.     After  cooling 


1  In  regard  to  the  division  of  the  amino-acids  among  the  three  chief  groups  of 
■organic  compounds  we  refer  to  pages  85-86. 

2  Meddelelser,  fraa  Carlsberg-laboratoriet,  6,  1905. 

3  Ber,  d.  d.  chem.  Gesellsch.,  34. 

4Ch.    Fischer,    Zeitschr.    f.    physiol.   Chem.,    19;   Spiro,   ibid.,    28;   Gonnermann, 
Pfliiger's  Arch.,  59. 


140  THE  PROTEIN   SUBSTANCES. 

the  benzoic  acid  is  removed,  the  filtrate  concentrated,  the  remaining 
benzoic  acid  removed  by  extracting  with  ether,  the  sulphuric  acid  pre- 
cipitated by  BaCOs,  and  the  filtrate  evaporated  to  the  point  of  crys- 
tallization. (In  regard  to  its  preparation  from  protein  substances  see 
below.) 

CH3 
d-Alanine(a-aminopropionic  acid),  C3H7N02  =  CH(NH2).     The  d-alanine 

COOH 
is  obtained  in  relatively  small  amounts  from  the  true  proteids,  but  in 
larger  quantities  from  the  albuminoids,  especially  from  fibroin,  spider- 
silk  and  elastin. 

d-alanine  has  been  prepared  from  Z-serine  by  E.  Fischer  and  K. 
Raske,1  and  Fischer  has  also  obtained  it  from  racemic  alanine  by  split- 
ting the  benzoyl  combination,  or  from  Z-alanine  by  splitting  with  yeast 
by  Walden's  reversion. 

Alanine  generally  crystallizes  in  needles  or  oblique  rhombic  columns. 
It  is  very  readily  soluble  in  water,  having  a  sweetish  taste,  and  dissolves 
cupric  hydroxide  on  boiling,  producing  a  deep  blue  solution  of  a  crystalliza- 
ble  copper  salt.  Alanine  is  insoluble  in  absolute  alcohol.  The  rota- 
tion of  alanine  at  20°  C.  in  aqueous  solution  is  (a)D=+2.7°  and  for  a 
solution  in  hydrochloric  acid  (9-10  per  cent  solution)  is  (ct)D= +10.3°. 

The  /3-naphthalene-sulpho-d-alanine  melts,  when  dry,  at  about  123° 
and  sinters  at  117°  C.  The  phenylisocyanate  melts  at  168°  and  the 
a-naphthylisocyanate  alanine  melts  at  198°.  On  putrefaction  alanine 
yields   propionic    acid. 

CH3CH3 

V 

ClT 
d- Valine      (a-amino-valeric     acid),     C5HnN02  =  ^TT,ATTT  N       ,  u 

n  CH(NH2),     has     been 

COOH 
detected  several  times  among  the  cleavage  products  of  protein  sub- 
stances, although  only  in  small  quantities.  Kossel  and  Dakin  obtained 
4.3  per  cent  valine  from  salmine,  and  E.  Fischer  and  Dorpinghaus2 
5.7  per  cent  from  horn  substance.  The  largest  quantity  has  been  obtained 
from  casein  and  edestin,  namely,  7.20  and  5.6  per  cent  respectively. 
Because  of  the  difficulty  in  separating  valine  from  the  two  leucines3 
the  figures  given  are  somewhat  uncertain.  The  acid  isolated  by  H. 
and  E.  Salkowski  4  from  putrefying  proteid  or  gelatin  seems  to  have 
been  5-amino-n-valeric  acid. 

1  Ber.  d.  d.  chem.  Gesellsch.,  40. 

2  Kossel  and  Dakin,  Zeitschr.  f.  physiol.  Chem.,  41;  Fischer  and  Dorpinghaus, 
i\nd.,  .'{<>. 

-<•<•  Levene  and  v.  Slyke,  Journ.  of  Biol.  Chem.,  6. 
4  Ber.  d.  d.  chem.  Gesellsch.,  16  and  31. 


LEUCINE.  HI 

d-valine  can  be  obtained  aa  microscopic  crystalline  leaves.  It  iq 
rather  readily  soluble  in  water  and  the  solution  lias  a  faint  sweetish  taste 
and  at  the  same  time  somewhat  bitter.  The  solution  has  a  rotation 
of  (a)D= +6.42°.  The  hydrochloric  acid  solution  (20  per  cent)  shows, 
according  to  Fischer,  a  rotation  of  (r;)D=+28.8°.  The  copper  salt, 
which  forms  leaves  which  are  rather  soluble  in  water,  is  very  easily  soluble 
in  methyl  alcohol  (Schulz  and  Winterstein1). 

The  phenylisocvanate  melts  at  147°,  and  on  boiling  with  20  per  cent 
hydrochloric  acid  for  a  short  time,  it  is  changed  into  rf-phenylisopropyl 
hydantoin,  which  melts  at  131-133°  C. 

On  putrefaction  valine  yields  isobutylamine  and  isovaleric  acid. 
/-Leucine  (aminocaproic  acid,  or,  more  correctlv,  a-aminoisobutylacetic 
CH3CH3 

\y 

CH 

acid),  CeHi3N02=  CH2  ,  is  produced  from  protein  substances  in 

CH(NH2) 

COOH 
their  hydrclytic  cleavage  by  proteolytic  enzymes,  by  boiling  with  dilute 
acids  or  alkalies  or  by  fusing  with  alkali  hydroxides,  and  by  putre!:  ttion. 
There  are  also  observations  that  indicate  that  in  the  hydrolysis  besides 
the  ordinary  leucine  perhaps  also  normal  leucine  may  be  formed  (Heckel 
and  Samec2). 

Because  of  the  ease  with  which  leucine  (and  tyrosine)  are  formed 
in  the  decomposition  of  protein  substances,  it  is  difficult  to  decide  pos- 
itively wThether  these  bodies  when  found  in  the  tissues  are  constituents 
of  the  living  body  or  are  to  be  considered  only  as  decomposition  products 
formed  after  death.  Leucine,  it  seems,  has  been  found  as  a  normal 
constituent  of  the  pancreas  and  its  secretion,  in  the  spleen,  thymus,  and 
lymph  glands,  in  the  thyroid  gland,  in  the  salivary  glands,  in  the  kidneys 
and  in  the  liver.  It  also  occurs  in  the  wool  of  sheep,  in  dirt  from  the 
skin  (inactive  epidermis),  and  between  the  toes,  and  its  decomposition 
products  have  the  disagreeable  odor  of  the  perspiration  of  the  feet. 
It  is  found  pathologically  in  atheromatous  cysts,  ichthyosis  scales,  pus, 
blood,  liver,  and  urine  (in  diseases  of  the  liver  and  in  phosphorus  poison- 
ing). Leucine  often  occurs  in  invertebrates  and  also  in  the  plant  king- 
dom. On  hydrolytic  cleavage  various  protein  substances  yield  different 
amounts  of  leucine,  as  shown  in  the  tables  given  on  pages  106,  107,  115  and 
125.  From  the  figures,  there  given,  we  call  attention  to  the  following: 
Erlenmeyer  and  Schoffer  obtained  36-45  per  cent  leucine  from  the 
cervical  ligament.  E.  Fis^  her  and  Abderhalden  20  per  cent  from  haemo- 

1  Zeitschr.  f.  physiol.  Chem.,  35. 

2  Heckel,  Monatsh.  f.  Chem.,  29;  Samec,  ibid.,  29. 


142  THE  PROTEIN  SUBSTANCES. 

globin,  and  Fischer  and  Dorpinghaus  18.3  -per  cent  from  horn  sub- 
stance.1    . 

The  leucine  obtained  by  cleavage  of  protein  substances  is  generally 
Z-leucine,  which  is  levorotatory  in  water  solution  and  dextrorotatory 
in  acid  solution.  The  leucine  prepared  synthetically  by  Hufner  2 
from  isovaleraldehyde,  ammonia,  and  hydrocyanic  acid  is  optically 
inactive.  Inactive  leucine  may  also  be  prepared,  by  the  cleavage  of  pro- 
teins with  baryta  at  160-180°  C,  because  of  a  ready  racemation.  The 
e/-/-leucine  may  be  split  into  the  two  components  by  various  means,  espe- 
cially by  the  preparation  of  the  formal  combination.3 

On  oxidation  the  leucines  yield  the  corresponding  oxyacids  (leucinic 
acids).  Leucine  is  decomposed  on  heating,  evolving  carbon  dioxide, 
ammonia,  and  amylamine.  On  heating  with  alkalies,  as  also  in  putre- 
faction, it  yields  valeric  acid  and  ammonia.  On  putrefaction  it  yields 
isoamylamine  and  isocaproic  acid. 

Leucine  crystallizes  when  pure  in  shining,  white,  very  thin  plates, 
usually  forming  round  knobs  or  balls,  either  appearing  like  hyaline,  or 
with  alternating  light  and  dark  concentric  layers  which  consist  of  radial 
groups  of  crystals.  By  slow  heating,  leucine  melts  and  sublimes  into 
white  woolly  flakes,  which  are  similar  to  sublimed  zinc  oxide.  At  the 
same  time  an  odor  of  amylamine  is  developed.  Quickly  heated  in  a 
closed  capillary  tube,  it  melts  with  decomposition  at  293-295°. 

Leucine,  as  obtained  from  animal  fluids  and  tissues  is  always  impure, 
and  is  very  easily  soluble  in  water  and  rather  easily  in  alcohol.  Pure 
leucine  is  soluble  with  difficulty.  Pure  /-  and  d-leucine  dissolve  in  4C- 
46  parts  water,  more  readily  in  hot  alcohol,  but  with  difficulty  in  cold 
alcohol.  The  (/-/-leucine  is  much  less  soluble.  According  to  Haber- 
mann  and  Ehrexfeld  4  100  parts  of  boiling  glacial  acetic  acid  dissolve 
29.23  parts  of  leucine.  The  specific  rotation  of  /-leucine,  dissolved  in 
hydrochloric  acid  (20  per  cent  solution)  is  (a)D  = +15.6°  according  to 
Fischer  and  Warburg.  In  aqueous  solution  it  is  (a)D=  —10.40°, 
according  to  F.  Ehrlich  and  Wendel.5 

The  solution  of  leucine  in  water  is  not,  as  a  rule,  precipitated  by 
metallic  salts.  The  boiling-hot  solution  may,  however,  be  precipitated 
by  a  boiling-hot  solution  of  copper  acetate,  and  this  fact  is  made  use  of 
in  separating  leucine  from   other  substances.     If  the  solution  of  leucine 

1  Erlenmeyer  and  Schoffer,  cited  from  Maly,  Chem.  d.  Verdauungssiifte,  in  Her- 
mann's Handb.  d.  Physiol.,  5,  Theil  2,  p.  209;  Fischer  and  his  collaborators,  ibid.,  36. 

-Jouni.  f.  prakt.  Chem.  (N.  F.),  1. 

■  Fischer  and  Warburg,  Ber.  d.  d.  chem.  Gesellsch.,  38. 

*Zeitschr.  f.  physiol.  Chem.,  37. 

R  Fischer  and  Warburg,  Ber.  d.  d.  chem.  Gesellsch.,  38;  E.  Ehrlich  and  Wendel, 
Biochem.  Zeitschr.,  8. 


I80LEUCINE.  143 

is  boiled  with  sugar  of  lead  and  then  ammonia  be  elded  to  the  cooled 
solution,  shining  crystalline  leaves  of  leucine-lead  oxide  separate.  Leucine 
dissolves  cupric  hydroxide,  but  dors  not  reduce  on  boiling. 

Leucine  is  readily  soluble  in  alkalies  and  acids.  It  gives  crystalline 
compounds  with  mineral  acids.  If  Leucine  hydrochloride  is  boiled  with 
alcohol  containing  3-4  ]><•!■  cenl  BC1,  long  narrow  crystalline  prisms  of 
leucine-ethyl-ester  hydrochloride,  melting  at  134°  ('..  an-  formed.  The 
picrate  of  the  leucine  ester  melts  at  128°.  The  phenylisocyanate  of 
'/-/-leucine  melts  at  165°  and  its  anhydride  at  12o°  G.  The  o-naphthyl- 
isocyanate  leucine  melts  at  163.5°,  the  naphthalene-sulpho-Meucine 
at     68°  C. 

Leucine  is  recognized  under  the  microscope  by  the  appearance  of  bail- 
or knobs,  by  its  action  when  heated  (sublimation  test),  and  by  its 
compounds,  especially  the  hydrochloride  and  picrate  of  the  ethyl  ester 
and  the  phenylisocyanate  compound  of  the  racemic  leucine  obtained 
on  heating  with  baryta  water,  the  a-naphthylisocyanate  compound  and 
the  p-naphthalene-sulpho-leucine.  According  to  the  method  suggested 
by  Lippich  l  the  leucine  can  be  transformed  into  isobutylhydantoin, 
having  a  melting-point  of  205°,  by  boiling  with  an  excess  of  urea  and 
baryta  water.  For  the  preparation  and  separation  of  leucine  from  the 
other  amino-acids  of  the  leucine  fraction  special  methods  have  been 
suggested  by  F.  Ehrlich  and  Wendel,  Levene  and  v.  Slyke.2 

Leucinimide,  CuH^N^  =    "    9' •,  „  '  TTT  '%u  ~  „    ,  was  first  obtained  by  Ritt- 

HAU8EN  in  the  hydrolytic  cleavage  products  on  boiling  proteins  with  acids,  and 
subsequently  by  R.  Cohx.  Salaskix  3  obtained  it  in  the  peptic  and  tryptic 
digestion  oi  haemoglobin.  As  an  anhydride  of  leucine  (2.5-diacipiperazine)  it 
is  probably  formed  by  a  secondary  change,  from  leucine. 

It  crystallizes  in  long  needles  and  sublimes  readily.  The  melting-point  has 
not  been  found  constant  in  the  different  cases.  The  leucinimide  (3.6-diisobutyl- 
2.5-diacipiperazine)  prepared  synthetically  by  E.  Fischer  4  from  leucine-ethyl 
ester  melted  at  271°  C. 

i-Isoleucine  (/3-methyl-ethyl-a:-amino-propionic  acid), 

CH3C2H5 

v 

C6Hl3N°2  =  CHNH2 
COOH 


1  Ber.  d.  d.  chem.  Gesellsch.,  39. 

-  F.  Ehrlich  and  Wendel,  1.  o.;  Levene  and  v.  Slyke,  Journ.  of  Biol.  Chem..  6. 

3  Ritfhausen,   Die  Eiweisskorper  der  Getreidearten,   etc.,   Bonn,    1872;  R.  Cohn, 
Zeit?chr.  f.  physiol.  Chem.,  22  and  29;  Salaskin,  ibid.,  32. 

4  Ber.  d.  d.  chem.  Gepellsch.,  34. 


144  THE  PROTEiN   SUBSTANCES. 

is  an  isomer  of  leucine  discovered  by  F.  Ehrlich,1  who  first  isolated 
it  from  the  mother-liquor  after  removing  the  sugar  from  beet-sugar 
molasses.  He  also  found  it  in  the  hydrolysis  of  several  proteins,  and 
recently  it  has  been  found  by  others  among  the  products  of  hydrolysis 
of  the  proteins.  The  largest  amount  thus  far  found  was  2.6  per  cent 
by  Levene,  v.  Slyke  and  Birchard  2  in  a  heteroproteose.  It  seems 
to  be  associated  regularly  with  ordinary  leucine,  forming  mixed  crystals, 
which  give  an  impression  of  a  chemical  combination  and  which  are  dif- 
ficult to  separate.  On  this  account  the  earlier  claims  as  to  the  quantity 
of  leucine  are  somewhat  uncertain,  as  they  always  refer  to  leucine 
containing  isoleucine. 

The  constitution  of  isoleucine  has  been  explained  by  Ehrlich  through 
its  relation  to  d-amyl  alcohol.  Just  as  according  to  F.  Ehrlich  valine 
3'ields  the  isobutyl  alcohol  in  alcoholic  fermentation  so  isoleucine  yields 
"d-amyl  alcohol  in  the  fermentation  of  sugar  with  yeast.  On  the  other 
hand,  it  can  also  be  obtained,  in  a  manner  analogous  to  the  synthesis 
of  leucine,  from  d-amyl  alcohol  (as  a  mixture  of  isoleucine  and  alloiso- 
leucine,  the  latter  is  levogyrate  and  has  a  different  stereometric  configura- 
tion from  the  isoleucine).  The  synthesis  of  isoleucine  has  been  accom- 
plished in  other  ways  by  several  investigators.3  On  putrefaction  d-ca- 
proic  acid  and  d-valeric  acid  have  been  obtained  from  isoleucine.4 

Isoleucine  crystallizes  in  leaves  or  rods  and  plates  of  the  rhombic 
form.  It  is  more  soluble  in  water  than  leucine  (1:25.8).  Its  solutions 
have  a  bitter  taste  and  are  astringent.  It  is  dextro-rotatory  in  aqueous 
as  well  as  in  acid  solution.  In  aqueous  solution  it  has  a  specific  rotation 
0f  (ajD=+9.74°  and  in  20  per  cent  hydrochloric  acid  (a)D  =  +36.8°. 
Like  valine  its  copper  salt  is  readily  soluble  in  methyl  alcohol.  The 
benzoyl  combination  melts  at  116-117°,  the  benzene  sulphoisoleucine 
at  149-150°,  the  phenylisocyanate  combination  at  119-120°,  and  the 
naphthylisocyanate  combination  at  178°  C. 

In  the  leucine  fraction,  from  the  amino-acids  contained  in  nerve 
substance,  Abderhalden  and  Weil5  have  obtained  a  new  amino-acid, 
C6H13NO2  which  is  isomeric  with  leucine  and  which  seems  to  be  d-a- 
amino-n-caproic  acid  and  called  d-caprine  by  them.  When  crystallized 
from  water  it  forms  six-sided  plates  which  unite  to  tufts  having  a  faint 
sweet  taste.     At  280°  (uncorrected)   it  softens  and  at  285°  (uncorrected) 

1  Felix  Ehrlich,  Her.  d.  d.  chem.  Gesellsch.,  37. 

2Journ.  of  Biol.  Chem.,  8. 

1  Ehrlich,  Ber.  d.  d.  chem.  Gesellsch.,  40  and  41;  Brasch  and  Friedmann,  Hof- 
meister'e  Beitrage,  11;  Bouveault  and  Locquin,  Compt.  rend.,  141,  and  Bull.  soc. 
fhim.  (3),  :{•">:  Locquin,  Bull.  boc.  chim.  (4),  1. 

;  C.  Neuberg,  Bioch.  Zeitachr.,  37. 

*Zeitschr.  f.  physiol.  Chem.,  81  and  84. 


SERINE.  145 

it  sublimes.  Its  solubility  in  water  is  1.5:100;  at  20°  in  aqueous  so- 
lution (a)D+6.53°  and  in  20  per  cent  hydrochloric  acid-f-14.1°.  It  gives 
a  copper  salt  crystallizing  in  needles. 

CH2(OH) 
/-Serine    (a-amino-/3-oxypropionic    acid),  C3H7N03  =  CH(NH2),    was 

COOH 
obtained  by  Fischer  and  his  collaborators  as  a  cleavage  product  of 
several  proteins,  generally  only  in  small  quantities.  The  largest  quan- 
tity, 6.6  per  cent,  was  obtained  by  Fischer  and  Skita  from  sericine; 
Kossel  and  Dakin  l  obtained  a  still  larger  amount  from  salmine,  namely 
7.8  per  cent.  The  racemic  serine  is  the  one  generally  obtained.  From 
fibroin  Fischer  2  obtained  a  mixture  of  active  and  inactive  serine  anhy- 
dride from  which  he  finally  prepared  /-serine  by  hydrolysis.  Serine  has 
also  been  found  by  G.  Embden  and  Tachau  3  in  fresh  perspiration. 
Synthetically  c/-/-serine  has  been  prepared  by  Fischer  and  Leuchs 
from  ammonia,  hydrocyanic  acid  and  glycol  aldehyde,  and  also  in  other 
ways  by  others.4  Fischer  and  Jacobs5  have  prepared  /-serine  from 
(/-/-serine  by  the  preparation  of  the  alkaloid  salt  of  the  p-nitro-benzoyl 
combination.  On  reduction  serine  is  transformed  into  alanine,  and  on 
oxidation  with  nitrous  acid  it  yields  glyceric  acid.  The  relation  of  serine 
to  alanine,  lactic  acid  and  glyceric  acid  is  evident  from  the  following  for- 
mulae: 


CH2(OH) 

CH3 

CH3 

CH2(OH) 

CH(NH2) 

CH(NH2) 

CH(OH) 

CH(OH) 

COOH 

COOH 

COOH 

COOH 

Serine 

Alanine 

Lactic  acid 

Glyceric  acid 

The  /-serine  crystallizes  in  thin  leaves  or  crusts.  It  is  rather  readily 
soluble  in  water;  the  (/-/-serine  is  soluble  in  23  parts  water  at  20°  C. 
The  solution  of  /-serine  has  a  sweet  taste  with  an  insipid  after  taste. 
The  specific  rotation  in  aqueous  solution  at  20°  C.  is  (a)D=—  6.83° 
and  the  hydrochloric  acid  solution  at  25°  C.  is  (a)D= +14.45°.  The 
/3-napthalene-sulpho-serine  melts  at  220°  C.  when  anhydrous.  The 
/-serine  anhydride,  which  is  identical  with  that  obtained  from  fibroin, 
forms  thin,  colorless  needles  which  melt  at  247°  with  decomposition. 
Its  specific  rotation  in  aqueous  solution  at  25°  C.   (a)D= —67.46°. 


1  Fischer  and  Skita,  Zeitschr.  f.  physiol.  Chem.,  35;  Kossel  and  Dakin,  ibid.,  41. 

2  Ber.  d.  d.  chem.  Gesellsch.,  40. 

3  Bioch.  Zeitschr.,  28. 

4  Fischer  and  Leuchs,  Ber.  d.  d.  chem.  Gesellsch,  35;  Erlenmeyer  and  Stoop,  ibid., 
35;  Leuchs  and  Geiger,  ibid.,  39. 

6  Ber.  d.  d.  chem.  Gesellsch.,  39. 


146  THE  PROTEIN  SUBSTANCES. 

Isoserine  (£-amino-a-oxypropionic  acid)  has  been  prepared  by  Ellinger 
from  diamino-propionic  hydrobromide  and  silver  nitrite,  and  by  Neuberg  and 
Silbermann  from  the  hydrochloric  acid  combination  of  diamino-propionic  acid. 
Other  syntheses  have  been  made  by  Neuberg  and  Mayer  and  by  Neuberg 
and  Ascher.1 

COOH 
Z-Aspartic  acid  (aminosuccinic  acid),  C4H7N04=att  ,  has  been 

CH.2 

COOH 

obtained  on  the  cleavage  of  protein  substances  by  proteolytic  enzymes  as 
well  as  by  boiling  them  with  dilute  mineral  acids  in  comparatively  small 
quantities.  This  acid  also  occurs  in  secretions  of  sea-snails  (Henze  2) 
and  is  very  widely  diffused  in  the  vegetable  kingdom  as  the  amide 
Asparagine  (aminosuccinic-acid  amide),  which  seems  to  be  of  the 
greatest  importance  in  the  development  and  formation  of  the  proteins 
in  plants.  d-Z-Aspartic  acid  has  been  prepared  synthetically  from  fumaric 
acid  and  alcoholic  ammonia.  On  putrefaction  of  aspartic  acid,  propionic 
acid  and  succinic  acid  are  formed. 

/-Aspartic  acid  dissolves  in  256  parts  water  at  10°  C.  and  in  18.6  parts 
boiling  water,  and  on  cooling  crystallizes  as  rhombic  prisms,  and  its 
4  per  cent  solution  acidified  with  HC1  has  the  rotation  («)d  =  +25.7°; 
in  alkaline  solution  the  acid  is  levo-rotatory.  It  forms  with  copper 
oxide  a  crystalline  compound  which  is  soluble  in  boiling-hot  water  and 
nearly  insoluble  in  cold  water,  and  which  may  be  used  in  the  prepara- 
tion of  the  pure  acid  from  a  mixture  with  other  bodies. 

The  benzoyl-Z-aspartic  acid  melts  at  184-185°.  For  identification 
we  make  use  of  the  analysis  of  the  free  acid  and  the  copper  salt,  as  well 
as  of  the  specific  rotation. 

COOH, 
CH(NH2) 
^/-Glutamic    acid    (a-aminoglutaric    acid),    CsH9N04  =  CH2  ,  is 

CH2 
COOH 
obtained  from  the  protein  substances  under  the  same  conditions  as  the 
other  monamino-acids  (see  tables  on  pages  106,  107,  115  and  125)  and 
from  the  peptones  (Siegfried).  It  is  absent  in  the  protamines  and  in  the 
varieties  of  silk,  it  occurs  only  in  small  amounts  with  the  exception  of  spi- 
der's web.  Hlasiwetz  and  Habermann  obtained  29  per  cent  from  casein 
by  cleavage  with  hydrochloric  acid,  while  Kutscher  could  obtain  only 
1.8   per  cent  glutamic  acid    by  cleavage    with    sulphuric    acid.     Other 


'Ellinger,   Ber.   d.   d.   chem.   Gesellsch.,   37;   Neuberg  and  Silbermann,  ibid.,   37;, 
Neuberg  and  Mayer,  Biochem.,  Zeitschr  3;  Neuberg  and  Ascher,  ibid.,  6. 
2  Ber.  d.  d.  chem.  Gesellsch.,  34. 


GLUTAMIC   ACID.  147 

investigators  such  as  Abderhalden  and  Ft  nk  and  Skraup  and  rl  i'KK 
have  shown  that  the  same  quantities  of  glutamic-  acid  can  be  obtained 
by  the  use  of  the  two  mineral  acids.  Skraup  and  Turk  obtained  on  the 
hydrolysis   of   casein   20.3-22.3   per   cent   glutamic   acid    hydrochloride 

corresponding  to  about  17  per  cent  glutamic  acid.  Abderhalden  and 
Sasaki  1  obtained  13.6  per  cent  glutamic  acid  from  meat  syntonin.  It 
occurs  most  abundantly  In  the  plant  proteins  where  the  quantity  may 
lie  more  than  40  per  cent.  Levene  and  Mandel2  have  obtains i  a  strik- 
ingly large  quantity  of  glutamic  acid,  namely  25  per  cent,  from  a  nucleo- 
protein  of  the  spleen. 

On  heating  glutamic  acid  to  180-190°  it  is  converted  into  pyrrolidon- 
carboxylie  acid,  which  latter  can  be  retransforn  ed  into  glutamic  acid 
by  HO  gas;  therefore,  a  formation  of  pyrrolidon-cnrbow  lie  acid  at  the 
same  time,  or  in  place  of  glutamic  acid,  in  the  hydrolases,  is  not  excluded. 

On  putrefaction  glutamic  acid  gives  7-aminobutyric  acid,  n-butyric 
acid  and  succinic  acid. 

tf-Glutamic  acid  crystallizes  in  rhombic  tetrahedra  or  octahedra  or 
in  small  leaves.  It  dissolves  in  100  parts  water  at  16°  C,  and  the  solu- 
tion has  an  acid  taste  with  a  peculiar  after-taste.  It  is  insoluble  in 
alcohol  and  in  ether. 

In  water  it  has  a  rotation  of  (a)D= +12.04°.  Strong  acids  increase 
the  rotation,  and  a  5  per  cent  solution  of  glutamic  acid  containing  9  per 
cent  HO  has  a  rotation  (a)D  = +31.7°,  while  that  obtained  by  heating 
with  barium  hydroxide  is  optically  inactive.  (/-Glutamic  acid  forms 
a  beautifully  crystalline  combination  with  hydrochloric  acid,  which  is 
almost  insoluble  in  concentrated  hydrochloric  acid.  This  compound 
is  used  in  the  isolation  of  glutamic  acid.  On  boiling  with  cupric  hydroxide 
a  beautiful  crystalline  copper  salt,  which  is  soluble  with  difficulty,  is 
obtained.3  The  benzoyl-d-glutamic  acid  melts  at  130-132°  C.  The 
hydrochloride,  the  a-naphthylisocyanate  of  glutamic  acid,  which  melts 
at  23(3-237°  C,  the  analysis  of  the  free  acid,  and  the  specific  rotation 
are  used  in  its  detection. 

As  previously  stated  monamino-oxydicarboxylic  acids  have  also 
been  found  among  the  cleavage  products  of  the  proteins.  To  these  belong 
the  following: 

That  oxyaminosuccinic  acid,  CuH-XOi  occurs  among  the  hydrolytic  cleavage 
products  of  proteids  has  been  shown  to  be  probable  by  Skraup.     This  acid  has 

1  Hlasiwctz  and  Habermann,  Annal.  d.  chem.  u.  Pharm.,  159;  Kutscher.  Zeitschr., 
f.  physiol.  Chem.,  28;  Abderhalden  and  Funk,  ibid.,  53;  with  Sasaki,  ibid.,  51;  Skraup 
and  Turk,  Monatseh.  f.  Chem.,  30. 

2  Bioch.  Zeitschr,  5. 

3  Several  salts  of  £lutamic  acid  have  been  prepared  and  studied  by  Abderhalden 
and  Kautzsch,  Zeitschr.  f.  physiol.  Chem.,  64,  68,  and  78. 


.148  THE  PROTEIN  SUBSTANCES. 

been  prepared  synthetically  by  Neuberg  and  Silbermann  from  diaminosuccinic 
acid  and  barium  nitrite  in  sulphuric  acid  solution.  Oxyaminosuberic  acid, 
C8HisN06,  has  been  detected  by  Wohlgemuth  l  in  the  cleavage  products  of  a 
liver  nucleoprotein. 

/-Cystine,  C6H12N2S2O4  (a-cliamino-/3-dithiolactolic  acid),  the  disulphide 

CH2— S— S— CH2 
of  cysteine  (a-amino-|3-thiolactic  acid),  CH(NH2)       CH(NH2),  was  first 

COOH  COOH 

obtained,  with  certainty,  as  a  cleavage  product  of  protein  substances  by 
K.  Morner,  and  then  also  by  Embden.  Kulz  2  obtained  it  once  as  a 
product  of  tryptic  digestion  of  fibrin.  The  quantities  found  by  Morner 
and  Buchtala  in  the  various  proteins  are  given  in  the  tables  on  pages 
106,  107,  115  and  125. 

According  to  Neuberg  and  Mayer3  two  kinds  of  cystine  occur  in  nature, 
namely,     stone-cystine,  designated  /3-cystine,  and  protein-cystine,  called  a-cystine 

CH2NH2        CH2NH» 
Stone-cystine  is  the  disulphide  of  /3-amino-a-thiolactic  acid,  CH — S — S — CH 
c  COOH  COOH 

The  protein-cj'stine  has  been  chiefly  obtained  from  the  protein  substance, 
but  also  from  calculi,  while  the  stone-cystine  has  been  obtained  from  urinary 
calculi  only. 

Many  objections  have  been  raised  from  many  sides  as  to  the  correctness  of 
this  assumption.  Rothera  could  not  find  any  difference  between  the  stone- 
cystine  and  the  cystine  prepared  from  hair,  and  Fischer  and  Suzuki,  and  recently 
also  Abderhalden,4  arrived  at  similar  results,  which  seems  to  place  the  exist- 
ence of  stone-cystine  in  doubt.  The  occurrence  of  two  structurally  isomeric 
cystines  is  not  improbable,  from  certain  observations  of  Morner,  but  Friedmann 
and  Baer5  have  shown  that  these  observations  do  not  lead  to  this  assumption 
and  at  the  present  time  we  cannot  admit  of  the  occurrence  of  two  different  cystines; 

Cystine  probably  occurs  normally  as  traces  in  the  urine.  In  rare 
cases,  in  cystinuria,  it  occurs  in  larger  quantities  in  the  urine,  the  sediment 
or  in  calculi.  Traces  have  also  been  found  in  the  ox-kidney,  in  the  liver 
of  the  horse  and  dolphin,  and  in  the  liver  of  a  drunkard.  Abder- 
halden 6  has  found  cystine  in  the  urine  and  also  abundantly  in  the 
organs  (spleen)  in  a  case  of  parental  cystine  diathesis. 

The  constitution  of  cystine  has  been  explained  by  Friedmann,7  and 

1  Skraup,  Zeitschr.  f.  physiol.  Chem.,  42;  Neuberg  and  Silbermann,  ibid.,  44; 
Wohlgemuth,  ibid.,  44. 

2  K.  Morner,  ibid.,  28,  34,  and  42;  Embden,  ibid.,  32;  Kulz,  Zeitschr.  f.  Biologie, 
27. 

3  Zeitschr.  f.  physiol.  Chem.,  44. 

4  Rothera,  Journ.  of  Physiol.,  32;  Fischer  and  Suzuki,  Zeitschr.  f.  physiol.  Chem., 
45;  Abderhalden,  ibid.,  51. 

Friedmajon,  Hofmeister's  Beitrage,  3.     With  Baer,  ibid.,  8. 
•  Zeitschr.  f.  physiol.  Chem.,  38. 
7  Hofmeister's  Beitrage,  3. 


CYSTINE.  149 

he  has  also  established  the  relation  between  cystine  and  taurine.  Cys- 
tine is  the  disulphide  of  cysteine,  which  is  a-amino-/3-thiola<t ic  acid. 
From   cysteine    by    oxidation   Friedmann    obtained    cysteinic   acid, 

CH2SO2OH 
C3H7NS06=CH(NH2),  from  which  taurine  CH2(S02OH)  is  produced'by 

COOH  CH2(NH2) 

splitting  off  C02. 

Cystine  has  also  been  prepared  synthetically  in  several  ways.  For 
example,  Fischer  and  Raske  *  have  prepared  cystine  from  a-amino- 
/S-chlorpropionic  acid  (obtained  from  Z-serine)  by  the  action  of  barium 
hydrosulphide  and  a  subsequent  oxidation  in  the  air. 

/-Cystine  crystallizes  in  thin,  colorless,  hexagonal  plates.  It  is  not 
soluble  in  water,  alcohol,  ether,  or  acetic  acid,  but  dissolves  in  mineral 
acids  and  oxalic  acid.  It  is  also  soluble  in  alkalies  and  ammonia,  but 
not  in  ammonium  carbonate.  Cystine  is  optically  active,  being  levorota- 
tory.  Morner  found  it  to  be  (a)D= —224.3°.  On  heating  with  hydro- 
chloric acid  it  can,  according  to  Morner,  be  changed  into  a  modifica- 
tion crystallizing  in  needles  and  with  a  weaker  levorotatory  power,  or 
indeed  dextrorotatory,  composed  of  a  mixture  of  the  two  optically  active 
cystines.  On  heating  with  HC1  to  165°  for  12-15  hours  Neuberg  and 
Mayer  obtained  inactive  cystine.  By  fungus  fermentation  with  Asper- 
gillus niger  they  obtained  dextrorotatory  cystine.  Cystine  has  no 
melting-point  but  slowly  decomposes  at  258-261°.  On  boiling  cystine 
with  caustic  alkali  it  decomposes  and  yields  alkali  sulphide,  which  can 
be  detected  by  lead  acetate  or  sodium  nitroprusside.  According  to  Mor- 
ner2 75  per  cent  of  the  total  sulphur  is  separated.  Cystine  treated 
with  tin  and  hydrochloric  acid  develops  only  a  little  sulphureted 
hydrogen,  and  is  converted  into  cj^steine.  Cystine  yields  sulphureted 
hydrogen  and  methyl  mercaptan  on  putrefaction. 

On  heating  upon  platinum-foil  cystine  ignites  and  burns  with  a  bluish- 
green  flame,  with  the  generation  of  a  peculiar  sharp  odor.  When  warmed 
with  nitric  acid  it  dissolves  with  decomposition,  and  leaves  on  evapora- 
tion a  reddish-brown  residue,  which  does  not  give  the  murexid  test. 

Cystine  is  gradually  precipitated  from  its  sulphuric  acid  solution  by 
phosphotungstic  acid.  Cystine  forms  crystalline  salts  with  mineral 
acids  and  with  bases.  For  isolating  and  separating  cystine  the  precipita- 
tion with  mercuric  acetate  is  especially  suited.  The  benzoyl  cystine 
(Baumann  and  Goldmann3)  melts  at  180-181°;  the  phenylisocyanate 
compound  at  160°.     On  boiling  with  25  per  cent  hydrochloric  acid  this 

1  See  Erlenmeyer  and  Stoop,  Ber.  d.  d.  chem.  Gesellsch.,  36;  Gabriel,  ibid.,  38;  Fischer 
and  Raske,  ibid.,  41. 

2  Zeitschr.  f.  physiol.  Chem.,  34. 

3  Ibid.,  12. 


150  THE  PROTEIN  SUBSTANCES. 

compound  passes  to  the  anhydride,  which  is  a  hydantoin  melting  at 
119°  C.  By  the  action  of  potassium  cyanide  Mauthner  l  obtained 
a-amino-jS-suphocyanpropionic  acid,  CH2(SCN).CH(NH2)COOH. 

Stone-cystine,  according  to  Neuberg  and  Mayer,  differs  in  many  respects 
from  the  ordinary  cystine,  among  which  the  following  may  be  mentioned:  The 
optically  active  stone-cystine  crystallizes  in  needles,  the  specific  rotation  is 
(a)D=-206°;  it  melts' at  190-192°  with  marked  swelling  up.  The  benzoyl 
compound  melts  at  157-159°;  the  phenylcyanate  compound  melts  at  170-172°, 
and  it  is  not  changed  on  boiling  with  hydrochloric  acid, 

In  the  detection  and  identification  of  cystine  we  make  use  of  the 
crystalline  form,  the  behavior  on  heating  on  platinum-foil,  and  the  sul- 
phur reaction  after  boiling  with  alkali.  As  to  its  preparation  from  protein 
substances  see  K.  Morner  and  Folin  2.  In  regard  to  the  detection 
of  cystine  in  the  urine  see  Chapter  XIV. 

CH2.SH 

Cysteine  (a-amino-/3-thiolactic  acid),  C3H7NS02=CH(NH2),    is    formed   from 

COOH 
cystine  by  reduction  with  tin  and  hydrochloric  acid.      It  is  also  produced  in  the 
cleavage  of  protein  substances  not  as  Embden  believes  as  a  primary  formation 
but  according  to  Morner  and  Patten  3  as  a  secondary  formation.     Cysteine 
can  be  easily  converted  into  cystine  by  oxidation. 

According  to  V.  Arnold4  cysteine  occurs  as  a  constituent  of  the  press-juice 
or  extracts  of  various  animal  organs.  He  has  found  it  especially  in  the  hair  and 
he  considers  it  as  a  primary  cell  constituent. 

Toward  alkalies  and  lead  acetate  it  acts  like  cystine.  With  sodium  nitro- 
prusside  and  alkali  it  gives  a  deep  purple-red  coloration;  with  ferric  chloride 
the  solution  gives  an  indigo-blue  coloration  which  quickly  disappears. 

CH3 

Thiolactic  acid  (a-thiolactic  acid),  C3K6S02=CH(SH),  has  been  found  once 

COOH 
as  a  cleavage  product  of  ox-horn  by  Baumann  and  Sitter.  Morner,  Fried- 
mann  and  Baer  obtained  it  from  cystine.  It  has  been  shown  by  Friedmann 
that  this  acid  is  a  regular  cleavage  product  of  keratin  substances,  and  that  it 
can  also  be  obtained  from  the  proteins.  Frankel  5  obtained  the  acid  from 
haemoglobin.  The  pyroracemic  acid  obtained  by  Morner  as  a  decomposition 
product  from  several  protein  substances  originates,  according  to  Morner,  only 
in  part  from  the  cystine. 

Taurine  (aminoethylsulphonic  acid),  C2H7NS03  =  Act- (SO  Vym  '  ^as 
not  been  obtained  as  a  cleavage  product  of  protein  substances;    still  its 

1  Zeitschr.  f.  physiol.  Chem.,  78. 

2  Morner,  Zeitschr.  f.  physiol.  Chem.,  34;  Folin,  Journ.  of  Biol.  Chem.,  8. 

3  See  foot-note  2,  rage  80. 
♦Zeitschr.  f.  physiol.  Chem.,  70. 

5  Morner,  Zeitschr.  f.  physiol.  Chem.,  42;  Suter,  Zeitschr.  f.  physiol.  Chem.,  20; 
Friedmann,  Hofmeister's  Beitrage,  3;  with  Baer,  ibid.,  8;  Frankel,  Sitzungsber.  d. 
Wien.  Akad.,  112,  II,  b,  1903. 


TAURINK.  151 

origin  from  proteins  has  been  shown  by  Friedmann  by  the  close  rela- 
tion that  taurine  bears  to  cysteine;  and  this  is  the  reason  why  it  is 
treated  here  in  connection  with  the  amino-acids. 

Taurine  is  especially  known  as  a  cleavage  product  of  taurocholic 
acid,  and  may  occur  to  a  slight  extent  in  the  intestinal  contents.  Taurine 
has  also  been  found  in  the  lungs  and  kidneys  of  oxen  and  in  the  blood 
and  muscles  of  cold-blooded  animals. 

Taurine  crystallizes  in  colorless,  often  in  large,  shining,  4-  or  6-sided 
prisms.  It  dissolves  in  15-16  parts  of  water  at  ordinary  temperatures, 
but  rather  more  easily  in  warm  water.  It  is  insoluble  in  absolute  alcohol 
and  ether;  in  cold  alcohol  it  dissolves  slightly,  but  better  when  warm. 
Taurine  yields  acetic  and  sulphurous  acids,  but  no  alkali  sulphides, 
on  boiling  with  strong  caustic  alkali.  The  content  of  sulphur  can  be 
determined  as  sulphuric  acid  after  fusing  with  saltpeter  and  soda. 
Taurine  combines  with  metallic  oxides.  The  combination  with  mercuric 
oxide  is  white,  insoluble,  and  is  formed  when  a  solution  of  taurine  is  boiled 
with  freshly  precipitated  mercuric  oxide  (J.  Lang  1).  This  compound 
may  be  used  in  detecting  the  presence  of  taurine.  Taurine  is  not  pre- 
cipitated b}r  metallic  salts. 

The  preparation  of  taurine  from  ox-bile  is  very  simple.  The  bile 
is  boiled  a  few  hours  with  hydrochloric  acid.  The  filtrate  from  the 
dyslysin  and  choloidic  acid  is  concentrated  well  on  the  water-bath,  and 
filtered  hot  so  as  to  remove  the  common  salt  and  other  substances  which 
have  separated.  The  solution  is  evaporated  to  dryness  and  the  residue 
dissolved  in  5  per  cent  hydrochloric  acid,  and  precipitated  with  10  vols. 
95  per  cent  alcohol.  The  crystals  are  readily  purified  by  recrystalliza- 
tion  from  water. 

The  acid  alcoholic  solution  can  be  used  for  the  preparation  of  glycocoll.  After 
the  evaporation  of  the  alcohol,  the  residue  is  dissolved  in  water,  treated  with  a 
solution  of  lead  hydroxide,  filtered,  the  lead  removed  by  H2S,  and  the  filtrate 
strongly  concentrated.  The  crystals  which  separate  are  dissolved  and  decolor- 
ized by  animal  charcoal  and  the  solution  then  evaporated  to  crystallization. 

Though  taurine  shows  no  positive  reactions,  it  is  chiefly  identified 
by  its  crystalline  form,  by  its  solubility  in  water  and  insolubility  in 
alcohol,  by  its  combination  with  mercuric  oxide,  by  its  non-precipitability 
by  metallic  salts,  and  above  all  by  its  sulphur  content. 

/-Phenylalanine  (phenyl-a-aminopropionic  acid), 

C6H5 
CH2. 
C9HnN02  =  CH(NH2)i 

COOH 


1  See  Maly's  Jahresber,  6. 


152  THE  PROTEIN  SUBSTANCES. 

was  first  found  by  E.  Schulze  and  Barbieri  l  in  etiolated  lupin  sprouts. 
It  is  produced  in  the  acid  cleavage  of  protein  substances  in  quantities 
rarely  above  5-6  per  cent.  It  has  been  prepared  synthetically  in  several 
ways  by  Erlenmeyer,  Jr.,  Sorensen  and  E.  Fischer,  Wheeler  and 
Hoffman.2 

The  /-phenylalanine  crystallizes  in  small,  shining  leaves  or  fine  needles 
which  are  rather  difficultly  soluble  in  cold  water  but  readily  soluble  in 
hot  water.  The  solution  has  a  faint  bitter  taste.  A  5-per  cent  solution 
acidified  with  hydrochloric  acid  or  sulphuric  acid  is  precipitated  by 
phosphotungstic  acid,  while  a  more  dilute  solution  is  not  precipitated. 
On  putrefaction,  phenylalanine  yields  phenylacetic  acid.  On  heat- 
ing with  potassium  dichromate  and  sulphuric  acid  (25  per  cent)  an  odor 
of  phenylacetaldehyde  is  produced  and  benzoic  acid  is  formed.  In 
aqueous  solution  it  has  a  rotation  of  (a)D  =  —  35.1°.  The  phenyliso- 
cyanate-1-phenylalanine  melts  at  about  182°  C. 

/-Tyrosine  (p-oxyphenyl-a-aminopropionic  acid), 

C6H4(OH) 

C9HiiN03  = 


CH2 


CH(NH2)' 
COOH 

is  produced  from  most  protein  substances  under  the  same  conditions 
as  leucine,  which  it  habitually  accompanies.  The  largest  quantity  of 
tyrosine  obtained  from  animal  proteins  was  about  10-13  per  cent  (see 
tables,  pages  106,  107,  115  and  125).  In  gelatin  and  a  few  keratins 
tyrosine  is  absent.  It  is  especially  found  with  leucine,  in  large  quantities, 
in  old  cheese  (Tupos),  from  which  it  derives  its  name.  Tyrosine  has  not 
been  found  with  certainty  in  perfectly  fresh  organs.  It  occurs  in  the 
intestine  during  the  digestion  of  protein  substances,  and  it  has  about 
the  same  physiological  and  pathological  importance  as  leucine. 

Tyrosine  was  prepared  by  Erlenmeyer  and  Lipp  from  p-amino- 
phenylalanine  by  the  action  of  nitrous  acid,  and  according  to  another 
method  by  Erlenmeyer  and  Halsey.3  On  fusing  with  caustic  alkali 
it  yields  p-oxybenzoic  acid,  acetic  acid,  and  ammonia.  On  putrefaction 
it  may  yield  oxyphenylethylamine,  oxyphenylpropionic  acid,  oxyphenyl- 
acetic  acid,  p-cresol  and  phenol. 

1  Ber.  d.  d.  chem.  Gesellsch.,  14,  and  Zeitschr.  f.  physiol.  Chem.,  12. 

2  Erlenmeyer,  Annal.  d.  Chem.  u.  Pharm.,  275;  Sorensen,  Zeitschr.  f.  physiol. 
Chem.,  44;  E.  Fischer,  Ber.  d.  d.  chem.  Gesellsch.,  37;  Wheeler  and  Hoffman,  Amer. 
Chem.  Journ.,  45. 

3  Erlenmeyer  and  Lipp,  Ber.  d.  d.  chem.  Gesellsch.,  15;  Erlenmeyer  and  Halsey, 
ibid.,  80. 


TYROSINE.  153 

Naturally  occurring  tyrosine  and  that  obtained  by  the  cleavage  of 
protein  substances  by  acids  or  enzymes,  is  generally  /-tyrosine,  while 
that  obtained  by  decomposition  with  baryta-water  or  prepared  syn- 
thetically is  inactive,  v.  Lippmann  l  has  obtained  d-tyrosine  from 
beet-sprouts.  The  statements  as  to  specific  rotation  of  tyrosine  are 
somewhat  variable.  For  tyrosine  from  proteins  E.  Fischer  has  found 
a  rotation  of  (a)D=— 12.56  to  13.2°  for  the  hydrochloric  acid  solution, 
while  Schulze  and  Winterstein  2  obtained  higher  results  using  tyrosine 
from  plants,  namely,  (a)D=— 16.2°. 

Tyrosine  in  a  very  impure  state  occurs  in  the  form  of  balls  similar 
to  leucine.  The  purified  tyrosine,  on  the  contrary,  appears  as  colorless, 
silky,  fine  needles  which  are  often  grouped  into  tufts  or  balls.  It  is  diffi- 
cultly soluble  in  water,  being  dissolved  by  2454  parts  of  water  at  20°  C, 
and  154  parts  boiling  water,  separating,  however,  as  tufts  of  needles  on 
cooling.  It  dissolves  more  easily  in  the  presence  of  alkalies,  ammonia, 
or  a  mineral  acid.  It  is  difficultly  soluble  in  acetic  acid.  Crystals  of 
tyrosine  separate  from  an  ammoniacal  solution  on  the  spontaneous 
evaporation  of  the  ammonia.  One  hundred  parts  glacial  acetic  acid 
dissolve  on  boiling  only  0.18  part  tyrosine,  and  by  this  means,  especially 
on  adding  an  equal  volume  of  alcohol  before  boiling,  the  leucine  can  be 
quantitatively  separated  from  the  tyrosine  (Habermann  and  Ehren- 
feld  3) .  The  Z-tyrosine-ethyl-ester  crystallizes  in  colorless  prisms  which 
melt  at  108-109°  C.  The  naphthylisocyanate-Z-tyrosine  melts  at  205- 
206°.  Tyrosine  can  be  oxidized  with  the  formation  of  dark-colored 
products  by  various  plant  as  well  as  animal  oxidases,  so-called  tyro- 
sinases (see  Chapters  XV  and  XVI).  In  alcoholic  fermentation  of  sugar 
the  tyrosine  present  at  the  same  time  is  transformed  according  to  F. 
Ehrlich4  into  tyrosol  (p-oxyphenylethyl  alcohol),  C8H10O2.  Tyrosin  is 
identified  by  its  crystalline  form  and  by  the  following  reactions: 

Piria's  Test.  Tyrosine  is  dissolved  in  concentrated  sulphuric  acid 
by  the  aid  of  heat,  by  which  tyrosine-sulphuric  acid  is  formed;  it  is 
allowed  to  cool,  diluted  with  water,  neutralized  by  BaCC>3,  and  filtered. 
On  the  addition  of  a  solution  of  ferric  chloride  the  filtrate  gives  a  beautiful 
violet  color.  This  reaction  is  disturbed  by  the  presence  of  free  mineral 
acids  and  by  the  addition  of  too  much  ferric  chloride. 

Hofmann's  Test.  If  some  water  is  poured  on  a  small  quantity  of 
tyrosine  in  a  test-tube  and  a  few  drops  of  Millon's  reagent  added  and 

1  Ber.  d.  d.  chem.  Gesellsch.,  17. 

2  See  Hoppe-Seyler-Thierfelder,-Handb.  d.  physiol.  u.  pathol.  chem.  Analyse,  8. 
Aufl.,  1909.  Also  E.  Fischer,  Ber.  d.  d.  chem.  Gesellsch.,  32;  Schulze  aud  Winter- 
stein, Zeitschr.  f.  physiol.  Chem.,  45. 

3  Zeitschr.  f.  physiol.  Chem.,  37. 

4  Ber.  d.  d.  chem.  Gesellsch.,  44. 


154  THE  PROTEIN  SUBSTANCES. 

then  the  mixture  boiled  for  some  time,  the  liquid  becomes  a  beautiful 
red  and  then  yields  a  red  precipitate. 

Deniges'  Test,  modified  by  C.  Morner,  is  performed  as  follows: 
To  a  few  cubic  centimeters  of  a  solution  consisting  of  1  vol.  formaline, 
45  vols,  water,  and  55  vols,  concentrated  sulphuric  acid  add  a  little 
tyrosine  in  substance  or  in  solution  and  heat  to  boiling.  A  beautiful 
permanent  green  coloration  is  obtained. 

Folin  and  Denis's  test.  The  reagent  consists  of  a  solution  containing 
10  per  cent  sodium  tungstate,  2  per  cent  phosphomolybdic  acid  and 
10  per  cent  phosphoric  acid.  In  performing  the  test  mix  1-2  cc.  of  the 
reagent  with  an  equal  volume  of  the  tyrosine  solution  and  then  add  3-10 
cc.  saturated  sodium  carbonate  solution  when  a  beautiful  blue  color 
results.  Its  delicacy  is  1:1000000.  The  reagent  can  also  be  used  for  the 
colorimetric  quantitative  estimation  of  tyrosine  in  proteins.  According 
to  Abderhalden  and  Fuchs  and  to  Abderhalden1  the  reagent  suggested 
by  Folin  and  Denis  for  tyrosine  also  gives  a  blue  coloration  with  trypto- 
phane, oxytryptophane  and  Z-oxyproline  and  the  value  of  this  reagent  for 
quantitative  tyrosine  determinations  requires  further  testing. 

H2C — CH2 

I       I 
/-Proline  (a-pyrolidine  carboxylic  acid),  C5H9N02  =  H2C     CH.COOH, 


NH 

was  first  obtained  by  E.  Fischer  and  then  by  Fischer  and  collabora- 
tors from  several  proteins  as  a  primary  cleavage  product  (Abder- 
halden and  Kautzsch2).  The  proline  here  obtained  was  generally 
the  laevo-rotatory  modification.  The  largest  quantity  of  proline  was 
secured  from  the  vegetable  proteins  hordein  and  gliadin,  namely,  13.7 
per  cent  and  13.2  per  cent,  and  also  from  gelatin,  7.7  per  cent  (see  table 
pages  106,  107,  115  and  125).  Kossel  and  Darin3  obtained  11  per  cent 
from  salmine.  Proline  also  occurs  in  scombrine  and  clupeine,  but  not  in 
sturine,  which,  according  to  Kossel,  seems  to  contradict  the  view  as 
to  the  common  origin  of  ornithine  and  proline. 

Sorensen,4  by  means  of  a  general  method  of  preparing  a-amino- 
acids  synthetically,  has  prepared  a-amino-5-cxy valeric  acid  from  phthali- 
midemalonic  ester  and  has  obtained  proline  from  this  by  evaporating  with 


1  Deniges,  Compt.  rend.,  130;  C.  Th.  Morner,  Zeitschr.  f.  physiol.  Chem.,  37;  Folin 
and  Denis,  Journ.  of  Biol.  Chem.,  12;  Abderhalden  and  Fuchs,  Zeitschr.  f.  physiol. 
Chem.,  83  and  Abderhalden,  ibid.,  85. 

2E.  Fischer,  Zeitschr.  f.  physiol.  (.'hem.,  33  and  35.  See  also  footnote  2,  p.  86, 
and  Abderhalden  and  Kautzsch,  Zeitschr.  f.  physiol.  Chem.,  78. 

1  Zeitschr.  f.  physiol.  Chem.,  41. 

4  Zeitschr.  f.  physiol.  Chem.,  44;  with  A.  C.  Anderson,  ibid.,  56. 


TRYPTOPHANE.  155 

hydrochloric  acid,  at  the  same  time  splitting  off  water.  Recently  he 
has  suggested  another  method  which  yields  good  rasults.  Other  syn- 
theses of  proline  have  also  been  performed  by  E.  Fischer  and  Will- 
statter.1  By  the  reduction  of  the  ethyl  ester  of  pyrrolidon  carboxylic 
acid  (see  glutamic  acid)  E.  Fischer  and  Boehner2  have  obtained  racemic 
a-proline.  On  putrefaction  proline  yields  5-amino-valeric  acid  and  n- 
valeric  acid  (Neuberg  and  Ackermann3). 

/-Proline  crystallizes  in  flat  needles.  It  is  readily  soluble  in  water 
and  alcohol.  The  solution  has  a  sweet  taste;  the  specific  rotation  at 
20°  C.  is  (a)D=  —77.40°.  The  solution  acidified  with  sulphuric  acid  is 
precipitated  by  phosphotungstic  acid.  In  the  detection  of  this  acid 
we  make  use  of  the  copper  salt,  the  anhydride  of  the  phenylisocyanate 
compound  (melting-point  14-i°),  and  the  picrate.  The  inactive  acid  and 
its  compounds  show  somewhat  different  properties. 

Oxyproline  (oxy-a-pyrolidine  carboxylic  acid),  C5H9NO3.  This  acid, 
whose  constitution  is  not  understood  was  first  obtained  by  E.  Fischer 
on  the  hydrolysis  of  casein  and  of  gelatin.  It  dissolves  readily  in  water; 
has  a  specific  rotation  of  (a)D=  —81.04°,  and  the  solution  has  a  sweet 
taste.  Oxyproline  crystallizes  in  beautiful  colorless  plates  and  gives 
a  readily  soluble  copper  salt.  The  constitution  of  natural  oxyproline 
has  recently  been  explained  by  Leuchs  and  Brewster.4  They  find 
that  the  natural  oxyproline  is  a  7-oxy-derivative  of  pyrrolidine-a-carbox- 
ylic  acid.  Leuchs  found  the  specific  rotation  of  ^-oxyproline  to  be 
(a)D=_76°  at  20°  C. 

/-Tryptophane  (indol-a-aminopropionic  acid), 

C.CH2.CH(NH2)COOH 
CnHi2N202  =  C6H4<^CH 
NH 

is  one  of  the  cleavage  products  of  the  proteins  formed  in  tryptic  diges- 
tion and  other  deep  decompositions  of  the  proteins,  such  as  putrefaction, 
cleavage  with  baryta-water  or  sulphuric  acid.  It  gives  a  reddish-violet 
product  with  chlorine  or  bromine  which  is  called  yroteinochrome.  Nencki  5 
considered  tryptophane,  which  name  is  generally  given  to  this  acid, 
as  the  mother-substance  of  various  animal  pigments. 

1  Ber.  d.  d.  chem..  Gesellsch.,  S3. 

2  Ber.  d.  d.  Chem.,  Gesellsch.,  44. 

'  Neuberg,  Bioch.  Zeitschr,  37;  Ackermann,  Zeitschr.  of  Biol.,  57. 

4  Fischer,  Ber.  d.  d.  chem.  Gesellsch.,  35  and  36;  Leuchs  and  Brewster,  Ber.  d.  d. 
Chem.,  Gesellsch..  46. 

5  In  regard  to  tryptophane,  see  Stadelmann,  Zeitschr.  f.  Biologic,  26;  Neumeister, 
ibid.,  26;  Nencki,  Ber.  d.  d.  chem.  Gesellsch.,' 28;  Beitler,  ibid.,  31;  Kurajeff,  Zeitschr. 
f.  physiol.  Chem..  26;  King,  Pfliiger's  Arch.,  86. 


156  THE  PROTEIN  SUBSTANCES. 

Tryptophane  was  first  prepared  in  a  pure  form  by  Hopkins  and 
Cole,1  and  they  considered  it  as  skatolaminoacetic  acid.  After  Ellin- 
ger  showed  that  skatolcarbonic  acid  (Salkowski)  and  skatolacetic 
acid  (Nencki)  were  indolacetic  acid  and  indolpropionic  acid  respectively, 
and  after  the  synthesis  of  d-Z-tryptophane  by  Ellinger  and  Flam  and,2 
the  nature  of  this  substance  as  indolaminopropionic  acid  was  established. 

By  condensation  of  /3-indolaldehyde  with  hippuric  acid  Ellinger  and  Flamand 
prepared  the  azlactone  (lactimide) : 

N 
CsHeNCHO+^Qg  6    6  =C8H6N.CH  ;  C      C  .C6H5+2H20. 

CO— O 

On  boiling  with  dilute  caustic  soda,  with  the  taking  up  of  water,  the  sodium 
salt  of  indoxyl-a-benzoylaminoacrylic  acid, 

C8H6N.CH  :  C.NH.COCeH, 
COONa 

is  obtained,  from  which  by  reduction  and  splitting  off  of  the  benzoyl  group  by 
the  action  of  sodium  alcoholate  the  tryptophane  is  obtained : 

C8H6N.CH  :  C.NH.COC6H5 

|  +H2+H20  =C8H6N.CH2.CH.NH2+C6H5COOH. 

COOH  COOH 

The  trytophane  formed  in  digestion  is  /-tryptophane,  which  is  lsevoro- 
tatory  in  aqueous  solution  (Hopkins  and  Cole).  Racemic  ^-trypto- 
phane has  also  been  obtained  by  digestion  in  certain  cases  by  Allers 
and  Neuberg,  this  is  probably  formed  from  the  /-tryptophane  (Abder- 
halden  and  L.  Baumann3),  which  very  readily  undergoes  racemization. 

Tryptophane  crystallizes  in  silky  rhombic  or  six-sided  leaves.  It 
does  not  have  a  sharp  melting-point,  and  according  to  the  rapidity  of  heat- 
ing melts  at  252°,  273°  and  289°,  according  to  various  authorities. 
Tryptophane  is  readily  soluble  in  hot  water,  difficultly  soluble  in  cold 
water,  and*  only  slightly  soluble  in  alcohol.  The  solution  of  d-Z-trypto- 
phane  has  a  faintly  sweetish  taste,  and  /-tryptophane  a  faintly  bitter  taste. 
The  statements  as  to  the  optical  behavior  of  tryptophane  differ  some- 
what, which,  according  to  Abderhalden,  is  probably  due  to  the  readiness 
with  which  it  undergoes  racemization.  According  to  Abderhalden 
and  L.  Baumann,4  at  20°  C.  the  aqueous  solution  has  a  rotation  of 

1  Journ.  of  Physiol.,  27. 

2  Ellinger,  Ber.  d.  d.  Chem.  Gesellsch.,  37  and  38.  With  Flamand,  ibid.,  40,  and 
Zeitschr.  f.  physiol.  Chem.,  55. 

3  R.  Allers,  Biochem.  Zeitschr.,  6;  C.  Neuberg,  ibid.,  6;  Abderhalden  and  Baumann, 
Zeitschr.  f.  physiol.  Chem.,  55.     (Literature  on  the  specific  rotation.) 

*  See  Abderhalden  and  Baumann,  Zeitschr.  f.  physiol.  Chem.,  55  (literature). 


INDOL  AND  SKATOL.  157 

(«)d=  —  30.33°.     Hopkins  and  Cole  give  (a)D=-33°  for  the  watery 

N       N  N 

solution.     It  is  dextrorotatory  in  —  or  —  NaOH  as  well  as  in  —  HC1. 

\       z  1 

Tryptophane  yields  indol  and  skatol  when  sufficiently  heated.  It 
gives  the  Adamkiewicz-Hopkins  *  reaction  and  a  rose-red  color  on  the 
addition  of  chlorine  or  bromine  water  (tryptophane  reaction).  The 
brom-tryptophane  is  readily  soluble  in  amyl  alcohol  or  acetic  ether 
and  on  shaking  with  these  solvents  the  reaction  is  more  delicate.2  If 
a  pine  stick  previously  moistened  with  hydrochloric  acid  and  washed 
with  water  is  introduced  into  a  concentrated  tryptophane  solution, 
it  becomes  purple  (pyrrole  reaction)  on  drying.  The  melting-points 
of  the  benzoylsulphotryptophane,  the  /3-naphthalenesulphotryptophane 
and  the  naphthylisocyanatetryptophane  are  according  to  Ellinger 
and  Flamand,3  185°,  180°  and  158°  C.  respectively.  Several  compounds 
of  tryptophane  have  been  prepared  by  Abderhalden  and  Kempe.4 
Among  these  we  will  mention  the  tryptophane  chloride  hydrochloride, 
because  it  is  used  as  the  starting  material  for  the  synthesis  of  trypto- 
phane polypeptides.  In  the  alcoholic  fermentation  of  sugar,  as  found 
by  F.  Ehrlich  5  the  tryptophane  present  is  transformed  into  tryptophol 
(/3-indoxylethyl  alcohol). 

In  regard  to  the  rather  complicated  method  for  preparing  trypto- 
phane we  must  refer  to  the  original  work  of  Hopkins  and  Cole,  of 
Neuberg,  and  of  Abderhalden  and  Kempe.  Fasal  6  has  suggested  a 
quantitative  colorimetric  method  for  estimating  tryptophane  based 
upon  the  Adamkiewicz-Hopkins  reaction. 

As  shown  by  Hopkins  and  Cole,7  tryptophane  on  anaerobic  putre- 
faction yields  indolpropionic  acid  and  indolacetic  acid,  and  indol  and 
skatol  on  aerobic  putrefaction.  Among  these  putrefactive  products  the 
indol  and  skatol  will  be  specially  discussed. 

CH 

Indol,     C8H7N  =  C6H4<^\CH,     and     Skatol,     or     /3-methylindol, 
NH 


1  In  regard  to  this  reaction  see  also  Dakin,  Journ.  of  Biol.  Chem.  2,  and  O.  Rosen- 
heim, Biochem.  Journ.,  1. 

2  Neuberg,  Bioch.  Zeitschr.,  24. 
»1.  c. 

4  Zeitschr.  f.  physiol.  Chem.,  52,  and  Ber.  d.  d.  chem.  Gesellsch.,  40. 

5  Ber.  d.  d.  chem.,  Gesellsch.,  45. 

6  Hopkins  and  Cole.  Journ.  of  Physiol.,  27  and  29;  Neuberg  and  Popowsky,  Biochem. 
Zeitschr.,  2;  Abderhalden  and  Kempe,  Zeitschr.  f.  physiol.  Chem.,  52;  Fasal,  Bioch. 
Zeitschr.,  44. 

7  Journ.  of  Physiol.,  29. 


158  THE  PROTEIN  SUBSTANCES. 

C.CH3 
C9HgX  =  C6H4<^  /CH,    are    formed    in    variable    quantities   from   pro- 


NH 

tein  compounds  under  different  conditions.  Hence  they  occur  habitually 
in  the  human  intestinal  canal,  and,  after  oxidation  into  in  doxy  1  and 
skatoxyl  respectively,  pass,  at  least  partly,  into  the  urine  as  the  cor- 
responding ethereal  sulphuric  acids,  and  also  as  glucuronic  acids. 

Indol  and  skatol  crystallize  in  shining  leaves,  and  their  melting- 
points  are  52°  and  95°  C.  respectively.  Indol  has  a  peculiar  excremen- 
titious  odor,  while  skatol  has  an  intense  fetid  odor.  Both  bodies  are  easily 
volatilized  by  steam,  skatol  more  easily  than  indol.  They  may  both  be 
removed  from  the  watery  distillate  by  ether.  Skatol  is  the  more  insoluble 
of  the  two  in  boiling  water.  Both  are  easily  soluble  in  alcohol  and  give 
with  picric  acid  a  compound  crystallizing  in  red  needles.  If  a  mixture 
of  the  two  picrates  be  distilled  with  ammonia,  they  both  pass  over  with- 
out decomposition;  while  if  they  are  distilled  with  caustic  soda,  the  indol 
but  not  the  skatol  is  decomposed.  The  watery  solution  of  indol  gives 
with  fuming  nitric  acid  a  red  liquid  and  then  a  red  precipitate  of  nitroso- 
indol  nitrate  (Nencki1).  It  is  better  first  to  add  two  or  three  drops 
of  nitric  acid  and  then  a  2-per  cent  solution  of  potassium  nitrite,  drop 
by  drop  (Salkowski  2).  Skatol  does  not  give  this  reaction.  An  alcoholic 
solution  of  indol  treated  with  hydrochloric  acid  colors  a  pine  chip  cherry- 
red.  Skatol  does  not  give  this  reaction.  Indol  gives  a  deep  reddish-violet 
color  with  sodium  nitroprusside  and  alkali  (Legal's  reaction).  On 
acidifying  with  hydrochloric  acid  or  acetic  acid  the  color  becomes  pure 
blue.  Skatol  does  not  act  the  same.  The  alkaline  solution  is  yellow 
and  becomes  violet  on  acidifying  with  acetic  acid  and  boiling.  With  a 
few  drops  of  a  4-per  cent  formaline  solution  and  concentrated  sulphuric 
acid  indol  gives  a  beautiful  violet  color  while  skatol  gives  a  yellow  or 
brown  color  (Kondo3).  On  warming  skatol  with  sulphuric  acid  a 
beautiful  purple-red  coloration  is  obtained  (Ciamician  and  Magnanini  4). 
According  to  Sasaki  skatol,  in  methyl  alcohol  free  from  aldehyde, 
gives  with  concentrated  sulphuric  acid  containing  ferric  salt  a  violet- 
red  ring  at  the  juncture  of  the  two  liquids.  Indol  and  tryptophane 
do  not  give  this  reaction.  Deniges  has  carefully  studied  the  behavior 
of  these  two  bodies  with  Ehrlich's  reagent,  dimethylaminobenzaldehyde, 
or  with  cinnamic  aldehyde  and  vanillin.     Comparative  investigations  on 

1  Ber.  <1.  d.  deutsch.  chem.  Gesellsch.,  8,  727,  and  ibid.,  722  and  1517. 

2  Zeit.^flir.  f.  phyBiol.  Chem.,  8,  447.  In  regard  to  newer  reactions  for  indol  and 
skatol,  see  Bteensma,  ibid.,  47,  and  Deniges,  Compt.  rend.  soc.  biol.,  64. 

3  Zeitschr.  f.  physiol.  C\\em.,  48. 

*  Ber.  (1.  d.  chem.  Gesellsch.,  21,  1928. 


HISTIDINE.  159 

the  behavior  of  indol  and  skatol  with  the  aromatic  aldehydes  have  been 
carried  out  by  Blumenthal.1 

For  the  detection  of  indol  and  skatol  in,  and  their  preparation  from, 
faeces  and  putrefying  mixture-,  the  main  points  of  the  usual  method  are 
as  follows:  The  mixture  is  distilled  after  acidifying  with  acetic  acid; 
the  distillate  is  then  treated  with  alkali  (to  combine  with  any  phenols 
which  may  lie  present)  and  again  distilled.  From  this  second  distillate 
the  two  bodies,  after  the  addition  of  hydrochloric  acid,  are  precipitated 
by  picric  acid.  The  precipitated  picrate  is  then  distilled  with  ammonia. 
The  two  bodies  are  obtained  from  the  distillate  by  repeated  shaking 
with  ether  and  evaporation  of  the  several  ethereal  extracts.  The  residue, 
containing  indol  and  skatol,  is  dissolved  in  a  very  small  quantity  of 
absolute  alcohol  and  treated  with  8-10  vols,  of  water.  Skatol  is  precip- 
itated, but  not  the  indol.  The  further  treatment  necessary  for  their 
separation  and  purification  will  be  found  in  other  works.2 

Skatosine,  GoHieNoOo,  is  a  base  first  obtained  by  Baum  in  the  pancreas  auto- 
digestion  and  later  studied  by  Swain.  It  develops  an  indol-  or  skatol-like  odor 
on  fusing  with  potassium  hydroxide.  Langstein  3  obtained  a  substance  which  is 
perhaps  identical  with  skatosine,  in  the  very  lengthy  peptic  digestion  of  blood 
proteins.  ' 

/-Histidine,  C6H9X3O2,  is  j3-imidazol-a-aminopropionic  4 

CH-NHX 

C N^CH 

acid,    =CHo 

CH(NH2) 

COOH 
Histidine  was  first  discovered  by  Kossel  in  the  cleavage  products 
of  sturine.  It  was  found  at  the  same  time  by  Hedin  in  the  cleavage 
products  of  proteins  by  acid  hydrolysis,  and  by  Kutscher  among  the 
products  of  tryptic  digestion,  and  finally  also  as  a  cleavage  product  of 
many  different  animal  and  plant  protein  substances.  It  does  not  occur 
in  the  protamines,  with  the  exception  of  sturine.  Of  the  protein  bodies 
globin    (from   horse-haemoglobin)    seems   to   be   richest    in   histidine,    as 


'Sasaki,  Bioch.  Zeitschr.  23,  29;  Deniges,  Compt.  rend.  soc.  biol.,  64;  Blumenthal, 
Bioch.  Zeitschr.,  19. 

2  For  quantitative,  colorimetric  determinations  of  indol  in  feces  see  Einhorn  and 
Hiibner,  Salkowski's  Festschrift,  Berlin,  1904;  C.  A.  Herter  and  Foster,  Journ.  of 
biol.  Chem.,  2. 

3  Baum,  Hofmesister's  Beitrage,  3;  Swain,  ibid.;  Langstein,  see  Hofmeister,  Ueber 
Bau  und  Gruppierung  der  Eiweisskorper,  in  Ergebnisse  der  Physiologie,  I,  Abt.  1, 
1902. 

1  Sec  Pauly,  Zeitschr.  f.  physiol.  Chem.,  42;  Knoop  and  Windaus,  Hofmeister's 
Beitrage,  7  and  8;  Knoop,  ibid.,  10;  Ackermann,  Zeitschr.  f.  physiol.  Chem.,  65. 


16a  THE  PROTEIN   SUBSTANCES. 

Abderhalden  found  10.96  per  cent.     It  also  occurs  in  germinating  plants 
(E.  Schulze  x). 

Histidine  has  been  prepared  synthetically  by  Pyman.2  4  (5)  chlormethyl 
glyoxalin  yields  with  sodium  chlormalonic  ester  the  glyoxalinmethylchlormalonic 
ester. 

CH.NHx 

II  > 

C N^ 

CH2.CC1(C02.C2H6)2 


>CH  ,  which  on 


hydrolysis    gives    cW-a-chlor-/3-glyoxalin-4    (5)    propionic    acid, 

CH— NHs 


> 


>CH 

C N 

CH2CHCI.COOH 

This  latter  treated  with  NH3  yields  d-Z-histidine,  which  is  changed  into  the  active 
forms  by  means  of  tartaric  acid. 

In  the  anaerobic  putrefaction  of  histidine,  /3-imidazolylethylamine 
and  imidazolylpropionic  acid  are  formed  (Ackermann  3). 

Histidine  crystallizes  in  colorless  needles  and  plates  and  is  readily 
soluble  in  water,  but  less  soluble  in  alcohol,  and  has  an  alkaline  reaction. 
It  is  precipitated  by  phosphotungstic  acid,  but  this  precipitate  is  soluble 
in  an  excess  of  the  precipitant  (Frankel).  With  silver  nitrate  alone 
the  aqueous  solution  is  not  precipitated;  on  the  careful  addition  of 
ammonia  or  baryta-water  an  amorphous  precipitate,  which  is  readily 
soluble  in  an  excess  of  ammonia,  is  obtained.  Histidine  can  be  pre- 
cipitated by  mercuric  chloride,  or,  still  better,  by  the  sulphate  acidified 
with  sulphuric  acid,  and  can  in  this  way  be  separated  from  the  other 
diamino-acids  (Kossel  and  Patten).  The  hydrochloride  crystallizes  in 
beautiful  plates  (Bauer),  dissolves  rather  readily  in  water,  but  is  insolu- 
ble in  alcohol  and  ether.  With  hydrochloric  acid  and  methyl  alcohol 
it  gives  the  dihydrochloride  of  histidine  methyl  ester,  which  melts  at 
196°.  Histidine  is  lsevorotatory,  (a)D= —39.74°,  while  its  solution  in 
hydrochloric  acid  is  dextrorotatory.  On  warming  it  gives  the  biuret  test 
(Herzog),  and  it  also  gives  Weld  el's  reaction  if  performed  as  sug- 
gested by  Fischer  (see  Xanthine,  Chapter  V)  (Frankel4).     On  adding 

1  Kossel,  Zeitsflir.  f.  physiol.  Chem.,  22;  Hedin,  ibid.,  Kutscher,  ibid.,  25;  Wetzel, 
ibid.,  26;  Lawrow,  ibid.,  28,  and  Ber.  d.  d.  chem.  Gesellsch.,  34;  Kossel  and  Kutscher, 
Zeitschr.  f.  physiol.  Chem.,  31;  Hart,  ibid.,  33;  Abderhalden,  ibid.,  37;  Schulze,  ibid.r 
24  and  28. 

-  Cited  from  Chem.  Centralbl.,  1911,  2,  p.  760. 

3  Zeitsr-lir.  f.  physiol.  Chem.,  65. 

*  Kossel  and  Patten,  Zeitschr.  f.  physiol.  Chem.,  38;  Bauer,  ibid.,  22;  Herzog,  ibid.y 
37;  Frankel,  Sitz.-Ber.  d.  Wiea.  Akad.,  112,  II.  B.,  1903,  and  Hofmeister's  Beitrage,  8. 


ARGININE.  161 

sufficient  bromine  water  and  warming,  a  reddish  coloration  ensues 
which  turns  deep  wine-red,  later  becoming  cloudy,  due  to  the  forma- 
tion of  dark  amorphous  particles  (F.  Knoop  ]).  It  gives  a  very  beautiful 
diazo-reaction  with  diazobenzenesulphonic  acid,  in  solutions  made  alkaline 
with  sodium  carbonate,  which  according  to  Pauly  is  deep  cherry-red 
in  dilutions  of  1:20000  and  still  markedly  red  in  1:100000  (tyrosine 
gives  a  similar  reaction). 

Several  salts  of  histidine  are  known;  H.  Pauly2  has  especially 
studied  the  iodized  derivatives  of  histidine  and  imidazole. 

On  feeding  ^-/-histidine  to  rabbits  Abderhalden  and  Weil3  obtained 
from  the  urine  tf-histidine  which  was  crystalline,  was  as  sweet  as  sugar 
and  showed  a  specific  rotation  (a)D= +40.15°  at  20°  C. 

Histidine  is  sometimes  classified  in  a  group,  with  the  two  diamino- 
acids,   arginine  and  lysine  which   Kossel  has  called  the  hexone  bases. 

(/-Arginine  ( 5-guanido-a-aminovaleric  acid), 

(HN)C<NH2CH2 
C6H14N402=  (CH2)2       , 

CH(NH2) 
COOH 

first  discovered  by  Schulze  and  Steiger  in  etiolated  lupin-  and  pumpkin- 
sprouts,  has  later  been  found  in  other  germinating  plants,  in  tubers  and 
roots.  Gulewitsch  has  found  arginine  in  the  ox-spleen,  and  Totani 
and  Katsuyama  have  found  it  in  ox-testicles.  It  was  first  found 
by  Hedin  as  a  cleavage  product  of  horn  substance,  gelatin,  and  several 
proteins,  and  then  by  Kossel  and  his  pupils  as  a  general  cleavage  prod- 
uct of  protein  substances  as  a  class.  The  greatest  quantity  was  obtained 
from  the  protamines;  but  the  histones  and  certain  plant  proteins, 
edestin  and  the  protein  from  pine  seeds  and  especially  excelsin  (14.14 
per  cent),  also  yield  abundant  arginine.  Arginine  also  occurs  among 
the  products  of  tryptic  digestion  (Kossel  and  Kutscher4). 

On  boiling  with  baryta-water,  as  well  as  by  the  action  of  an  enzyme, 
arginase,  discovered  bv  Kossel  and  Dakin,5  arginine  yields  urea  and 
ornithine. 


1  Hofmeister's  Beitrage,  1 1 . 

2  Ber.  d.  d.  chem.  Gesellsch.,  43. 
1  Zeitschr.  f.  physiol.  Chem.,  77. 

4  Schulze  and  Steiger,  Zeitschr.  f.  physiol.  Chem.,  11;  Schulze  and  Castoro,  ibid.,  41; 
Gulewitsch,  ibid.,  30;  Totani  and  Katsuyama,  ibid.,  64;  Hedin,  ibid.,  20  and  21;  Kossel 
and  Kutscher,  ibid.,  22,  25,  26. 

6  Zeitschr.  f.  physiol.  Chem.,  41,  and  Dakin,  Journ.  of  biol.  Chem.,  3. 


162  THE  PROTEIN   SUBSTANCES. 

Arginine  has  been  prepared  synthetically  from  ornithine  (a-5-diamino- 
valeric  acid)  and  cyanamide  by  Schulze  and  Winterstein.  Recently 
Sorensen  and  Hoyrup  x  have  prepared  oM-arginine  from  ornithuric 
acid.     The    a-monobenzoyl    ornithine    obtained    by    splitting  ornithuric 

N 
acid   with    —    barium    hydrate    yields   a-benzoylamino-5-guanido-valeric 

5 
acid  with  cyanamide  and  this  on  boiling  with  hydrochloric  acid  gave 
S-guanido-a-aminovaleric  acid  (eW-arginine) . 

Arginine  crystallizes  in  rosette-like  tufts,  plates,  or  thin  prisms,  is  readily 
soluble  in  water  with  alkaline  reaction  and  almost  insoluble  in  alcohol. 
With  several  acids  and  metallic  salts  it  forms  crystalline  salts  and  double 
salts  respectively.  Its  acidified  watery  solution  is  precipitated  by  phos- 
photungstic  acid.  The  most  important  salts  are  the  copper-nitrate 
(C6Hi4N402)2.Cu(N03)2+3H20  and  the  silver  salts 

C6Hi4N402.HN03+AgN03 

(the  more  readily  soluble)  and  CeHuN^.AgNOa+^O  (the  more 
difficultly  soluble),  and  its  compound  with  picrolonic  acid  (Steudel  2). 

Arginine  is  dextrorotatory.  For  arginine-chloride  in  watery  solu- 
tion with  excess  of  hydrochloric  acid,  Gulewitsch3  found  (a)D  =  +21.25° 
at  20°  C.  The  arginine  obtained  by  Kutscher  in  the  tryptic  digestion 
of  fibrin  was  racemic  arginine.  As  found  by  Kossel  and  Weiss  (see 
page  112)  arginine  or  more  properly  the  ornithine  is  very  easily 
racemerized  within  the  protein  molecule  by  the  action  of  alkali.  The 
racemic  arginine  can,  as  Riesser  4  has  shown,  during  cleavage  by  means 
of  arginase,  yield  Z-arginine,  which  is  an  asymmetric  change.  In^putre- 
faction    arginine    yields    ornithine,    guanidine,    putrescine  and   5-amino- 

valeric  acid. 

/NH2 
Agmatine     (guanidobutylamine),   C6H14N4  =  HN.C<; 

\NH.CH2(CH2)2.CH2NH2, 

is  a  base  obtained  by  Kossel  in  the  hydrolysis  of  herring  sperm,  and  later  by 
Kutscher  and  Engeland  5  from  ergot.  Kossel  has  also  obtained  it  synthetically 
from  cyanamide  and  tetramethylendiamine,  and  in  this  manner  proved  its  con- 
stitution. It  is  produced  from  arginine  by  splitting  off  C02  and  bears  the! same 
relation  to  arginine  that  putrescine  does  to  ornithine  and  cadeverine  does  to 
lysine  (see  below).  Agmatine  gives  several  crystalline  salts  as  described  by 
Kossel.     It  is  precipitated  by  phosphotungstic  acid. 

Schulze  and  Winterstein,  Ber.  d.  d.  chem.  Gesellseh.,  32  and  Zeitschr.  f.  physiol- 
Chem.,  34;  Sorensen  and  Hoyrup,  Ber.  d.  d.  chem.  Gesellseh,  43  and  Zeitschr.  f.  physiol- 
Chem.,  76. 

2  Zeitschr.  f.  physiol.  Chem.,  37  and  44. 

3  Ibid.,  27. 

4  Kutscher,  Zeitschr.  f.  physiol.  Chem.,  28  and  32;  Riesser,  ibid.,  49. 

6  Kossel,  ibid.,  66  and  68;  Engeland  and  Kutscher,  Centralbl.  f.  Physiol.,  24,  479. 


ORNITHINE  AND   LYSINE.  163 

CH2.(NH2) 

(('IT  ^ 

(/-Ornithine  (a-8-diaminovaleric  acid),  CsHtsNjOa'  /...V.,.  kl  is  not  a  primary 

Crl(.\  rl^) 

COOH 
cleavage  product  of  proteins,  but  is  formed  from  arginine  on  boiling  with  baryta- 
water.    Jaffe,1  who  first   discovered  this  body,  obtained  it  as  a  cleavage  product 

from  ornithinic  acid,  which  is  found  in  the  urine  of  hens  fed  with  benzoic  acid. 
The  ornithine  which  E.  Fischer  and  later  Sorensen,2  have  prepared  syn- 
thetically yields,  as  shown  by  Ellinger,  putrescine  (tetramethylene diamine), 
(\H,iX1Ij)j,  on  putrefaction.  A.  Loewy  and  Neuberg  3  have  shown  that 
ornithine  is  split  into  putrescine  and  CO>  in  the  organism  of  cystinuria  patients. 

Ornithine  is  a  non-crystalline  substance  which  dissolves  in  water,  giving  an 
alkaline  reaction,  and  yields  several  crystalline  salts.  It  is  precipitated  by 
phosphotungstic  acid  and  several  metallic  salts,  but  not  by  silver  nitrate  and 
baryta-water  (differing  from  arginine).  Ornithine  hydrochloride  is  dextrorotatory; 
the  synthetically  prepared  one  is  inactive.  On  shaking  ornithine  with  benzoyl 
chloride  and  caustic  soda  it  is  converted  into  dibenzoylornithine  (ornithuric 
acid).  On  splitting  artificially  prepared  racemic  ornithuric  acid  Sorensen  has 
shown  that  the  naturally  occurring  ornithuric  acid  is  identical  with  the  dextro- 
rotatory a-5-dibenzoyldiaminovaleric  acid.  Salts  and  derivatives  of  ornithine 
have  been  described  by  Kossel  and  his  collaborators4  and  they  have  given  a 
method  for  its  isolation  from  mixtures. 

Diaminoacetic  acid,  C,jH6X..Oj=CH(XHj)oCOOH  was  obtained  by  Drechsel  5 
as  a  cleavage  product  of  casein  by  boiling  with  tin  and  hydrochloric  acid.  It 
crystallizes  in  prisms  and  gives  a  monobenzoyl  compound  which  is  not  very  soluble 
in  cold  water  and  is  almost  insoluble  in  alcohol,  and  can  be  usi'd  in  the  isolation 
of  the  acid. 

CH2(NH2) 

d-Lysine   (a-c-diaminocaproicacid),  C6Hi4N20o=  ;ltJ^L  \,  was  first 

C  rl(J\ri2/ 

COOH 

obtained  by  Drechsel  as  a  cleavage  product  of  casein.     Later  he  and 

his  pupils,  as  well  as  Kossel  and  others,  found  it  among  the  cleavage 

products  of  various  proteins.     It  has  not  been  detected  in  some  vegetable 

proteins  such  as  the  prolamines  (page  1C6).     E.  Schulze  found  lysine 

in  germinating  plants  of  the  Lupinus  luteus,  and  Winterstein   found 

it  in  ripe  cheese.     It  has  been  obtained  in  largest  amounts  (28.8  per  cent) 

by  Kossel  and  Dakin  from  the  protamine  a-cyprinine.     From  a  gliadin 

which  was  not  contaminated  and  which  they  considered  as  a  unit  substance 

although  obtained  from   different   fractions  having  different  solubilities 

in  alcohol,  Osborne  and  Leavenworth  6  found  a  small  amount  of  lysine 

1  Ber.  d.  d.  chem.  Gesellsch.,  10  and  11. 

2  Fischer,  Ber.  d.  d.  chem.  Gesellsch,  34;  Sorensen,  Zeitschr.  f.  physiol.  Chem.,  44. 

3  Ellinger,  Zeitschr.  f.  physiol.  Chem.,  29;  Loewy  and  Neuberg,  ibid.,  43. 

4  Kossel  and  Weiss,  Zeitschr.  f.  physiol.  Chem.,  68. 

5  Ber.  d.  k.  sachs.  Gesellsch.  d.  Wiss.,  44 

6  Drechsel,  Arch.  f.  (Anat.  u.)  Physiol.,  1891,  and  Ber.  d.  d.  chem.  Gesellsch.,  25; 
Siegfried,  Arch.  f.  (Anat.  u.)  Physiol.,  1891,  and  Ber.  d.  d.  chem.  Gesellsch.,  24;  Hedin, 
Zeitschr.  f.  physiol.  Chem.,  21;  Kossel,  ibid.,  25;  Kossel  and  Mathews,  ibid.,  25;  Kossel 


164  THE   PROTEIN  SUBSTANCES. 

{0.07  and  0.15  per  cent  in  two  different  fractions).  The  generally 
accepted  view  that  lysine  is  completely  absent  in  gliadin  is  still  doubtful. 
They  could  not  detect  lysine  in  zein  by  the  same  method. 

Lysine  has  been  synthetically  prepared  by  E.  Fischer  and  Weigert.1 
This  lysine  was  racemic,  while  that  prepared  from  protein  is  always 
optically  active  and  dextrorotatory.  The  rotation  depends  upon  the 
concentration  and  degree  of  acidity;  for  the  hydrochloride  a  rotation  of 
(«)d=  +14°  to  17.25°  has  been  found.  .  On  heating  with  barium  hydroxide 
it  is  converted  into  the  racemic  modification.  According  to  Ellinger 
lysine  yields  cadaverine  (pentamethylenediamine),  CsHio(NH~2)2,  on 
putrefaction,  and  this  base  is  formed  from  the  lysine  in  the  organism 
of  those  with  cystinuria  and  at  the  same  time  CO2  is  split  off  (A.  Loewy 
and  Neuberg).2 

Lysine  is  readily  soluble  in  water  but  is  not  crystallizable.  The  aque- 
-ous  solution  is  precipitated  by  phosphotungstic  acid,  but  not  by  silver 
nitrate  and  baryta-water  (differing  from  arginine  and  histidine).  It 
gives  two  hydrochlorides  with  hydrochloric  acid,  and  with  platinum 
chloride  a  chloroplatinate  which  is  precipitable  by  alcohol  and  has  the 
composition  CoHn^C^.H^PtCle-r^HsOH.  It  gives  two  silver  salts 
with  AgNOs;  one  has  the  formula  AgN03+CeHi4N202  and  the  other 
AgN03+CGHi4N202.HN03.  With  benzoyl  chloride  and  alkali,  lysine 
forms  an  acid,  lysuric  acid,  C6Hi2(C7H50)2N202  (Drechsel),  which 
is  homologous  with  ornithuric  acid,  and  whose  difficultly  soluble  acid 
barium  salt  may  be  used  in  the  separation  of  lysine.3  The  rather 
insoluble  picrate,  which  is  precipitated  from  a  not  too  dilute  solution 
of  the  hydrochloride  by  sodium  picrate,  may  also  be  used  in  the  detec- 
tion of  lysine. 

Kutscher  and  Lohmann  4  have  found  a  lysine  having  somewhat  different 
properties  in  the  final  products  of  pancreas  autolysis. 

In  the  preparation  of  the  so-called  hexone  bases  we  can  first  precipitate 
all  the  bases  by  phosphotungstic  acid,  when  the  monamino-acids  remain  in 
solution.  The  precipitate  is  then  decomposed  in  boiling  water  by  barium 
hydroxide  and  the  bases  obtained  as  silver  compounds  from  this  filtrate. 
In  regard  to  further  details  and  the  methods  of  separating  the  various 

and  Kutscher,  ibid.,  31;  Kutscher,  ibid.,  29;  Schulze,  ibid.,  28;  Winterstein,  cited  in 
Schulze  and  Winterstein,  Ergebnisse  der  Physiologic,  I,  Abt.  1,  1902;  Kossel  and 
Dakin,  Zeitschr.  f.  physiol.  Chem.,  40;  Osborne  and  Leavenworth,  Journ.  of  biol. 
Chem.,  14 

1  Ber.  «l.  d.  chem  Gesellsch.,  35. 

2  See  footnote  3,  p.  163. 

3  Drechsel,  Ber.  d.  d.  chem.  Gesellsch.,  28;  see  also  C.  Willdenow,  Zeitschr.  f.  physiol. 
Chem..  2.V 

*  Zeitschr.  f.  physiol.  Chem.,  41. 


DIAMINO-ACIDS.  165 

bases  we  will  refer  to  Steudel  in  Abderhalden's  Handbuch  der  biochem- 
ischen  Arbeitsmethoden,  Bd.  2,  II,  s.  498. 

We  give  below  a  tabulation  of  the  amounts  of  the  three  hexone  bases 
found  in  certain  protein  substances  (in  weight  per  cent): 

Arginine.  Lysine.  HLatidine. 

Sturine1 58.2  L2.0  12.9 

Cyprinine  (a)6 1  9  is  s  0.0 

Other  protamines » 62.5—87.4  0.0  0.0 

Histories1 14.36—15.52  7.7  —8.3  1.21—2.34 

Casein- 4.70—4.84  1.92—5.80  2.53—2.59 

Syntonin  (from  meat)  « 5.06  3  26  2.66 

Heterosyntonose  * 8.53  3.08—7.03  0.37— 11 2 

Protosyntonose 2 4.55  3.08  3.35 

Edestin3 11.0—14.07  1 .3  117 

Proteid  from  conifera:  seeds  3 10 . 9—11 .3  0 .  25—0  79  0 .  62—0  78 

Gluten  casein  J 4  4  2.15  1.16 

Gluten  proteins  « 2.75—  3.13  0.0  0.43—1.53 

Gelatin  '  and  2 7.62—9.3  2.49—6.0  0.40 

Elastin4 0.3  +  0.027 

Of  the  oxydiamino-acids  found  on  the  hydrolysis  of  proteins  we  will 
mention  the  following: 

Oxydiaminosebacic  acid,  (?)  CwHmNjOb,  has  been  isolated  by  Wohlgemuth  « 
from  a  nucleoprotein  of  the  liver.  The  free  acid  was  obtained  as  small  white 
plates.  It  is  soluble  with  difficulty  in  hot  water,  insoluble  in  cold  water  and 
in  alcohol.  It  was  optically  inactive  in  hydrochloric  acid.  The  beautifully 
crystalline  phenylcyanate  compound  had  a  melting-point  of  206°. 

Dioxydiaminosuberic  acid,  C8H,6X>06.  has  been  obtained  by  Skraup  7  on  the 
hydrolysis  of  casein  with  hydrochloric  acid.  The  copper  salt  crystallizes  in 
beautiful  deep  bluish-violet  rosettes  which  are  composed  of  long,  irregular,  right- 
angled  plates.  It  is  quite  soluble  in  cold  water.  The  free  acid  crystallizes  in 
fern-like  formations.  Besides  this  acid  Skraup  obtained  two  other  acids  which 
he  calls  caseanic  acid,  C9H16N2O7,  and  caseinic  acid,  C12H24N0O5.  The  caseanic 
acid  crystallizes,  melts  at  190-191°,  is  tribasic,  and  is  probably  an  oxydiamino- 
acid.  The  caseinic  acid  is  dibasic  and  occurs  in  two  modifications.  The  one 
which  melts  at  228°  is  faintly  dextrorotatory;  the  other  modification  melts 
at  245°  and  is  optically  inactive.  Both  crystallize,  but  the  inactive  form  does 
not  yield  well-defined  crystals.  Caseinic  acid  seems  also  to  be  an  oxydiamino- 
acid. 

Diaminotrioxydodecanoic  acid,  Ci2H26N>05,  is  an  acid  obtained  by  Fischer  and 
Abderhalden  8  on  the  hydrolysis  of  casein  and  seems  to  stand  close  to  Skraup's 
caseinic  acid,  but  differs  from  it  in  its  optical  properties.  This  acid  is  faintly 
levorotatory:  (a)D  =about— 9°.     It  crystallizes  in  plates,  which  grow  into  rosettes 

1  Kossel  and  Kutscher,  Zeitschr.  f.  physiol.  Chem.,  31. 
*  Hart,  ibid.,  33. 

3  Schulze  and  Winterstein,  ibid.,  33;  see  also  Kossel,  Ber.  d.  d.  chem.  Gesellsch., 
34,  3236. 

4  Kossel  and  Kutscher,  Zeitschr.  f.  physiol.  Chem.,  25,  and  Richards  and  Gies, 
Amer.  Journ.  of  Physiol.,  7. 

5  Kossel  and  Dakin,  Zeitschr.  f.  physiol.  Chem.,  40. 

6  Ber.  d.  d.  chem.  Gesellsch.,  37,  and  Zeitschr.  f.  physiol.  Chem.,  44. 

7  Zeitschr.  f.  physiol.  Chem.,  42. 

8  Zeitschr.  f.  physiol.  Chem..  42. 


166  THE  PROTEIN  SUBSTANCES. 

or  spherical  aggregations.  It  has  a  faint  bitter  taste,  gives  a  crystalline  hydro- 
chloride which  is  slightly  soluble  in  strong  hydrochloric  acid,  and  gives  a  crys- 
talline copper  salt. 

After  describing  the  different  amino-acids  it  remains  for  us  to  call 
attention  to  certain  general  reactions  of  the  amino-acids. 

By  the  action  of  formaldehyde  the  amino  groups  are  changed  into 
methylene  groups  according  to  the  scheme: 

R.CH.NH2  R.CH.N  :  CH2 

+HCOH  =      I  +H20. 

COOH  COOH 

The  amino-acids  behave  like  neutral  bodies  while  the  methylene 
combinations  are  acids  and  on  this  behavior  is  based  Sorensen's  1 
formoltitration  which  serves  for  the  estimation  of  amino-acids  in  the 
urine  (Chapter  XI V)  as  well  as  to  follow  the  progress  of  proteolysis. 
As  the  proteolysis  progresses  and  imide  bindings  are  loosened  a  large 
number  of  atomic  complexes  with  free  NH2  and  COOH  groups  are  set 
free.  If  now  the  NH2  groups  are  fixed  as  methylene  groups  by  the  addi- 
tion of  formol,  the  complex  behaves  like  acids  and  the  number  of  their 

.  .       .  N 

COOH  groups  can  be  determined  by  titration  with  —  barium  or  sodium 

5 

hydroxide  solution,  using  phenolphthalein  or  thymolphthalein  as  in- 
dicator. With  the  presumption  that  for  every  COOH  group  set  free 
there  existed  a  free  NH2  group  the  extent  of  the  proteolysis  can  also 
be  expressed  in  milligrams  N  by  multiplying  the  number  of  cubic  centi- 

N 
meters  —  alkali  used  by  2.8. 
5 

Siegfried  has  found  that  amino-acids  in  the  presence  of  alkali  or 

alkaline  earths  de-ionize  carbon  dioxide  and  form  salts  of  the  type  of 

the  carbamino  salts,  Siegfried's  "  carbamino-reaction."     For  example 

glycocoll  in  the  presence  of  lime  yields  with  carbon  dioxide,   calcium 

carbamino-acetic  acid,  CH2.NH.COO 

I  I    • 

COO Ca 

If  the  nitrogen  is  determined  and  at  the  same  time  the  combined  carbon 

dioxide  estimated  by  means  of  the  calcium  carbonate  split  off  on  boil- 

CO 
ing  the  filtered  solution,  then  the  quotient  — —  gives  the  number  of  N 

atoms  for  every  molecule  CO2  taken  up.  This  quotient  is  equal  to  1 
for  glycocoll  and  the  aliphatic  amino-acids  because  these  go  over  quan- 

1  Sorensen,  Bioch.  Zeitschr.,  7;  with  Jessen  Hansen,  ibid.,  7;  with  V.  Henriques, 
Zeitschr.  f.  physiol.  Chem.,  63  and  64;  Henriques  and  Gjaldbak,  Ibid.,  67  and  75. 


COMPOUND  PROTEINS.  167 

titatively  into  carbaminoacids.  With  the  diamino-acid  arginine,  which 
contains  4  nitrogen  atoms,  it  is  on  the  contrary  only  one-fourth  because 
this  acid  reacts  with  only  one  amino  group,  that  of  the  a-amino  valeric 
acid  chain. 

The  reaction  which  has  been  developed  and  extensively  used  by 
Siegfried1  and  his  pupils  is  of  great  value  in  the  characterization  of  pep- 
tones, kyrines,  and  proteoses,  for  the  separation  and  fractional  pre- 
cipitation and  for  the  determination  of  their  constitution.  The  binding 
of  the  carbon  dioxide  as  carbamino-salts  seems  also  in  many  ways 
to  be  of  physiological  importance,  as  for  example,  the  solubility  of  cal- 
cium carbonate  in  alkaline  fluids  and  for  the  carbon  dioxide  binding  in 
blood,  etc. 

The  amino-acids  can  by  methylation  form  betaines,  for  example, 
trimethyl  glycocoll  or  betaine  CH2 — N(CH3)3.     Betaine  occurs  abundantly 

CO 0 

in  the  plant  kingdom.  In  the  animal  kingdom  such  bodies  have  been 
found  under  physiological  conditions  in  cold  blooded  animals  and  they 
belong  to  those  groups  of  bodies  which  have  been  called  "  aporrhegmas" 
by  Ackermann  and  Kutscher.2  As  "  aporrhegmas "  they  designate 
all  those  fractions  of  amino-acids  from  the  protein,  which  can  be  pro- 
duced from  the  proteins  in  a  physiological  manner  and  indeed  in  the  life 
of  animals  as  well  as  the  plants.  These  bodies  are  essentially  the  same 
as  have  been  observed  in  the  putrefaction  of  the  amino-acids  and  which 
have  been  specially  mentioned  with  every  amino-acid  described. 

The  behavior  of  the  amino-acids  in  yeast  fermentation  will  be  dis- 
cussed in  Chapter  III. 

In  regard  to  the  methods  for  separating  and  preparing,  in  a  pure  form, 
the  various  amino-acids  and  other  products  of  protein  hydrolysis  which 
have  not  been  given  in  the  preceding  pages,  we  must  refer  to  Abderhal- 
den's  Handbuch  der  biochemischen  Arbeitsmethoden,  1909-1910  Bd.  2. 

II.     Compound  Proteins.3 

We  designate  as  compound  proteins  those  bodies  which  yield,  on 
cleavage,  proteins  (with  their  decomposition  products)  and  other  bodies 
such  as  carbohydrates,  nucleic  acids,  or  pigments. 

The  compound  proteins  known  at  present  can  be  divided  into  three 
groups:    glycoproteins,   nucleoproleins  and  chromoproteins.     Of  these   the 

1  In  regard  to  the  literature  see  Siegfried  in  Ergebnisse  d.  Physiol.  Bd.  9. 

2  Zeitschr.  f.  physiol.  Chem.,  69.     See  also  Engeland,  ibid.,  69. 

3  Hoppe-Seyler  has  given  the  name  proteide  to  these  compound  proteids,  but  as 
this  term  is  misleading  in  English  we  do  not  use  it  in  English  classifications  in  this 
sense. 


168  THE  PROTEIN  SUBSTANCES. 

last-mentioned  group  (haemoglobin  and  haemocyanine)  will  be  discussed 
in  a  subsequent  chapter  (Chapter  V  on  the  blood). 

A.     Glycoproteins   (glucoproteins) . 

Glycoproteins  *  are  those  compound  proteins  which  on  decomposi- 
tion yield  a  protein  on  the  one  side,  and  a  carbohydrate  or  derivatives 
of  this  on  the  other,  but  no  purine  bodies.  Some  glycoproteids  are  free 
from  phosphorus  (mucin  substances,  chondroproteins,  and  hyalogens), 
and  some  contain  phosphorus  (phosphoglycoproteins). 

The  glycoproteins  free  from  phosphorus  may,  as  regards  the  nature 
of  the  carbohydrate  groups  split  off,  be  divided  into  two  chief  groups, 
the  mucin  substances  and  the  chondroproteins.  The  first  yield  on  hydrolytic 
cleavage  an  amino-sugar,  which  has  been  shown  to  be  glucosamine  in 
all  but  a  few  exceptions.2  In  the  chondroproteins,  on  the  contrary,  the 
protein  is  united  to  chondroitin-sulphuric  acid. 

1.     Mucin  Substances. 

Compared  with  the  simple  proteins  the  mucin  substances  are  poorer 
in  nitrogen  and  as  a  rule  also  have  considerably  less  carbon.  The  carbo- 
hydrate complex,  whose  nature  has  been  shown  by  the  investigations 
cf  Fr.  Muller  3  and  his  pupils,  occurs,  so  it  seems,  in  the  mucin  sub- 
stances as  a  polysaccharide  related  to  chitosan,  which  on  hydrolytic 
cleavage  yields  glucosamine  (chitosamine),  and,  at  least  in  most  cases, 
acetic  acid  also.  The  mucin  substances  differ  very  markedly  among 
themselves,  hence  we  divide  them  into  two  groups,  the  mucins  and  the 
mucoids. 

The  true  mucins   are   characterized  by  the  fact  that  their  natural 

1  Abderhalden  (Lehrb  d.  physiol.  Chem.,  1909,  p.  191)  has  proposed  dropping  the 
name  glycoproteids  entirely  and  to  consider  these  bodies  as  simple  proteins,  because 
it  has  not  been  shown  that  the  carbohydrate  groups  occupy  the  same  relationship 
to  the  protein  component  that  the  haemin  or  the  nucleic  acid  bears  to  the  haemo- 
globin or  the  nuclooprotein  molecule.  It  is  possible  that  this  proposition,  which  is 
not  applicable  to  the  entire  group  (including  the  proteins  containing  chondroitin- 
sulphuric  acid)  but  applies  only  to  the  mucin  group,  will  be  found  in  the  future  to  be 
correct.  It  is  the  opinion  of  Hammarsten  that  it  is  better  to  wait  for  further  research 
in  this  direction  before  we  drop  the  generally  accepted  nomenclature  and  the  usual 
subdivisions  of  the  proteins. 

2  See  Schulz  and  Ditthorn,  Zeitschr.  f.  physiol.  Chem.,  29;  A.  v.  Ekenstein  and 
Blunksma,  Chem.  Centralbl.,  1907.  2.  When  both  carbohydrate  groups  are  simul- 
taneously combined  in  one  body,  then  probably  we  are  not  dealing  with  a  chemical 
individual,  but  rather  with  a  mixture. 

3  See  Fr.  Muller,  Zeitschr.  f.  Biologie,  42,  which  contains  all  the  pertinent  litera- 
ture, and  also  L.  Langstein,  Die  Bildung  von  Kohlenhydraten  aus  Eiweiss,  Ergebnisse 
der  Physiologie,  Jahr.  I,  Abt.  1. 


MUCINS.  1(39 

solutions,  or  solutions  prepared  by  the  aid  of  a  traee  of  alkali,  arc  mucilagi- 
nous, ropy,  and  give  a  precipitate  with  acetic  acid  which  is  insoluble  in 
excess  of  acid  or  soluble  only  with  great  difficulty.  The  mucoids  do  not 
show  these  physical  properties,  and  have  other  solubilities  and  precipit- 
ation properties.  As  we  have  intermediate  steps  between  different  pro- 
tein 1  todies,  so  also  we  have  such  between  true  mucins  and  mucoids,  and 
a  sharp  line  cannot  be  drawn  between  these  two  groups. 

It  is  just  as  difficult  at  present  to  draw  a  sharp  line  between  the  pro- 
teins and  the  mucins  or  mucoids,  since  we  have  been  able  to  split  off 
carbohydrate  complexes  from  several  proteins,  and  as  proteins  have 
been  isolated  from  white  of  egg  which  yield  more  or  less  glucosamine. 
The  very  variable  amounts  of  glucosamine  obtained  under  various  con- 
ditions from  the  crystalline  ovalbumin  seem  to  indicate  that  we  are 
dealing  with  a  contamination  with  a  glycoprotein. 

True  mucins  are  secreted  by  the  larger  mucous  glands,  by  certain 
mucous  membranes,  and  by  the  skin  of  snails  and  other  animals.  True 
mucin  also  occurs  in  the  navel-cord.  Sometimes,  as  in  snails  and  in 
the  membrane  of  the  frog-egg  (Giacosa)  and  perch-eggs  (Hammarsten  l), 
a  mother-substance  of  mucin,  a  mucinogen,  has  been  found  which  may 
be  converted  into  mucin  by  alkalies.  Mucoid  substances  are  found  in 
certain  cysts,  in  the  cornea,  the  crystalline  lens,  white  of  egg,  and  in 
certain  ascitic  fluids.  '  The  so-called  tendon-mucin,  which,  according 
to  the  investigations  of  Levene  and  of  Cutter,  and  Gies,2  contains 
chondroitin-sulphuric  acid  or  a  related  substance,  cannot  be  classified 
as  a  mucin,  but  must,  like  the  chondromucoid  and  the  osseomucoid, 
be  classified  as  chondroprotein.  As  the  mucin  question  has  not  been 
sufficiently  studied,  it  is  at  the  present  time  impossible  to  give  any  positive 
statements  in  regard  to  the  occurrence  of  mucins  and  mucoids,  especially 
as  without  doubt  in  many  cases  non-mucinous  substances  have  been 
described  as  mucins. 

True  Mucins.  Thus  far  we  have  been  able  to  obtain  only  a  few 
mucins  in  a  pure  and  unchanged  condition,  because  of  the  reagents  used. 
The  elementary  analyses  of  these  mucins  have  given  the  following  results: 

c         h         n         s 
Mucin  from  mucous  membrane  (air- 
passages)  48.26  6.91  10.70  1.40  (Fr.  Muller  «) 

Mucin  from  submaxillary 48.84  6.80  12.32  0.84  (Hammarsten5) 

Mucin  from  snail 50 .  32  6 .  84  13 .  65  1 .  75  (Hammarsten  j) 

Synovial  mucin 51.05  6.53  13.01  1.34  (v.  Holst  *) 

1  Giacosa,  Zeitschr.  f.  physiol.  Chem.,  7;  Hammarsten,  Pfluger's  Archiv.,  36,  and 
Skand,  Arch.  f.  Physiol.,  17. 

2  Levene,  Zeitschr.  f.  physiol.  Chem.,  31;  Cutter  and  Gies,  Amer.  Journ.  of  Physiol.,  6. 

3  Fr.  Muller,  Zeitschr.  f.  Biologie,  42;  Hammarsten,  Zeitschr.  f.  physiol.  Chem..  12, 
.and  Pfluger's  Arch.,  36. 

4  Zeitschr.  f.  physiol.  Chem.,  43. 


170  THE  PKOTEIN  SUBSTANCES. 

Muller  obtained  35  per  cent  glucosamine  from  mucous-membrane 
mucin  and  23.5  per  cent  from  the  submaxillary  mucin. 

On  boiling  mucin  with  dilute  mineral  acids,  acid  albuminate  and 
bodies  similar  to  proteoses  are  obtained,  besides  a  reducing  substance 
which  is  not  free  glucosamine  (Steudel1).  By  the  action  of  strong 
acids  upon  mucins  or  mucoids  Otori  2  obtained  several  of  the  cleavage 
products  of  the  proteins,  such  as  leucine,  tyrosine,  glycocoll,  glutamic 
acid,  oxalic  acid,  guanidine,  arginine,  lysine,  and  humus  substances, 
and  also  carbohydrate  cleavage  products,  such  as  levulinic  acid.  Cer- 
tain mucins,  as  the  submaxillary  mucin,  are  easily  changed  by  very 
dilute  alkalies,  as  lime-water,  while  others,  such  as  tendon-mucin,  are 
not  affected.  If  a  strong  caustic-alkali  solution,  such  as  a  5-per  cent 
KOH  solution,  is  allowed  to  act  on  submaxillary  mucin,  we  obtain  alkali 
albuminate,  bodies  similar  to  proteoses  and  peptones  and  one  or  more 
substances  of  an  acid  reaction  which  have  strong  reducing  powers. 

On  peptic  digestion  proteoses  and  peptone-like  bodies,  still  con- 
taining the  carbohydrate  group,  are  produced.  On  tryptic  digestion 
still  simpler  cleavage  products  are  formed,  namely,  leucine,  tyrosine, 
and  tryptophane  (Posner  and  Gies3).  The  glucosamine,  so  far  as  we 
know,  is  not  split  off  by  proteolytic  enzymes,  but  only  after  strong 
hydrolysis  with  acids. 

In  one  or  another  respect  the  various  mucins  act  somewhat  dissimilarly. 
For  example,  the  snail  and  sputum  mucins  are  insoluble  in  dilute  hydro- 
chloric acid  of  1-2  p.  m.,  while  the  mucin  of  the  submaxillary  gland  and 
the  navel-cord  is  soluble.  The  former  become  flaky  with  acetic  acid, 
while  the  submaxillary  mucin  is  precipitated  in  more  or  less  fibrous, 
tough  masses.     Still  all  the  mucins  have  certain  reactions  in  common. 

In  the  dry  state  mucin  forms  a  white  or  yellowish-gray  powder.  When 
moist  it  forms,  on  the  contrary,  flakes  or  yellowish-white  tough  lumps 
or  masses.  The  mucins  are  acid  in  reaction.  They  give  the  color  reac- 
tions of  the  proteins.  They  are  not  soluble  in  water,  but  may  give  a 
neutral  solution  with  water  with  the  aid  of  small  amounts  of  alkali.  Such 
a  solution  does  not  coagulate  on  boiling,  but  acetic  acid  gives  at  the 
normal  temperature  a  precipitate  which  is  nearly  insoluble  in  an  excess 
of  the  precipitant.  If  5-10  per  cent  NaCl  be  added  to  a  mucin  solution, 
it  can  be  carefully  acidified  with  acetic  acid  without  giving  a  pre- 
cipitate. Such  acidified  solutions  are  copiously  precipitated  by  tan- 
nic acid;  with  potassium  ferrocyanide  they  give  no  precipitate,  but  on 
sufficient  concentration  they  become  thick  or  viscous.  A  neutral  solu- 
tion of  alkali  mucin  is  precipitated  by  alcohol  in  the  presence  of  neutral 

1  Zeitschr.  f.  physiol.  Chem.,  34. 

2  Ibid.,  42  and  43. 

'■'■  Amer.  Journ.  of  Physiol.,  11. 


HYALOGENS.  171 

salts;  it  is  also  precipitated  by  several  metallic  salts.  If  mucin  is  heated 
on  the  water-bath  with  dilute  hydrochloric  acid  of  about  2  per  cent, 
the  liquid  gradually  becomes  a  yellowish  or  dark  brown,  and  reduces 
copper  salts  in  alkaline  solutions. 

The  mucin  most  readily  obtained  in  large  quantities  is  the  submax- 
illary mucin,  which  may  be  prepared  in  the  following  way:  The  filtered 
watery  extract  of  the  gland,  free  from  form-elements  and  as  colorless 
as  possible,  is  treated  with  25  per  cent  hydrochloric  acid,  so  that  the 
liquid  contains  1.5  p.  m.  HC1.  On  the  addition  of  the  acid  the  mucin 
is  immediately  precipitated,  but  dissolves  on  stirring.  If  this  acid  liquid 
is  immediately  diluted  with  2-3  vols,  of  water,  the  mucin  separates  and 
may  be  purified  by  redissolving  in  1-5  p.  m.  acid,  and  diluting  with 
water  and  washing  therewith.  The  mucin  of  the  navel-cord  may  be 
prepared  in  the  same  way.  As  a  rule  the  mucins  can  be  prepared  by 
precipitation  with  acetic  acid  and  repeated  solution  in  dilute  lime-water 
or  alkali,  and  reprecipitation  with  acetic  acid.  Finally  they  are  treated 
with  alcohol  and  ether.  In  the  preparation  of  sputum  mucin  the  method 
is  very  complicated  (Fr.  Muller). 

Mucoids  or  Mucinoids.  In  this  group  we  must  include  those  non- 
phosphorized  glycoproteins  which  are  neither  true  mucins  nor  chondro- 
proteids,  although  they  show  among  themselves  such  differences  in 
behavior  that  they  can  be  divided  into  several  subgroups  of  mucoids. 
To  the  mucoids  belong  pseudomucin,  the  probably  related  body  colloid, 
ovomucoid,  and  other  bodies,  which  on  account  cf  their  differences  will  be 
best  treated  individually  in  their  respective  chapters. 

Hyalogens.  Under  this  name  Krukenberg  l  has  designated  a  number  of 
different  bodies,  which  are  characterized  by  the  following:  By  the  action  of 
alkalies  they  change,  with  the  splitting  off  of  sulphur  and  some  nitrogen,  into 
soluble  nitrogcnized  products  called  by  him  hyalines,  and  which  yield  a  pure  car- 
bohydrate by  further  decomposition.  We  find  that  very  heterogeneous  sub- 
stances are  included  in  this  group.  Certain  of  these  hyalogens  seem  undoubtedly 
to  be  glycoproteins.  Neossin 2  of  the  Chinese  edible  swallow's-nest,  membranin* 
of  Descemet's  membrane  and  of  the  capsule  of  the  crystalline  lens,  and  spiro- 
graphin  4  of  the  skeletal  tissue  of  the  worm  Spirographs,  seem  to  act  as  such. 
Others,  on  the  contrary,  such  as  hyalin  5  of  the  walls  of  hydatid  cysts,  and  arm- 
phin*  from  the  tubes  of  Onuphis  tubicola,  do  not  seem  to  be  compound  proteins. 
The  so-called  mucin  of  the  holothuria,  7  and  chondrosin  8  of  the  sponge,  Chondrosia 

1  Verh.  d.  physik.-med.  Gesellsch.  zu  Wurzburg,  1883;  also  Zeitschr.  f.  Biologie,  22. 

2  Krukenberg,  Zeitschr.  f.  Biologie,  22. 

3  C.  Th.  Morner,  Zeitschr.  f.  physiol.  Chem.,  18. 

4  Krukenbere,  Wurzburg,  Verhandl.,  1883:  also  Zeitschr.  f.  Biologie,  22. 

5  A.  Lucke,  Virchow's  Arch.,  19;  also  Krukenberg,  Vergleichende  physiol.  Stud., 
Series  1  and  2,  1881. 

6  Schmiedeberg,  Mitth.  aus  d.  zool.  Stat,  zu  Neapel,  3,  1882. 

7  Hilger.  Pfliiger's  Archiv,  3. 

8  Krukenberg,  Zeitschr.  f.  Biologie,  22. 


172  THE  PROTEIN  SUBSTANCES. 

reniformis,  and  others  may  also  be  classed  with  the  hyalogens.  As  the  various 
bodies  designated  by  Krunkenberg  as  hyalogens  are  very  dissimilar,  it  is  not 
of  much  advantage  to  arrange  these  in  special  groups. 


2.     Chondroproteins. 

Chondroproteins  are  those  glycoproteins  which  as  primary  cleav- 
age products  yield  protein  and  an  ethereal  sulphuric  acid,  the  chondroitin- 
sulphuric  acid.  Chondromucoid,  occurring  in  cartilage,  is  the  best  example 
of  this  group.  Amyloid  occurring  under  pathological  conditions  also 
belongs  to  this  group.  On  account  of  the  property  of  chondroitin-sul- 
phuric  acid  of  precipitating  protein,  it  is  also  possible  that  under  certain 
circumstances  combinations  of  this  acid  with  protein  may  be  precipitated 
from  the  urine  and  be  considered  as  chondroproteins. 

The  chondromucoid,  the  so-called  tendon-mucin,  and  the  osseomucoid 
have  greatest  interest  as  constituents  of  cartilage,  of  the  connective 
tissues,  and  the  bones,  and  on  this  account  these  bodies  and  their  cleavage 
product,  chondroitin-sulphuric  acid,  will  be  treated  in  a  following  chap- 
ter (IX).  On  the  contrary,  amyloid,  which  has  always  been  considered 
in  connection  with  the  protein  substances,  will  be  described  here. 

Amyloid,  so  called  by  Virchow,  is  a  protein  substance  appear- 
ing under  pathological  conditions  in  the  internal  organs,  such  as  the  spleen, 
liver  and  kidneys,  as  infiltrations;  and  in  serous  membranes  as  granules 
with  concentric  layers.  It  probably  also  occurs  as  a  constituent  of 
certain  prostate  calculi.  The  chondroprotein  occurring  under  physio- 
logical conditions  in  the  walls  of  the  arteries  is,  perhaps,  according  to 
Krawkow,  very  closely  related  to  the  amyloid  substance,  but  not  iden- 
tical with  it,  as  shown  by  Neuberg.1 

Recently  O.  Hanssen  has  studied  the  mechanically  isolated  amy- 
loid obtained  from  the  so-called  "  sago  kernels  "  of  an  amyloid  spleen, 
and  could  not  detect  any  conjugated  sulphuric  acid  in  it.  According  to 
his  investigations  true  amyloid  is  not  a  chondroprotein.  Mayeda  2 
has  also  prepared  an  amyloid  substance  free  from  chondroitin-sulphuric 
acid.  On  the  other  hand,  Hanssen  has  found  that  amyloid  organs 
(liver  and  spleen)  are  much  richer  in  sulphuric  acid  that  splits  off  than 
normal  organs,  and  it  is  not  improbable  that  the  amyloid  formation  goes 
hand  in  hand  with  the  formation  of  a  chondroprotein. 

The  amyloid  prepared  by  Krawkow  and  Neuberg  had  about  the 
same  composition:    C  49.0-50.1;    H  7-7.2;    N   14-14.1,  and  S  1.8-2.8 


1  Krawkow,  Arch.  f.  exp.  Path.  u.  Pharm.,  40,  which  contains  the  literature;  Neu- 
berg, Verhandl.  d.  d.  Pathol.  Gesellsch.,  1004. 

2  Hanssen,  Bioch.  Zeitschr.,  13;  Mayeda,  Zeitschr.  f.  physiol.  Chem.,  58. 


AMYLOID.  173 

per  cent.  The  aorta  amyloid  of  man  and  of  the  horse  contained  respect- 
ively C  49.6  and  50.5;  H  7.2;  N  14.4  and  13.8;  S  2.3  and  2.5  per  cent. 
As  we  cannot  tell  whether  the  amyloid  analyzed  was  pure  or  not  the 
results  are  of  questionable  value. 

According  to  older  investigations  amyloid  splits,  by  the  action  of 
alkali,  into  protein  and  chondroitin-sulphuric  arid  (see  Chapter  IX), 
and  according  to  Krawkow  it  is  therefore  a  firm,  perhaps  ester-like 
combination  of  this  acid  with  protein.  The  protein,  from  the  investiga- 
tions of  Neuberg,  is  of  a  basic  nature  and  most  comparable  to  the 
histones.  The  investigations  of  Mayeda  do  not  coincide  with  this  view 
as  the  amyloid  protein  obtained  by  him  did  not  behave  like  a  histone. 
Its  content  of  hexone  bases  was  not  greater  than  that  of  the  proteins 
of  the  normal  organs  and  this  amyloid  protein  did  not  yield  any  his- 
tone-peptone.  To  all  appearances,  different  investigators  have  worked 
with  different  substances  and  it  is  possible  that  in  the  amyloid  degenerated 
organs  partly  chondroproteins  and  partly  amyloid  proteins  may  occur, 
both  of  which  give  the  color  reactions. 

Amyloid  is  an  amorphous  white  substance,  insoluble  in  water,  alcohol, 
ether,  dilute  hydrochloric  and  acetic  acids.  It  is  soluble  in  concen- 
trated hydrochloric  acid  or  caustic  alkali  with  decomposition.  On  boil- 
ing with  dilute  hydrochloric  acid  it  yields  sulphuric  acid  and  a  reducing 
substance.  It  is  not  dissolved  by  gastric  juice,  according  to  Krawkow, 
which  agrees  with  most  of  the  older  reports.  It  is  nevertheless  changed 
so  that  it  is  soluble  in  dilute  ammonia,  while  the  typical  amyloid  is 
insoluble  therein.  Neuberg  finds  on  the  contrary  that  amyloid  (from 
liver)  is  digested  by  pepsin  as  well  as  by  trypsin,  although  more  slowly 
than  fibrin,  and  that  it  is  also  destroyed  in  autolysis,  so  that  in  life  an 
absorption  is  possible.  The  amyloid  from  the  "  sago  "  spleen  studied 
by  Hanssen  showed  the  same  behavior  with  gastric  juice  as  Krawkow 
found,  while  trypsin,  as  well  as  autolysis  for  months,  was  without  action. 
Mayeda's  amyloid  was  gradually  dissolved  by  gastric  juice. 

Amyloid  gives  the  xanthoproteic  reaction  and  the  reactions  of  Mil- 
lon  and  Adamkiewicz-Hopkins.  Its  most  important  property  is  its 
behavior  with  certain  coloring  matters.  It  is  colored  reddish-brown 
or  a  dingy  violet  by  iodine;  a  violet  or  blue  by  iodine  and  sulphuric 
acid;  red  by  methylaniline  iodide,  especially  on  the  addition  of  acetic 
acid;  and  red  also  by  aniline  green.  Of  these  color  reactions  those  with 
aniline  dyes  are  the  most  important.  The  iodine  reaction  appears  less 
constant  and  is  greatly  dependent  upon  the  physical  condition  of  the 
amyloid.  The  color  reactions  are  due  to  the  presence  cf  the  chondroitin- 
sulphuric  acid  component,  but  this  stands  in  opposition  to  the  behavior 
of  the  intact  amyloid  obtained  by  Hanssen  from  the  "  sago  "  spleen 
and  the  amyloprotein  of  Mayeda. 


174  THE  PROTEIN  SUBSTANCES. 

In  preparing  amyloid,  extract  the  finely  divided  organs  with  very 
dilute  ammonia.  The  undissolved  amyloid  in  the  residue,  if  it  does  not 
resist  pepsin  digestion,  can  be  directly  extracted  by  dilute  barium  hydrate 
solution  and  then  precipitated  from  the  filtrate  by  hydrochloric  acid. 
Otherwise  the  above  mentioned  residue  is  digested  for  several  days  with 
pepsin.  The  digestion  residue  is  dissolved  in  dilute  ammonia,  filtered, 
the  amyloid  precipitated  by  dilute  hydrochloric  acid,  the  precipitate 
dissolved  in  baryta-water,  when  the  nucleins  remain  behind,  the  barium 
filtrate  precipitated  with  hydrochloric  acid  and  purified,  if  necessary 
by  repeated  solution  in  ammonia  and  precipitating  with  hydrochloric 
acid,  washing  and  treating  with  alcohol  and  ether. 

Phosphoglycoproteins.  This  group  includes  the  phosphorized  glycoproteins. 
They  yield  no  purine  bases  (nuclein  bases)  as  cleavage  products.  They  are  not 
nucleoproteins  and  therefore  they  must  not  be  mistaken  for  them.  On  pepsin 
digestion  they  may,  like  certain  nucleoalbumins,  yield  pseudonuclein,  but  they 
differ  from  the  nucleoalbumins  in  that  they  yield  a  reducing  substance  on  boil- 
ing with  dilute  acid.  They  differ  from  the  nucleoproteins,  which  also  yield  reduc- 
ing carbohydrates,  in,  as  above  stated,  not  yielding  any  purine  bases. 

Only  two  phosphorized  glycoproteins  are  known  at  the  present  time,  namely, 
ichthvlin,  occurring  in  carp  eggs  and  studied  by  Walter,1  and  which  was  con- 
sidered as  a  vitellin  for  a  time.  Ichthulin  has  the  following  composition:  C  53.52; 
H  7.71;  N  15.64;  S  0.41;  P  0.43;  Fe  0.10  per  cent.  In  regard  to  solubilities  it 
is  similar  to  a  globulin.  Walter  has  prepared  a  reducing  substance  from  the 
pseudonuclein  of  ichthulin  which  gave  a  highly  crystalline  compound  with 
phenylhydrazine. 

Another  phosphoglycoprotein  is  helicoproteid,  obtained  by  Hammarsten  2 
from  the  glands  of  the  snail  Helix  pomatia.  It  has  the  following  composition: 
C  46.99;  H  6.78;  N  6.08;  S  0.62;  P  0.47  per  cent.  It  is  converted  into  a  gummy, 
levorotatory  carbohydrate,  called  animal  sinistrin,  by  the  action  of  alkalies. 
On  boiling  with  an  acid  it  yields  a  dextrorotatory  reducing  substance. 

The  compound  protein  found  by  Shultz  and  Ditthorn  3  in  the  spawn  of 
the  frog  probably  belongs  to  this  group,  but  instead  of  glucosamine  it  gives 
galactosamine  on  cleavage. 

B.    Nucleoproteins. 

By  this  name  we  designate  those  compound  proteins  which  yield 
protein  and  nucleic  acid  on  cleavage.  The  nucleoproteins  seem  to  be 
widely  diffused  in  the  animal  body.  They  occur  chiefly  in  the  cell- 
nuclei,  but  they  also  often  occur  in  the  protoplasm.  They  may  pass 
into  the  animal  fluids  on  the  destruction  of  the  cells,  hence  nucleopro- 
teins have  also  been  found  in  blood  serum  and  other  fluids. 

The  nucleoproteins  may  be  considered  as  combinations  of  a  pro- 
tein with  a  side  chain,  which  Kossel  calls  the  prosthetic  group.  This 
side  chain,  which  contains  the  phosphorus,  may  be  split  off  as  nucleic 
acid  on  treatment  with  alkali.     The  protein  may  be  of  different  kinds. 

1  Zeitschr.  f.  physiol.  Chem.,  15. 

2  IIammar.st.en,  Pfltiger's  Arch.,  36. 

3  Zeitschr.  f.  physiol.  Chem.,  29. 


NUCLE0PE0TEIN8.  175 

In  certain  cases  this  is  histone,  and  the  combinations  between  nucleic 
acid  and  protamines  are  also  sometimes  classified  as  nucleoproteins. 
The  combination  between  protamine  and  nucleic  acid  is,  it  seems,  a 
salt-like  combination,  and  entirely  different  from  the  combination  of  the 
proteins  with  nucleic  acid  in  the  nucleoproteins.  The  following  facts, 
given  in  connection  with  the  nucleoproteins,  do  not  apply  to  the  nucleo- 
protamines.  The  nucleoproteins  differ  not  only  according  to  the  protein 
component  they  contain,  but  also  as  to  the  nucleic  acids,  which  vary 
among  themselves.  There  are  essentially  different  nucleic  acids,  some 
among  which  contain  a  pentose  carbohydrate  while  others  contain  a 
hexose  carbohydrate.  The  nucleic  acids  also  differ  in  regard  to  the 
amount  of  purine  and  pyrimidine  bases  they  contain  (sec  below). 

The  native  nucleoproteins  contain  a  variable,  but  not  a  high  percentage 
of  phosphorus,  which  in  most  of  the  nucleoproteins  investigated,  ranges 
between  0.5  and  1.6  per  cent.  They  also  regularly  contain  iron,  and  in 
Octopodes,  Henze  i  has  observed  an  iron-free  nucleoprotein  with  0.96 
per  cent  copper.  The  nucleoproteins  behave  like  weak  acids,  especially 
those  having  considerable  protein  in  the  molecule.  They  therefore 
give  the  ordinary  protein  reactions  and  behave  in  this  regard  like  the 
proteins.  The  nucleoproteins  prepared  from  organs  rich  in  cell  nuclei 
seem  to  be  characterized  by  containing  more  phosphorus  and  having  a 
stronger  acid  character.  All  nucleoproteins  are  bodies  that  are  insoluble 
in  water,  but  whose  alkali  combination  is  soluble  in  water.  From 
such  a  solution  the  nucleoprotein  can  be  precipitated  by  acetic  acid,  and 
in  an  excess  of  the  acid,  the  precipitate  dissolves  with  more  or  less 
difficulty  and  in  some  cases  not  at  all.  It  dissolves,  on  the  contrary,  in 
very  dilute  hydrochloric  acid.  In  this  respect  nucleoproteins  are  similar 
to  the  nucleoalbumins  and  the  mucin  substances,  but  differ  from  these 
two  groups  in  that  they  yield  purine  bases  on  hydrolysis.  According 
to  Plimmer  and  Scott2  the  nucleoproteins  differ  from  the  nucleo-albu- 
mins  by  the  fact  that  with  sodium  hydroxide  in  1  per  cent  solution  the 
nucleoalbumins  split  off  phosphoric  acid  while  the  nucleoproteins  do  not. 
The  nucleoproteins  give  the  color  reactions  of  the  proteins,  but  those 
which  have  been  investigated  are  dextrorotatory  and  not  laevorotatory 
(Gamgee  and  Jones3). 

The  nucleoproteins  are  readily  modified.  The  alkali  combination 
scluble  in  water  suffers  a  decomposition  on  heating  its  solution,  when 
as  neutral  as  possible,  and  coagulated  protein  separates  while  a  protein 
rich  in  phosphorus  and  poor  in  protein  with  strong  acid  character  remains 


1  Zeitschr.  f.  physiol.  Chem.,  55. 

2  Plimmer  and  Scott,  cited  in  Biochem.  Centralbl.,  8,  p.  109. 

3  Hofmeister's  Beitrage,  4. 


176  THE  PROTEIN   SUBSTANCES. 

in  solution.  By  the  action  of  weak  acids  and  by  gastric  juice  a  similar 
cleavage  takes  place,  whereby  the  protein  split  off  goes  into  solution 
while  the  nucleoprotein  rich  in  phosphorus,  so-called  nuclein  (Miescher, 
Hoppe-Seyler  *)  or  true  nuclein,  remains  undissolved.  As  the  nuclein 
is  probably  nothing  but  a  partly  modified  nucleoprotein  poorer  in  pro- 
tein, having  a  composition  varying  with  the  intensity  of  the  cleavage, 
it  seems  unnecessary  to  give  the  name  nuclein  thereto.  On  the  other 
hand,  the  nucleins  have  other  properties  than  the  nucleoproteins,  and 
as  the  nucleins  bear  the  same  relation  to  the  nucleoproteins  that  the 
pseudonuclein  does  to  the  nucleoalbumins,  we  will  here  give  a  short 
description  of  the  nucleins  as  well  as  the  pseud'o-  or  paranucleins. 

Nucleins  or  true  nucleins  are  formed,  as  above  stated,  from  nucleo- 
proteins in  their  peptic  digestion  or  by  treatment  with  dilute  acids.  It 
must  be  remarked  that  the  nucleins  are  not  entirely  resistant  toward 
gastric  juice,  and  also  that  at  least  one  nucleoprotein,  namely,  the  one 
obtained  from  the  pancreas,  completely  dissolves,  leaving  no  nuclein 
residue  on  treatment  with  gastric  juice  (Umber,  Milroy2).  The 
nucleins  are  rich  in  phosphorus,  containing  in  the  neighborhood  of  5  per 
cent.  According  to  Liebermann,3  metaphosphoric  acid  can  be  split 
off  from  true  nucleins  (yeast  nuclein).  The  nucleins  are  decomposed 
into  protein  and  nucleic  acid  by  caustic  alkali,  and  as  different  nucleic 
acids  exist,  so  also  there  exist  different  nucleins.  As  previously  stated 
proteins  may  be  precipitated  in  acid  solutions  by  nucleic  acids,  and  in 
this  way,  as  shown  by  Milroy,  combinations  of  nucleic  acid  and  pro- 
teins may  be  prepared  which  behave  quite  like  true  nucleins.  All  nucleins 
yield  purine  bases  (so-called  nuclein  bases)  on  boiling  with  dilute  acids. 
They  act  like  rather  strong  acids. 

The  nucleins  are  colorless,  amorphous  and  insoluble  or  only  slightly 
soluble  in  water.  They  are  insoluble  in  alcohol  and  ether.  They  are 
more  or  less  readily  dissolved  by  dilute  alkalies.  The  nucleins  give  the 
biuret  test  and  Millon's  reaction.  They  show  a  great  affinity  for  many 
dyes,  especially  the  basic  ones,  and  take  these  up  with  avidity  from  watery 
or  alcoholic  solutions.  On  burning  they  yield  an  acid  residue  which 
is  very  difficult  to  incinerate  and  which  contains  metaphosphoric  acid. 
On  fusion  with  saltpeter  and  soda  the  nucleins  yield  alkali  phosphates. 

To  prepare  nucleins  from  cells  or  tissues,  first  remove  the  chief  mass 
of  proteins  by  artificial  digestion  with  pepsin-hydrochloric  acid,  lixiviate 
the  residue  with  very  dilute  ammonia,  filter,  and  precipitate  Avith  hydro- 
chloric   acid.     The   precipitate   is    further    digested    with    gastric    juice, 

1  Hoppe-Seyler,  Med.  chem.  Unters.,  452. 

5  ["ruber,  Zeitsclir.  f.  klin.  Med.,  34;  Milroy,  Zeitschr.  f.  physiol.  Chem.,  22. 

3  Pfluger's  Arch.,  47. 


FSEUDONUCLEINS.  177 

washed  and  purified  by  alternately  dissolving  in  very  faintly  alkaline 
water  and  reprecipitating  with  an  acid,  washing  with  water,  and  treating 
with  alcohol  and  ether.  A  nuclein  may  be  prepared  more  simply  by  the 
digestion  of  a  nucleoprotein.  In  the  detection  of  nucleins  we  make  use 
of  the  above-described  method,  testing  for  phosphorus  in  the  product 
after  fusing  with  saltpeter  and  soda.  Naturally  the  phosphates  and 
phosphatides  must  first  be  removed  by  treatment  with  acid,  alcohol, 
and  ether,  respectively.  No  exact  methods  are  known  for  the  quanti- 
tative estimation  of  nucleins  in  organs  or  tissues. 

Pseudonucleins  or  Paranucleins.  These  bodies  are  obtained  as 
an  insoluble  residue  on  the  digestion  of  certain  nucleoalbumins  or  phospho- 
glycoproteins  with  pepsin-hydrochloric  acid.  Attention  is  called  to 
the  fact  that  the  pseudonuclein  may  be  dissolved  by  the  presence  of  too 
much  acid  or  by  a  too  energetic  peptic  digestion.  If  the  relation  between 
the  degree  of  acidity  and  the  quantity  of  substance  is  not  properly  selected, 
the  formation  of  pseudonucleins  may  be  entirely  overlooked  in  the 
digestion  of  certain  nucleoalbumins.  Pseudonucleins  contain  phosphorus, 
which,  as  shown  by  Liebermann,1  is  split  off  as  metaphosphoric  acid 
by  mineral  acids. 

The  pseudonucleins  are  amorphous  bodies  insoluble  in  water,  alcohol, 
and  ether,  but  readily  soluble  in  dilute  alkalies  and  barium  hydroxide 
solution.  They  are  readily  split  by  barium  hydroxide  solution  with  the 
splitting  off  of  phosphoric  acid,  and  according  to  Giertz  2  they  differ 
in  this  regard  from  the  true  nucleins,  which  are  neither  dissolved  nor 
decomposed  by  baryta.  They  are  not  soluble  in  very  dilute  acids,  and 
may  be  precipitated  from  their  solution  in  dilute  alkalies  by  adding 
acid.  They  give  the  protein  reactions  very  strongly,  but  do  not  yield 
purine  bases. 

In  preparing  a  pseudonuclein,  dissolve  the  mother-substance  in  hydro- 
chloric acid  of  1-2  p.  m.,  filter  if  necessary,  add  pepsin  solution,  and 
allow  the  mixture  to  stand  at  the  temperature  of  the  body  for  about 
twenty-four  hours.  The  precipitate  is  filtered  off,  washed  with  water, 
and  purified  by  alternately  dissolving  in  very  faintly  alkaline  water  and 
reprecipitating  with  acid. 

Cleavage  Products  of  the  Xucleoproteins. 

1.     The  Nucleic  Acids. 

All  nucleic  acids  are  rich  in  phosphorus  and  yield  phosphoric  acid, 
purine  bases  and  a  carbohydrate  or  carbohydrate  derivative  as  cleavage 
products;     most    of    them    also    contain    pyrimidine  bases.     The  older 

1  Ber.  d.  d.  chem.  Gesellsch.,  21,  and  Centralbl.  f.  d.  med.  Wissensch.,  1889. 

2  Zeitschr.  f.  physiol.  Chem.,  28. 


178  THE  PROTEIN  SUBSTANCES. 

statements  as  to  the  occurrence  of  more  than  two  purine  bases  in  a  nucleic 
acid  are  not  correct  and  depend  upon  the  fact  that  the  two  purine  bases 
xanthine  and  hypoxanthine  can  be  secondarily  formed  from  guanine  and 
adenine.  There  is  no  doubt  that  the  most  thoroughly  studied  nucleic 
acids,  such  as  the  thvmus-nucleic  acids,  the  closely  related  or  perhaps 
identical  acids  of  the  salmon  sperm  (salmo-nucleic  acid),  of  the  herring 
sperm  and  burbot  sperm,  and  of  the  pancreas,  do  not  contain  more  than 
two  purine  bases,  namely,  guanine  and  adenine. 

Of  the  known  nucleic  acids  we  have  two,  the  guanylic  acid  and  inosinic 
acid,  which  contain  only  one  purine  base,  namely,  guanine  and  hypoxan- 
thine, respectively.  These  two  acids  do  not  contain  any  pyrimidine 
bases,  which  are  found  thus  far  in  all  carefully  investigated  nucleic  acids. 
The  occurrence  of  pyrimidine  bases  is  somewhat  different  in  the  various 
nucleic  acids.  In  one  group  of  animal  nucleic  acids  (thymonucleic  acids) 
thymine,  cytosine  and  uracil  are  found,  the  uracil  being  produced  second- 
arily from  the  cytosine.  The  plant  nucleic  acids  (the  triticonucleic  acid 
and  the  yeast  nucleic  acid,  which  may  perhaps  be  identical  with  it)  do 
not  contain  any  thymine  and  yields  as  nitrogenous  cleavage  products 
besides  the  two  purine  bases  only  cytosine  and  uracil. 

All  nucleic  acids,  as  above  stated,  contain  a  carbohydrate  group. 
In  the  plant  nucleic  acids  and  in  two  animal  ones,  the  guanylic  and  inosinic 
acids,  the  carbohydrate  is  a  pentose.  In  the  remaining  animal  nucleic 
acids  it  is  on  the  contrary  a  hexose  or  at  least  a  hexacarbohydrate. 

The  nature  of  this  hexacarbohydrate  has  not  been  determined  and 
the  nature  of  the  pentoses  occurring  in  the  nucleic  acids  is  also  a  disputed 
point.  Based  upon  the  investigations  of  Neuberg  we  have  considered 
the  pentose  of  guanylic  acid  and  of  inosinic  acid  as  /-xylose.  The  correct- 
ness of  this  view  is  disputed  by  others.  According  to  Levene  and  Jacobs 
the  pentose  of  all  nucleic  acids  containing  pentose,  is  d-ribose.  Haiser 
and  Wenzel  who  for  a  time  considered  the  pentose  of  inosinic  acid  as 
'/-xylose  are  now  of  the  view  that  it  is  probably  d-ribose.  The  view  of 
Levene  and  .Jacobs,  that  the  pentose  of  the  guanylic  acid  is  d-ribose  has 
received  important  support  by  the  investigations  of  Schulze  and  Trier 
on  the  identity  of  the  plant  guaninpentoside  vcrnine  with  the  guanin- 
pentoside  (see  below)  prepared  by  Levene  and  Jacobs.  Still  we  have 
no  explanation  why  Neuberg  and  Rewald  l  obtained  only  /-xylose 
from  the  pancreas  on  the  hydrolysis  of  the  entire  organ,  and  Levene 
and  Jacobs  on  the  contrary  only  r/-ribose. 


1  Neuberg  and  Brahn,  Bioch.  Zeitschr.,  5;     see  also  Ber.  d.  d.  chem.  Gesellsch 
41  and  42:    Levene  and  Jacobs,  Ber.  d.  d.  chem.  Gesellsch.,  42  and  43;  Haiser  and 
Wenzel,  Monatah.  f.  Chern.,  61;  Schulze  and  Trier,    Zeitschr.  f.  physiol.  Chem.,  70; 
lid,  Ber.  d.  d.  chem.  Gesellsch.,  42. 


NUCLEIC  ACIDS.  179 

All  nucleic  acids  contain  phosphoric  acid.  The  relation  between  ph<  >- 
phorus  and  nitrogen  is  as  1 :  1  in  the  inosinic  acid  and  as  1:5  in  the  guanylic 
acid.  In  the  thymus-  and  the  salmo-nucleic  acids  the  relation  accord- 
ing to  Schmibdeberg  is  4:14  and  according  to  Steudel  4:15.  In  the 
triticonucleic  acid,  Osborne  and  Harris  found  the  relation  4:10;  in  the 
yeast  nucleic  acid,  Lkyene  and  Jacobs  found  it  was  equal  to  4:15. 

According  to  the  number  of  bases  contained  in  the  nucleic  acids 
we  can  differentiate  between  the  simple  nucleic  acid  with  only  one  base 
and  the  complex  nucleic  acids  with  several  bases.  Levene  and  Mandel  ' 
have  called  the  first  (inosinic  acid,  guanylic  acid)  nucleotides  or  mono- 
nucleotides and  the  last  polynucleotides. 

The  properties  and  the  constitution  of  the  nucleic  acids,  as  far  as  we 
know  them,  have  been  determined  essentially  by  the  work  of  Kossel 
and  his  pupils,  by  Schmiedeberg,  Steudel  and  Levene2  and  their 
collaborators. 

On  complete  acid  hydrolysis  the  nucleic  acids  are  split  into  the  three 
above  mentioned  components,  phosphoric  acid,  carbohydrate  and  bases. 
The  purine  bases  are  more  readily  split  off  than  the  pyrimidine  bases 
and  on  careful  acid  hydrolysis  of  thymus  nucleic  acid,  a  new  acid,  the 
ihyminic  acid  of  Steudel  and  Brigl  is  obtained.  This  acid  is  very 
similar  to  the  thyminic  acid  of  Kossel  and  Neumann  a  with  the  barium 
salt,  CioH23N3P20i2Ba,  and  the  nucleotinphosphoric  acid  of  Schmiede- 
berg.  This  acid  differs  probably  from  the  original  nucleic  acid  only 
by  the  absence  of  purine  bases.  By  the  action  of  strong  nitric  acid  in 
the  cold  we  can,  according  to  the  method  suggested  by  Steudel,  split 
off  the  purine  bases  while  nearly  all  the  phosphoric  acid  remains  in  organic 
combination  with  the  carbohydrate  complexes. 

The  hydrolyses  of  pentose  containing  nucleic  acids  as  carried  out  by 
Levene  and  Jacobs  in  neutral,  or,  if  the  pyrimidine  complexes  of  the 
plant  nucleic  acid  were  being  studied,  in  ammoniacal  reaction,  by  heating 
to  high  temperatures  in  the  autoclave  or  in  sealed  tubes,  are  of  special 
interest.     In  these  cases  the  binding  with  the  phosphoric  acid  was  rup- 

1  Ber.  d.  d.  chem.  Gesellsch.,  41. 

2  The  work  of  Kossel  and  his  pupils  on  the  nucleic  acids  can  be  found  in:  Arch. 
f.  (Anat.  u.)  Physiol.  1892,  1893  and  1894;  Sitz.  Ber.  d.  Berl.  Akad.  d.  Wiss.,  18,  1S94; 
Centralbl.  f.  d.  med.  Wiss.  1893;  Ber.  d.  d.  chem.  Gesellsch.,  26  and  27;  Zeitschr.  f. 
physiol.  Chem.,  23  and  38;  see  also  Neumann,  Arch.  f.  (Anat.  u.)  Physiol.,  1898  and  1899 
Suppl.;  Miescher,  Hoppe-Seyler's  Med.  chem.  Unters.,  p.  441  and  Arch.  f.  exp.  Path, 
u.  Pliann.,  37;  Schmiedeberg,  ibid.,  37,  43,  and  57;  Altnian.  Arch.  f.  (Anat.  u.)  Physiol., 
1889;  Steudel,  ibid.,  42,  43,  46.  49,  50,  52,  53,  55,  56,  70,  77;  Ascoli,  Zeitschr.  f.  j  1 
Chem.,  28  and  31;  Lev.  tie,  Orid.,  52,  37,  38,  39,  43,  45;  Levene  and  Mandel.  ibid.  4*i. 
47,  49,  50;  Inouye  and  Kotake,  ibid..  46;  Levene  and  Jacobs,  Ber.  d.  d.  i 
Gesellsch.,  42,  43,  44;  with  La  Forge,  ibid.,  43  and  45. 

3  Steudel  and  Brigl,  Zeitschr.  f.  physiol.  Chem.,  70;  Kossel  and  Neumann,  ibid.,  22. 


180  THE  PROTEIN  SUBSTANCES. 

tared  while  the  binding  between  the  pentose  and  purine  bases  remained 
intact.  In  this  manner  they  obtained  pentosides,  i.e.,  glucoside-like^com- 
bination  between  pentose  and  a  purine  base.  These  pentosides  have 
also  been  called  nucleosides  and  such  a  nucleoside  was  the  inosine, 
which  was  first  found  by  Haiser  and  Wenzel  l  and  which  is  the  pen- 
toside  of  inosinic  acid  and  is  a  combination  of  hypoxanthine  with  pentose 
(f?-ribose).  The  other  three  nucleosides  adenosine,  guanosine  and  xantho- 
sine  have  been  prepared  by  Levene  and  Jacobs. 

The  nucleosides  are  crystalline  bodies  which  give  crystalline  combina- 
tions. Of  special  interest  is  guanosine  because  it  is  identical  with  the  base 
vernine,  occurring  in  the  plant  kingdom  and  discovered  by  Schtjlze  2 
and  because  of  the  identity  of  the  pentose  occurring  in  both  has  been 
positively  proved.  The  guanosine  has  also  been  found  by  Levene  and 
Jacobs3  in  the  pancreas.  On  acid  hydrolysis  every  nucleoside  splits 
into  purine  base  and  pentose.  By  the  action  of  nitrite  and  glacial  acetic 
acid  the  guanosine  is  transformed  into  xanthosine  and  the  adenosine 
into  inosine. 

Mandel  and  Dunham  have  prepared,  from  acetone-yeast,  a  crystalline 
adenine-hexose  compound  corresponding  to  the  pentoside  but  whose 
relation  to  the  cleavage  products  of  nucleic  acids  is  not  known.  From 
thymus  nucleic  acid  Levene  and  Jacobs1  have  later  isolated  a  guanine 
hexoside. 

The  pyrimidine  complexes  corresponding  to  the  nucleosides  also 
contain  (in  the  plant  nucleic  acids)  pentose,  according  to  Levene  and 
La  Forge  5  but  in  much  firmer  bondage.  This  is  the  reason  why  they 
give  only  a  faint  orcin  reaction,  are  much  more  resistant  to  enzymes 
than  the  purine  nucleosides  and  give  off  furfurol  only  very  slowly  on 
distilling  with  hydrochloric  acid.  Still  they  contain  pentose  and  pyrimi- 
dine bases  in  equimolecular  proportions.  The  pyrimidine  complexes  are 
called  cytidine  and  uridine,  the  first  containing  cytosine  and  the  second 
uracil.  Uridine  is  crystalline;  the  cytidine  has  not  been  obtained  in  a  crys- 
talline form  but  it  gives  several  crystalline  salts.  The  uridine  is  claimed 
to  exist  pre-formed  in  the  yeast  nucleic  acid  and  not  produced  secondarily 
from  the  cytidine. 

Based  upon  the  investigations  carried  out  by  Steudel,  Levene  and 
Jacobs  we  can  for  the  present  represent  the  structure  of  the  nucleic  acids 
in  the  following  way: 


1  Monatsh.  f.  Chem.,  29. 

'  E.  Bchulze  and  Bosshard,  Zeitschr.  f.  physiol.  Chem.,  10;  with  Trier,  ibid.,  70. 

•  Bioch.  Zeitschr.,  28. 

4  Mandel  and  Dunham,  Journ.  of  biol.  Chem.,  11;  Levene  and  Jacobs,  ibid.,  12. 

5  Ber.  d.  d.  chem.  Gesellseh.,  45. 


NUCLEIC  ACIDS.  181 

The  simple  nucleic  acids  are  ester-like  combinations  between  phosphoric 
acid  and  a  purine  base-pentoside. 

The  complex  nuclei  acids  arc  complex  molecules  each  composed  of 
four  simple  nucleic  acids  (nucleotides).  In  regard  to  the  complex  nucleic 
acids  we  differentiate  between  two  groups. 

The  acids  of  the  thymonucleic  acid  group  are,  according  to  Steudel, 
tetrabasic  phosphoric  acid  ester  which  corresponding  to  each  phosphorus 
atom,  contains  a  hexose  group  and  one  of  the  four  bases,  guanine, 
adenine,  cystosinc  and  thymine.  From  the  name  of  this  group  we  infer 
that  these  acids  contain  thymine. 

The  plant  nucleic  acid  group  differs  from  the  preceding  by  the  follow- 
ing. They  do  not  contain  any  thymine  but  uracil  instead.  They  do  not 
contain  any  hexose  but  do  contain  pentose.  In  the  acids  of  this  group 
for  each  atom  of  phosphor  we  have  1  mol.  pentose  and  on  each  the 
purine  and  pyrimidine  bases  are  combined. 

It  must  be  remarked  that  the  complex  nucleic  acids  have  not  been 
prepared  from  isolated  component  proteins  but  generally  from  organs, 
namely  perhaps  from  a  mixture  of  different  nucleoproteins  and  that  for 
this  reason  we  do  not  know  whether  these  acids  are  chemical  individuals 
or  only  a  mixture  of  closely  related  simple  nucleic  acids.  On  the  other 
hand  it  is  also  possible  that  the  simple  nucleic  acids  originate  from  more 
complex  nucleic  acid  by  cleavage  because  such  cleavages  are  in  fact 
known.  Such  an  assumption  does  not  apply  at  least  for  the  guanylic 
acid  from  the  pancreas  as  it  is  obtained  from  a  compound  protein  with 
only  one  base,  namely  guanine. 

All  nucleic  acids  are  amorphous,  white,  and  have  an  acid  reaction. 
They  are  readily  soluble  in  ammoniacal  or  alkaline  water.  They  also 
dissolve  in  concentrated  acetic  acid  and  form  insoluble  salts  with  copper 
chloride  and  salts  of  the  heavy  metals,  and  as  a  rule  insoluble  basic 
salts  with  the  alkaline  earths.  Their  solubility  in  water  is  very  different. 
Inosinic  acid,  for  example,  is  very  readily  soluble  in  cold  water  while 
tf-guanylic  acid  is  soluble  with  difficulty.  The  complex  nucleic  acids 
are  also  soluble  with  difficulty  in  cold  water.  The  solution  of  their 
alkali  combination  is  not  as  a  rule  precipitated  by  acetic  acid  but  is 
precipitated  by  a  slight  excess  of  hydrochloric  acid,  especially  in  the  pres- 
ence of  alcohol.  The  nucleic  acids  soluble  in  dilute  acids  give  in  such 
solution  a  precipitate  with  proteins,  which  are  considered  as  nucleins.  All 
nucleic  acids  are  insoluble  in  alcohol  and  ether.  They  do  not  give  either 
the  biuret  test  or  Millon's  reaction.  The  nucleic  acids  are  optically 
active  and,  with  the  exception  of  inosinic  acid  (Gamgee  and  Jones) 
and  of  guanylic  acid  (Levene  and  Jacobs  l),  are  dextro-rotatory. 

1  Gamgee  and  Jones,  Proc.  Roy.  Soc,  72;  Levene  and  Jacobs,  Journ.  of  biol. 
Chem.,  12. 


182  THE  PJHOTEIN   SUBSTANCES. 

The  proteolytic  enzymes,  such  as  pepsin  and  trypsin,  decompose  the 
nucleoproteins  more  or  less;  the  nucleic  acids  are  apparently  not 
split  by  these  enzymes  or  at  least  not  as  far  as  phosphoric  acid  and 
purine  bases.  Such  a  cleavage  can,  on  the  contrary,  be  brought  about 
by  erepsin  (Nakayama)  or  by  other  closely  allied  enzymes  found  in 
various  organs  which  have  been  called  nucleases.  Micro-organisms  can  also 
bring  about  a  more  or  less  deep  cleavage  of  the  nucleic  acids  (Schit- 

TENHELM  and  SCHROTER1). 

Levene  and  Medigreceantt  2  differentiate  between  three  kinds  of 
nucleases  namely,  nucleinases,  nucleotidases  and  nucleosidases.  The 
nucleinases,  which  are  found  in  the  pancreatic  juice  and  all  organs 
investigated,  but  not.  in  gastric  juice,  acts  only  upon  the  complex  nucleic 
acids  and  splits  them  into  nucleotides.  The  nucleotidases,  which,  with 
the  exception  of  the  gastric  and  pancreatic  juices,  occurs  all  over  and 
especially  in  the  intestinal  mucosa,  split  the  simple  nucleic  acids  (mono- 
nucleotides) into  phosphoric  acid  and  the  corresponding  nucleoside 
(purine  pentoside).  The  nucleosidases,  which  are  not  found  in  the  gastric, 
pancreatic  or  intestinal  juices,  nor  in  the  blood  or  the  pancreas  but  in 
other  organs,  split  the  nucleosides  into  purine  base  and  pentose.  It  is 
unknown  how  the  cleavage  of  the  pyrimidine  and  hexose  complexes 
of  the  nucleic  acids  is  brought  about. 

According  to  W.  Jones  3  the  purine  bases  of  the  nucleic  acids  can  be 
deamidized  without  being  previously  split  off  as  free  base  from  the  acid. 
Thus  the  pig-pancreas  contains  an  adencsin-deamidase  which  deami- 
dizes  the  still  combined  adenine.  On  the  contrary  the  same  organ  also 
contains  a  guanase  which  deamiclizes  the  free  guanine  but  does  not 
contain  a  guanosine  deamidase.  The  pig  liver,  in  which  only  traces  of 
guanase  occur,  contain  on  the  contrary  a  guanosine-deamidase.  Recent 
investigations  of  Schittenhelm  and  K.  Wiener4  show  that  we  must 
also   admit    of   nucleoside-deamidases   besides   purine   deamidases. 

Inosinic  Acid,  C10H13N4PO8  was  first  isolated  by  Liebig  from  the 
flesh  of  certain  animals  and  then  closely  studied  by  Haiser.  It  is  obtained 
from  beef  extracts,  and  according  to  the  investigations  of  Neuberg  and 
Brahn,  Fr.  Bauer,  and  Levene  and  Jacobs  it  is  a  simple  nucleic  acid.5 


1  Nakayama,  Zeitschr.  f.  physiol.  Chem.,  41;  Iwanoff,  iUd.,  39;  Fr.  Sachs,  "  1st  die 
Nuklease  mit  'lorn  Trypsin  identisch?  "  Inaug.-Dissert,  Heidelberg,  1905;  Schitten- 
helm and  Schroter,  f.  physiol.  Zeitschr.  Chem.,  41. 

2  Journ.  of  biol.  Chem.,  9. 

3  Journ.  of  bio!.  Chem.,  9. 

4  Zeitschr.  f.  physiol.  Chem.,  77. 

Liebig,  Annul,  d.  chem.  u.  Pharm.,  62;  Haiser,  Monatsh.  f.  chem.,  16;  Neuberg 
and  Brahn,  Biochem.  Zeitschr.,  5  and  Ber.  d.  d.  chem.  Gesellsr-h.,  41,  p.  3376;  Bauer 
Hofmeister's  Beitriige,  10;  Levene  and  Jacobs,  Ber.  d.  d.  chem.  Gesellsch.,  41,  p.  2703. 


INOSINIC  AND  GUANYLIC  ACIDS.  183 

On    hydrolysis    it    yields    phosphoric    acid,    hypoxanthine   and    pentose, 
according  to  the  equation: 

CiaH18N4POiB+2HjO=H3P04+C8H4N40+C5HioO«. 

The  pentose,  whose  somewhat  disputed  nature  has  been  discussed 
on  page  178,  is  combined  with  hypoxanthine  in  a  glucoside-like  com- 
bination forming  the  pentoside  inosine,  which,  according  to  Levene  and 
Jacobs,  is  combined  with  the  phosphoric  acid,  like  an  ester  by  means 
of  the  5-carbon  atom  of  the  pentose  (ribose). 

Inosinic  acid  is  amorphous,  syrupy,  readily  soluble  in  water  and  pre- 
cipitable  by  alcohol.  It  is  lavo-rotatory;  for  the  Ba  salt  containing 
hydrochloric  acid  Neuberg  and  Brahn  found  (a)D=— 18.5°  at  10°  C. 
It  gives  several  crystalline  salts  among  which  the  barium  salt,  which  is 
soluble  with  difficulty  in  water,  must  be  mentioned. 

In  regard  to  the  preparation  of  this  acid  we  must  refer  to  the  works 
of  Haiser,  Neuberg  and  Erahn,  Levexe  and  Jacobs  mentioned  in 
footnote  5,  page  182. 

Guanylic  acid.  This  acid,  which  was  first  prepared  by  Bang  from 
the  pancreas  has  also  been  found  by  Jones  and  Rowxtree  in  the  spleen 
and  by  Levexe  and  Mandel  '  in  the  liver.  As  cleavage  products  it  yields 
guanine,  pentose  and  phosphoric  acid  and  therefore  its  simplest  formula 
is  ('10H14N5PO8.  This  formula  is  accepted  also  by  Steudel  and  Brigl 
and  by  Levexe  and  Jacobs,  while  Baxg  basing  his  views  on  the  results 
of  elementary  analysis  gives  the  formula  C44H6GN20P4O34.  Accord- 
ing to  this  formula  the  acid  would  contain  besides,  guanine,  pentose  and 
phosphoric  acid  also  an  unknown  residue,  C4H10O2,  and  according  to  Bang 
is  not  a  simple  nucleic  acid  but  would  occupy  an  intermediary  position 
between  the  inosinic  acid  and  the  thymus  nucleic  acid.  In  opposition 
to  this  it  must  be  remarked  that  Levene  and  Jacobs  2  have  recently 
prepared  the  crystalline  brucine  salt  of  the  acid  and  the  analysis  of  this 
salt  as  well  as  the  barium  salt  substantiates  the  first  mentioned,  simple 
formula.     In  regard  to  the  pentose  of  guanylic  acid  see  page  178. 

The  acid  first  described  by  Bang,  the  /3-acid,  is  soluble  with  great  diffi- 
culty in  cold  water  and  rather  readily  soluble  in  boiling  water.  It  is  easily 
precipitated  by  acetic  acid  from  the  solution  of  the  alkali  combination 
in  water.  The  /3-acid  may,  according  to  Bang,  be  derived  from  another 
guanylic   acid,   the  a-guanylic   acid,   by  the   action  of  the  alkali.     The 

1  Banc,  Zeitschr.  f.  physiol.  Chem.,  .26;  with  Raas-hou,  Hofmeister's  Beitrage.  4; 
Jones  and  Rowntree,  Journ.  of  biol.  chem.,  4;  Levene  and  Mandel,  Bioehem.  Zeitschr. 
10. 

:  Steudel  and  Brigl,  Zeitschr.  f.  physiol.  Chem.,  68;  Bang,  ibid.,  69  and  Bioch. 
Zeitschr.,  26;  Levene  and  Jacobs,  Journ.  of  biol.  Chem.,  12. 


184  THE  PROTEIN   SUBSTANCES. 

a-guanylic  acid  is  readily  soluble,  even  in  cold  water,  and  it  is  also  similar 
to  thymus  nucleic  acid  in  other  respects.  It  is  precipitated  from  the 
solution  of  its  salts  by  hydrochloric  acid  but  not  by  acetic  acid,  and  its 
solutions  precipitate  proteins.  Steudel  and  Brigl  believe  that  the 
jS-acid  is  a  potassium  salt  and  that  the  a-acid  is  the  actual  acid,  but  this 
view  Bang  disputes.  Levene  and  Jacobs  found  that  the  acid  con- 
taminated with  alkali  does  not  gelatinize  while  the  pure  acid  does.  The 
specific  rotation  of  the  latter  was  (o)d=  —1-27°  at  25°  C. 

In  regard  to  the  preparation  of  guanylic  acid  we  refer,  to  the  work 
of  Bang,  Levene  and  Jacobs.1 

Thymonucleic  Acids.  A.  Neumann  has  isolated  two  nucleic  acids, 
a-  and  /3-thymus  nucleic  acid,  from  the  thymus  gland.  The  a-acid  is 
soluble  with  difficulty,  and  in  proper  concentration  gives  a  sodium  salt 
which  gelatinizes  in  proper  concentration,  and  a  barium  salt  which  is 
precipitated  by  barium  acetate  in  substance  (Kostytschew) .  The 
barium  salt  of  the  /3-acid  is  not  precipitated  by  barium  acetate.  The 
a-acid  is  designated  as  anhydric  by  Schmiedeberg,2  and  the  /3-acid  as 
hydrate,  and  the  first  can  be  transformed  into  the  second  by  heating. 
This  transformation,  according  to  Kostytschew,  is  a  decomposition 
whereby  two-thirds  of  the  purine  bases  are  split  off. 

According  to  Schmiedeberg  the  thymus  nucleic  acid  is  identical 
with  the  salmo-nucleic  acid  (from  salmon  sperm),  and  also  according 
to  Steudel  probably  with  the  acid  from  the  herring  sperm.  Other 
nucleic  acids,  at  least  those  very  closely  related  to  this  nucleic  acid,  have 
been  prepared  from  the  sperm  of  the  burbot  (Lota  vulgaris)  by  Alsberg, 
of  the  sturgeon  (Noll)  and  of  the  sea-urchin  (Mathews),  also  from 
ox-sperm,  brain,  spleen  (Levene),  pancreas  (Levene,  v.  Furth  and 
Jerusalem,  Steudel),  mammary  glands  and  kidneys  (Levene  and  Man- 
del  3)  and  from  other  organs. 

At  the  present  time  we  arc  not  agreed  as  to  the  formula  for  the  most 
carefully  studied  thymonucleic  acids  (from  the  thymus,  herring  and  sal- 
mon sperms) .  According  to  the  numerous  analyses  of  Schmiedeberg  and 
his  collaborators  for  every  4  atoms  of  phosphorus  there  occur  14  atoms 
of  nitrogen.  The  relationship  of  C  to  P  was  40  to  4  and  the  relationship  of 
C  to  N  in  12  out  of  15  preparations  was  40  to  14,  and  only  in  3  prepara- 
tions 40  to   15.     From  these  facts  Schmiedeberg  has  given  the  acid 

1  See  footnotes  1  and  2,  p.  183. 

2  A.  Neumann,  Arch.  f.  (Anat.  u.)  physiol,  1898  and  1899;  Kostytschew,  Zeitschr. 
f.  physiol.  Chem.,  39;  Schmiedeberg,  1.  c. 

3  Alsberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  51;  Noll,  Zeitschr.  f.  physiol.  Chem.,  25; 
Mathews,  ihvl.,  23;  v.  Furth  and  Jerusalem  Hofmeister's  Birtrage,  10  and  11;  Steudel, 
Zeitschr.  f.  pi  ysiol.  Chem.,  53;  Levene  and  Mandel,  ibid.,  46,  47.  See  also  footnote  2, 
p.  179. 


PLANT   NUCLEIC  ACIDS.  185 

the  formula  C40HMN14O16.2P2O8.  According  to  Steudel  for  every  4 
atoms  of  phosphorus  we  have  15  atoms  nitrogen  and  from  this  he  has 
calculated  fche  formula  ( UaHeiN  15P4O34+9H2O  for  the  acid  containing 

water. 

The  probable  constitution  of  the  thymo-nucleic  acids  has  been  previ- 
ously indicated  and  as  positively  known  cleavage  products  we  have  at  least 
phosphoric  acid,  a  hexose  carbohydrate,  guanine,  adenine,  thymine  and 
cytosine. 

The  thymo-nucleic  acids  have  the  reactions  as  given  for  the  complex 
nucleic  acids.  They  are  amorphous,  dextro-rotatory,  and  soluble  in  cold 
water  with  difficulty.  They  form  soluble  salts  with  alkalies  and  the  acid 
is  precipitated  from  these  solutions  by  mineral  acid  but  not  by  acetic 
acid.  Tannic  acid  alone  does  not  cause  a  precipitate  but  does  in  the 
presence  of  sodium  acetate.  Proteins  precipitate  their  solutions  contain- 
ing acetic  acid.  The  two  special  thymo-nucleic  acids  differ  from  each 
other  by  the  different  behavior  of  their  salts  (see  above). 

The  preparation  of  the  nucleic  acids  is  based  in  the  first  place  always 
upon  the  cleavage  of  the  nucleoprotein  into  protein  and  nucleic  acid  by 
the  action  of  alkali  and  then  separating  the  nucleic  acids  from  the 
protein.  The  operations  necessary  for  purifying  the  nucleic  acids  from 
proteins  are  very  complicated  and  we  must  refer  to  the  works  of 
Schmiedeberg,  Neumann,  Levene,  and  others.1 

Plant  Nucleic  Acids.  The  two  best  known  acids  of  this  group  are 
the  yeast  nucleic  acid  and  the  triticonucleic  acid  isolated  from  the 
wheat  embryo.  The  identity  of  these  two  acids,  as  suggested  by  Osborne 
and  Harris  has  become  more  and  more  probable.  According  to  Kowa- 
lewskt2  the  yeast  nucleic  acid  contain  only  adenine,  guanine  and  cytosine, 
the  uracil  is  only  formed  secondarily  from  the  cytosine.  The  yeast  nucleic 
acid  may  perhaps  be  a  triphosphoric  acid  with  three  molecules  of  pentose 
each  with  a  molecule  of  adenine,  guanine  and  cytosine. 

This  view  stands  in  opposition  to  the  observations  of  Levene  and 
Jacobs  3  that  the  yeast  nucleic  acid  contains  one  molecule  of  pentose 
combined  with  adenine  and  guanine,  and  besides  this  it  contains  two 
pyrimidinehexose  complexes,  cytidine  and  uridine. 

The  triticonucleic  acid  yields  also,  as  Osborne  and  Heyl,  Wheeler 
and  Johnson  and  recently  Levene  and  La  Forge  4  have  shown,  the  same 
hydrolytic  products  as  the  yeast  nucleic  acid  and  both  contain  rf-ribose. 

Schmiedeberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  43  and  57;  Herlant,  ibid.,  44;  Neu- 
mann, Arch.  f.  (Anat.  u.)  Physiol.  1899  Supplb.;  Levene,  Zeitschr.  f.  physiol.  Chem., 
32  and  45;  Kostytschew,  1.  c. 

2  Osborne  and  Harris,  Zeitschr.  f.  physiol.  Chem.,  36;  Kowalewsky,  ibid.,  69. 

3  Ber.  d.  d.  chem.  Gesellsch.,  44. 

4  Osborne  and  Heyl,  Amer.  Journ.  of  Physiol.,  21;  Wheeler  and  Johnson,  Amer. 
Chem.  Journ.,  29;  Levene  and  La  Forge,  Ber.  d.  d.  Chem.,  Gesellsch.,  43. 


186  THE  PROTEIN  SUBSTANCES. 

The  somewhat  different  results  found  on  the  elementary  analysis  of  these 
two  acids  do  not  seem  to  be  of  very  great  importance  and  we  have  strong 
evidence  for  the  identity  of  these  acids.  Osborne  and  his  collaborators 
found  the  formula  C41H61N16P4O31  for  triticonucleic  acid. 

The  plant  nucleic  acids  have  the  general  reactions  of  the  complex 
nucleic  acids  but  can  be  precipitated  by  an  excess  of  acetic  acid.  They 
are  dextro-rotatory. 

In  regard  to  their  preparation  we  refer  to  the  works  of  Kossel,  Osborne 
and  Harris  and  to  Levene  and  co-workers.1 

Plasminic  acid  is  an  acid  which  was  prepared  by  Ascoli  and  Kossel  2  by 
the  action  of  alkali  upon  yeast.  It  contains  iron,  and  is  soluble  in  very  dilute 
hydrochloric  acid  (1  p.  m.).  It  is  still  a  question  whether  it  is  a  mixture  or  a 
chemical  individual. 

2.  Purine  Bases. 

The  cleavage  products  obtained  from  the  nucleic  acids,  the  nuclein 
bases,  which  are  also  called  alloxuric  bases  by  Kossel  and  Kruger,  are 
members  of  the  larger  group  of  purines,  to  which  also  belongs  the  uric 
acid  which  is  a  substance  occurring  in  the  animal  body.  The  constitu- 
tion of  these  bodies  has  been  explained  by  E.  Fischer,3  and  he  has 
prepared  many  of  the  bodies  synthetically.  They  can  all  be  derived  from 
the  synthetically  prepared  purine,  C5H4N4,  which  has  the  formula  given 
below  and  which  may  be  considered  as  a  combination  of  a  pyrimidine 
ring  with  an  imidazole  ring. 

N=CH  N=CH 

II  II  HC-NH> 


HC    C— NR  HC     CH  |  >CH 

II      II  >CH  ||      ||  HC N^ 

N— C N^  N— CH 

Purine  Pyrimidine  Imidazole 

The  different  purine  bodies  are  derived  therefrom  by  the  substitution  of  the 
various  hydrogen  atoms  by  hydroxyl,  amide,  or  alkyl  groups.  In  order  to  signify 
the  different  positions  of  substitution  Fischer  has  proposed  to  number  the  nine 
members  of  the  purine  nucleus  in  the  following  way: 

1N=C6 

I      I 
2C  5C— N7 


> 


.C8. 


3N— C— N9 

4 


1  See  footnote  2,  p.  179,  and  footnote  3  and  4,  p.  185. 

2  Ascoli,  Zeitschr.  f.  physiol.  Chem.,  28. 

3Sff  ]■'..  Kisr-her,  Untersuchungen  in  der  Purinfjrwppe  (1882-1906)  Berlin,  1907. 


IT  KINK   BASES.  187 

I IX— CO 

I      I 
For  example,  uric  acid,  OC     0 — NH\       ,  is  2,  P>,  S-trioxvpurine;  adenine, 

I     II         >co 

HN-C— XJT 
N=CHN,  •  IIX-CO 

II  M. 

HC     C — XI  k  ,  is  G-aminopurine,  and  heteroxanthine,  OC    C — X.CH3,  is 

H      II  >CH  I      II        >CH 

X - ( ' X^  II x-c-x/tn 

7-methyl-2,  6-dioxypurine,  etc. 

The  starting-point  used  by  Fischer  for  the   synthetical  preparation  of  the 

purine  bases  was  2,  6,  S-triehlorpurine,  which  is  obtained,  with  N-oxy-2,  (i-dichlor- 

purine  as  an  intermediary  product,  from  potassium  urate  and  phosphorus  oxychlo- 

ride. 

The  purine  bodies  or  alloxuric  bodies,  found  in  the  animal  body  or  its 

excreta  are  as  follows:    Uric  acid,  xanthine,  heteroxanthine,   \-methylxan- 

paraxanthine,     guanine,     epiguanine,     hypoxanthine,    episarkine, 

adenine.      The  bodies  theobromine,  theophylline,  and  caffeine,  occurring  in 

the  vegetable  kingdom,  stand  in  close  relation  to  this  group. 

The  composition  of  the  purine  bodies  most  important  from  a  physio- 
logical standpoint  is  as  follows: 

Uric  acid,  C5H4X4O3 2,  6,  8-trioxypurine 

Xanthine C5H4X4O2 2,  6-dioxypurine 

l-methylxanthine,  C6H6X402 1-methyl  " 

Heteroxanthine,  C6H6X402 7      "  " 

Theophylline,  C7H8X4()2 1,  3-dimethyl 

Paraxanthine,  C7H8X4O2 1.7-     " 

Theobromine  C7H8X4O2 3,  7- 

Caffeine,  C8H,oX402 1,3,  7-trimethyl 

Hypoxanthine,  Cs^X^O 6-oxypurine 

Guanine  C5H5X50 2-amino  "       " 

Epiguanine,  C6H7X50 7-methyl  "       "      "        " 

Adenine  CsHsX'j 6-aminoourine 

Episarkine,  C4H6X30(?) 

After  Salomon  !  had  shown  the  occurrence  of  xanthine  bodies  in 
young  cells,  the  importance  of  the  purine  bases  as  decomposition  prod- 
ucts of  cell  nuclei  and  of  nucleins  was  shown  by  the  pioneering  researches 
of  Kossel,  who  discovered  adenine  and  theophylline.  In  those  tissues 
in  which,  as  in  the  glands,  the  cells  have  kept  their  original  state,  the 
purine  bases  are  not  found  free,  but  in  combination  with  other  atomic 
groups  (nucleic  acids).  In  such  tissues,  on  the  contrary,  as  in  muscles, 
which  are  poor  in  cell  nuclei,  the  purine  bases  are  found  in  the  free  state. 
Since  the  purine  bases,  as  suggested  by  Kossel,  stand  in  close  relation- 
ship to  the  cell  nucleus,  it  is  easy  to  understand  why  the  quantity  of 
these  bodies  is  so  greatly  increased  when  large  quantities  of  nucleated 

1  Sitzungsber.  d.  Bot.  Verein  der  Provinz  Brandenburg,  1880. 


188  THE  PROTEIN   SUBSTANCES. 

cells  appear  in  such  places  as  were  before  endowed  in  a  relatively  poor 
manner.  As  an  example  of  this,  the  blood,  in  leucaemia,  is  extremely  rich 
in  leucocytes.  In  such  blood  Kossel  1  found  1.04  p.  m.  purine  bases, 
against  only  traces  in  the  normal  blood.  That  these  bases  are  also  inter- 
mediate steps  in  the  formation  of  uric  acid  in  the  animal  organism  is 
probable,  and  will  be  shown  later  (see  Chapter  XIV). 

Only  a  few  of  the  purine  bases  have  been  found  in  the  urine  or  in  the 
muscles.  Only  four  bases — xanthine,  guanine,  hypoxanthine,  and  ade- 
nine— have  been  obtained,  thus  far,  as  cleavage  products  of  nucleins, 
and  these  do  not  always  have  a  primary  origin.  In  regard  to  the  purine 
bodies  from  other  substances  we  refer  the  reader  to  their  respective 
chapters.  Only  the  above  four  bodies,  the  real  nuclein  bases,  will  be 
considered  at  this  time. 

Of  these  four  bodies,  xanthine  and  guanine  form  one  special  group 
and  hypoxanthine  and  adenine  another.  By  the  action  of  nitrous  acid 
guanine  is  converted  into  xanthine  and  adenine  into  hypoxanthine. 

C5H4N40.NH+HN02  =  C5H4N402+N2+H20; 

Guanine  Xanthine 

C5H4N4.NH  +  HNO2  +  C5H4N4O  +  N2  +  H2O. 

Adenine  Hypoxanthine 

Similar  transformation,  where  xanthine  and  hypoxanthine  are  pro- 
duced secondarily,  may  also  occur  in  the  hydrolysis  of  nucleic  acids  as 
well  as  in  putrefaction  and  by  the  action  of  special  enzymes.  The 
researches  of  Schittenhelm,  Levene,  Jones,  Partridge,  Winternitz, 
and  Burian  have  shown  that  in  various  organs  deamination  enzymes, 
such  as  guanase  and  adenase,  occur,  which  convert  guanine  and  adenine 
into  xanthine  and  hypoxanthine  respectively,  and  also  oxidases  which 
oxidize  hypoxanthine  into  xanthine  and  this  then  into  uric  acid.  This 
formation  of  uric  acid  from  the  purine  bases,  which  will  be  discussed  in 
detail  in  a  following  chapter  (XIV),  is  of  very  great  interest. 

The  nuclein  bases  form  crystalline  salts  with  mineral  acids,  which, 
with  the  exception  of  the  adenine  salts,  are  decomposed  by  water.  They 
are  easily  dissolved  by  alkalies,  while  with  ammonia  their  action  is  some- 
what different.  They  are  all  precipitated  from  acid  solution  by  phos- 
photungstic  acid;  they  also  separate  as  silver  compounds  on  addition 
of  ammonia  and  ammoniacal  silver-nitrate  solution.  These  precipitates 
are  soluble  in  boiling  nitric  acid  of  1.1  specific  gravity.  All  purine  bodies 
are  also  precipitated  by  Fehling's  solution  (see  Chapter  III)  in  the  pres- 
ence of  a  reducing  substance  such  as  hydroxylamine  (Drechsel  and 
Balke).     Copper  sulphate  and  sodium  bisulphite  may  also  be  used  to 

1  Zeitschr.  f.  physiol.  Chem..  7. 


XANTHINE.  189 

advantage  in  their  precipitation  (Kruger)1.  This  behavior  of  the 
purine  bases  serves  just  as  well  as  the  behavior  with  the  silver  solution 
for  their  precipitation  and  preparation. 

HN— CO 

I       I 
Xanthine,  C5H4N402,  =  OC     C— NHV         (2,  6-dioxypurine)  is  f 0Und 

I       II  >CH 

HN— C  —  W 

in  several  cellular  organs.  It  occurs  in  small  quantities  as  a  physio- 
logical constituent  of  urine,  and  it  occasionally  has  been  found  as  a  urinary 
sediment,  or  calculus.  It  was  first  observed  in  such  a  stone  by  Marcet. 
Xanthine  is  found  in  larger  amounts  in  a  few  varieties  of  guano  (Jarvis 
guano) . 

Xanthine  can  be  prepared,  according  to  E.  Fischer,  by  boiling  uric 
acid  with  25  per  cent  hydrochloric  acid  or,  according  to  Sundvik  2  by 
heating  uric  acid  with  anhydrous  oxalic  acid  in  glycerin  to  about  200°  C. 

Xanthine  is  amorphous,  or  forms  granular  masses  of  crystals,  or  may 
also,  according  to  Horbaczewski,3  separate  as  masses  of  shining,  thin, 
large  rhombic  plates  with  1  mol.  water  of  crystallization.  It  is  very 
slightly  soluble  in  water,  in  14,151-14,600  parts  at  16°  C,  and  in  1300- 
1500  parts  at  100°  C.  (Almen4).  It  is  insoluble  in  alcohol  or  ether,  but 
is  readily  dissolved  by  alkalies  and  with  difficulty  by  dilute  acids.  With 
hydrochloric  acid  it  gives  a  crystalline,  difficultly  soluble  combination. 
With  very  little  caustic  soda  it  gives  a  readily  crystallizable  compound, 
which  is  easily  dissolved  by  an  excess  of  alkali.  Xanthine  dissolved  in 
ammonia  gives  with  silver  nitrate  an  insoluble,  gelatinous  precipitate 
of  silver  xanthine.  This  precipitate  is  dissolved  by  hot  nitric  acid,  and 
by  this  means  an  easily  soluble  crystalline  double  compound  is  formed. 
Xanthine  in  aqueous  solution  is  precipitated  on  boiling  with  copper 
acetate.  At  ordinary  temperatures  xanthine  is  precipitated  by  mercuric 
chloride  and  by  ammoniacal  basic  lead  acetate.  It  is  not  precipitated 
by  basic  lead  acetate  alone. 

When  evaporated  to  dryness  in  a  porcelain  dish  with  nitric  acid, 
xanthine  gives  a  yellow  residue,  which  turns,  on  the  addition  of  caustic 
soda,  first  red,  and  after  heating,  purple-red.  If  we  place  some  chlorinated 
lime  with  some  caustic  soda  in  a  porcelain  dish  and  add  the  xanthine 


1  Balke,  Zur  Kenntnis  der  Xanthinkorper,  Inaug.-Diss.  Leipzig,  1893 ;  Kruger 
Zeitschr.  f.  physiol.  Chem.,  18. 

2  E.  Fischer,  Ber.  d.  d.  chem.  Gesellsch,  43;  Sundvik,  Zeitschr.  f.  physiol.Chem. 
76.  In  regard  to  the  synthesis  of  xanthine  and  other  purines  see  E.  Fischer,  footnote 
3,  p.  186. 

5  Zeitschr.  f.  physiol.  Chem.,  23. 
4  Journ.  f.  prakt.  Chem.,  96. 


190  THE  PROTEIN  SUBSTANCES. 

to  this  mixture,  at  first  a  dark  green  and  then  quickly  a  brownish  halo 
forms  around  the  xanthine  grains  and  finally  disappears  (Hoppe-Seyler). 
If  xanthine  is  warmed  in  a  small  vessel  on  the  water-bath  with  chlorine- 
water  and  a  trace  of  nit.'ic  pcid,  and  evaporated  to  dryness,  and  the 
residue  is  then  exposed  under  a  bell-jar  to  the  vapors  of  ammonia,  a 
red  or  purple-violet  color  is  produced  (Weidel's  reaction).  E.  Fischer  x 
has  modified  Weidel's  reaction  in  the  following  way:  He  boils  the  xan- 
thine in  a  test-tube  with  chlorine-water  or  with  hydrochloric  acid  and  a 
little  potassium  chlorate,  then  evaporates  the  liquid  carefully,  and  moistens 
the  dry  residue  with  ammonia. 

HN— CO 

!     I 

Guanine,  C5H5N50,=H2N.C     C— NH\  (2-amino-6-oxypurine) . 

II      II  >CH 

N— C—    W 

Guanine  is  found  in  all  organs  rich  in  cells.  It  is  further  found  in  the 
muscles  (in  very  small  amounts),  in  the  scales  and  in  the  air-bladder  of 
certain  fishes,  as  iridescent  crystals  of  guanine-lime;  in  the  retinal  epithe- 
lium of  fishes,  in  guano,  and  in  the  excrement  of  spiders  it  is  found  as 
chief  constituent.  It  also  occurs  in  human  and  pig  urine.  Under  patholog- 
ical conditions  it  has  been  found  in  leucsemic  blood,  and  in  the  muscles, 
ligaments,  and  articulations  of  pigs  with  guanine-gout. 

Guanine  is  a  colorless,  ordinarily  amorphous  powder,  which  may  be 
obtained  as  small  crystals  by  allowing  its  solution,  in  concentrated 
ammonia,  to  evaporate  spontaneously.  According  to  Horbaczewski  it  may 
under  certain  conditions  appear  in  crystals  similar  to  creatinine-zinc  chlor- 
ide. It  is  insoluble  in  water,  alcohol,  and  ether.  It  is  rather  easily  dissolved 
by  mineral  acids  and  readily  by  alkalies,  but  it  dissolves  with  great 
difficulty  in  ammonia.  According  to  Wulff2  100  cc.  of  cold  ammonia 
solution  containing  1,  3,  or  5  per  cent  NH3  dissolve  9,  15,  or  19  milli- 
grams of  guanine  respectively.  The  solubility  is  relatively  increased 
in  hot  ammonia  solution.  The  hydrochloride  readily  crystallizes,  and 
has  been  recommended  by  Kossel  3  for  the  microscopical  detection  of 
guanine,  on  account  of  its  behavior  toward  polarized  light.  The  sul- 
phate contains  2  molecules  of  water  of  crystallization,  which  is  completely 
expelled  on  heating  to  120°  C.,  and  this  fact,  as  well  as  the  fact  that 
guanine  yields  guanidine  on  decomposition  with  chlorine-water,  differ- 
entiates it  from  6-amino-2-oxypurine,  which  is  considered  as  an  oxida- 
tion  product   of  adenine  and  possibly  occurs   as   a  chemical  metabolic 

1  Ber   (1    deutsch.  chem.  Gesellsch.,  30,  2236. 

2  Zeitschr  f.  physiol.  Chem.,  17. 

3  Ueber  die  chem.  Zusammensetz.  der  Zelle,  Verh.  d.  physiol.  Gesellsch.    zu  Berlin 
1890-91,  Nop.  5  and  6. 


HYPOXANTHINE.  191 

product  (E.  Fischer).  The  G-amino-2-oxypurine  sulphate  contains 
only  1  molecule  of  water  of  crystallization,  which  is  not  expelled  at  120°  C. 
Very  dilute  guanine  solutions  arc  precipitated  by  both  picric  acid  and 
metaphosphoric  acid.  These  precipitates  may  be  used  in  the  quantita- 
tive estimation  of  guanine.  The  silver  compound  dissolves  with  difficulty 
in  boiling  nitric  acid,  and  on  cooling  the  double  compound  crystallizes 
out  readily.  Guanine  acts  like  xanthine  in  the  nitric-acid  test,  but  gives 
with  alkalies  on  heating  a  more  bluish-violet  color.  A  warm  solution 
of  guanine  hydrochloride  gives  with  a  cold  saturated  solution  of  picric 
acid  a  yellow  precipitate  consisting  of  silky  needles  (Capranica).  With 
a  concentrated  solution  of  potassium  bichromate  a  guanine  solution 
gives  a  crystalline,  orange-red  precipitate,  and  with  a  concentrated 
solution  of  potassium  ferricyanide  a  yellowish-brown,  crystalline  pre- 
cipitate (Capranica).  It  also  gives  a  compound  with  picrolonic  acid 
(Levene  l).     Guanine  gives  Weidel's  reaction. 

HN— CO 

I  I 

Hypoxanthine,  Sarkine,  C5H4N4O,  =HC     C — NPL       =  (6-oxypurine) . 

II  II  >CH 
N— C—  N  ^ 

This  body  has  been  found  in  all  cellular  organs  and  in  meat  extracts, 
and  as  a  cleavage  product  of  inosinic  acid.  It  is  especially  abundant  in 
the  sperm  of  the  salmon  and  carp.  Hypoxanthine  occurs  also  in  the  mar- 
row and  in  very  small  quantities  in  normal  urine,  and,  as  it  seems,  also  in 
milk.  It  is  found  in  rather  considerable  quantities  in  the  blood  and  urine 
in  leucsemia. 

Hypoxanthine  can  be  obtained  according  to  Sundvik's  2  method 
from  uric  acid  or  xanthine  by  heating  with  a  formate  or  more  simply 
by  heating  with  chloroform  in  alkaline  solution. 

Hypoxanthine  forms  very  small,  colorless,  crystalline  needles.  It 
dissolves  with  difficulty  in  cold  water,  but  the  claims  concerning 
solubility  therein  are  very  contradictory.3  It  dissolves  more  readily 
in  boiling  water,  in  about  70-80  parts.  It  is  almost  insoluble  in  alcohol, 
but  is  dissolved  by  acids  and  alkalies.  The  compound  with  hydrochloric 
acid  is  crystalline,  and  is  more  soluble  than  the  corresponding  xanthine 
derivative.  It  is  easily  soluble  in  ammonia.  The  silver  compound 
dissolves  with  difficulty  in  boiling  nitric  acid.  On  cooling,  a  mixture 
of  two  hypoxanthine  silver-nitrate  compounds  possessing  an  inconstant 
composition  separates  out.  On  treating  this  mixture  with  ammonia 
and   an   excess   of   silver  nitrate  and   heating,    a   silver  hypoxanthine   is 

1  Capranica,  Zeitschr.  f.  physiol.  Chem.,  4;  Levene,  Bioch.  Zeitsehr.,  4. 

5 1.  c.  and  Skand,  Arch.  f.  Physiol.,  25. 

'See  E.  Fischer,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30. 


192  THE  PROTEIN  SUBSTANCES. 

formed,  which  when  dried  at  120°  C.  has  a  constant  composition, 
2(C5HoAg2N40)H20,  and  is  used  in  the  quantitative  estimation  of 
hypoxanthine.  Hypoxanthine  picrate  is  soluble  with  difficulty,  but 
if  a  boiling-hot  solution  of  it  is  treated  with  a  neutral  or  only  faintly 
acid  solution  of  silver  nitrate  the  hypoxanthine  is  almost  quantitatively 
precipitated  as  the  compound  C5H3AgN40.C6H2(N02)30H.  Hypo- 
xanthine does  not  yield  an  insoluble  compound  with  metaphosphoric 
acid.  When  treated,  like  xanthine,  with  nitric  acid,  it  yields,  an  almost 
colorless  residue  which,  on  warming  with  alkali,  does  not  turn  red.  Hypo- 
xanthine does  not  give  Weidel's  reaction.  After  the  action  of  hydro- 
chloric acid  and  zinc  upon  a  hypoxanthine  solution,  followed  by  the 
addition  of  an  excess  of  alkali  a  ruby-red  color  develops,  followed  by 
a  brownish-red  color  (Kossel).  According  to  E.  Fischer1  a  red 
coloration  occurs  even  in  the  acid  solution. 
N— C.NH2 

Adenine,  CsHsNs^HC     C — NH\        (6-aminopurine),  was  first  found 
II      II  >CH 

N— C—  N  ^ 

by  Kossel  2  in  the  pancreas.  It  occurs  in  all  nucleated  cells,  but  in 
greatest  quantities  in  the  sperm  of  the  carp  and  in  the  thymus.  Adenine 
has  also  been  found  in  lucsemic  urine  (Stadthagen  3) .  It  may  be  obtained 
in  large  quantities  from  tea-leaves. 

Adenine  crystallizes  with  3  molecules  of  water  of  crystallization  in 
long  needles  which  gradually  become  opaque  in  the  air,  but  much  more 
rapidly  when  warmed.  If  the  crystals  are  warmed  slowly  with  a  quan- 
tity of  water  insufficient  for  solution,  they  suddenly  become  cloudy  at 
53°  C,  a  characteristic  reaction  for  adenine.  It  dissolves  in  1086  parts 
cold  water,  but  is  easily  soluble  in  warm.  It  is  insoluble  in  ether,  but 
somewhat  soluble  in  hot  alcohol  and  easily  so  in  acids  and  alkalies.  It 
is  more  easily  soluble  in  ammonia  solution  than  guanine,  but  less  soluble 
than  hypoxanthine.  The  silver  compound  of  adenine  is  soluble  with 
difficulty  in  warm  nitric  acid,  and  deposits  on  cooling  as  a  crystalline 
mixture  of  adenine  silver  nitrates.  With  picric  acid  adenine  forms  a 
compound,  CsHsNs.Ce^CNC^sOH,  which  is  very  insoluble  but 
separates  more  readily  than  the  hypoxanthine  picrate,  and  can  be 
used  in  the  quantitative  estimation  of  adenine.  We  also  have  an  adenine 
mercury-picrate.  Metaphosphoric  acid  with  adenine  gives  a  precipitate 
which  dissolves  in  an  excess  of  the  acid  if  the  solution  is  not  too  dilute. 
Adenine    hydrochloride    gives   with    gold   chloride    a    double    compound 

1  Kossel,  Zeitschr.  f.  physiol.  Chem.,  12,  252;  E.  Fischer,  1.  c. 

2  See  Zeitschr.  f .  physiol.  Chem.,  10  and  12. 
»  Virchow's  Arch.,  109. 


PYRIMIDINE  BASES.  193 

which  consists  in  part  of  leaf-shaped  aggregations  and  in  part  of  cubical 
or  prismatic  crystals,  often  with  rounded  corners.  This  compound  is 
used  in  the  microscopic  detection  of  adenine.  With  the  nitric-acid  test 
and  with  Weidel's  reaction  adenine  acts  in  the  same  way  as  hypoxan- 
thine.  The  same  is  true  for  i£s  behavior  with  hydrochloric  acid  and 
zinc  with  subsequent  addition  of  alkali. 

The  procedure  for  the  preparation  and  detection  of  the  four  above- 
described  purine  bases  is  as  follows:  Boiling  with  0.5-1  vol.  per  cent 
sulphuric  acid,  saturating  with  baryta-water,  removing  the  excess  of 
barium  with  CO2,  precipitating  all  the  bases  as  silver  compounds 
from  the  strongly  ammoniacal  filtrate  and  dissolving  them  in  boiling 
nitric  acid  when  the  xanthine  compound  remains  in  solution  on  cooling 
while  the  combination  with  the  other  three  bases  precipitate.  The  silver 
xanthine  may  be  precipitated  from  the  filtrate  by  the  addition  of  ammonia 
and  the  xanthine  set  free  by  means  of  sulphureted  hydrogen.  The 
three  above-mentioned  silver-nitrate  compounds  are  decomposed  by  sul- 
phureted hydrogen  and  the  guanine  separated  from  the  adenine  and 
hypoxanthine  by  treatment  while  hot  with  ammonia,  in  which  the 
guanine  is  soluble  with  difficulty.  When  the  above  filtrate  containing  the 
adenine  and  hypoxanthine,  which  has  been,  if  necessary,  freed  from 
ammonia  by  evaporation,  is  allowed  to  cool,  the  adenine  separates, 
while  the  hypoxanthine  remains  in  solution.  According  to  Balke  l — 
we  can  advantageously  precipitate  the  purine  bases  with  copper  sulphate 
and  hydroxylamine.  Details  for  the  above  methods  may  be  found  in 
complete  hand-books.  The  same  procedures  are  followed  in  the  quan- 
titative estimation  of  the  purine  bases  in  animal  organs.2 

3.  Pyrimidine  Bases. 

These   bodies   are   closely   related   to   the   purines,    and   pyrimidine, 
N=CH 

C4H4N2,  =  HC     CH,  may  be  considered  as  the  mother  substance  thereof. 

li      II 
N— CH 

The  pyrimidine  bases  contained  in  the  nucleic  acids  are  cytosine,  uracil 
and  thymine. 

N=CNH2 

Cytosine,  C4H5N3O,  =  OC     CH      (6  amino-2  oxypyrimidine),  wag  first 

HN— CH 

prepared  by  Kossel  and  Neumann  from  thymus  nucleic  acid,  and  then 
by  Kossel  and  Steudel  and  others  from  various  nucleic  acids.   Wheeler 


•See  footnote  1,  p.  190. 

2  See  Burian  and  Hall,  Zeitschr.  f.  physiol.  Chem.,  38;    Kossel  ibid.,   5-8,    Bruhns, 
ibid.,  14;  His  and  Hagen,  ibid.,  30. 


194  THE  PROTEIN  SUBSTANCES. 

and  Johnson  l  have  also  prepared  it  synthetically.  It  is  transformed 
into  uracil  by  the  action  of  nitrous  acid. 

The  free  base  is  soluble  with  difficulty  in  water  {129  parts)  and  crystal- 
lizes in  thin  leaves  with  a  mother-of-pearl  luster.  It  is  insoluble  in  ether 
and  soluble  with  difficulty  in  alcohol.  The  double  compound  with  platinum 
chloride,  the  crystalline  picrate,  the  nitrate,  and  the  picrolonate  are 
of  importance  in  the  detection  of  cytosine.  This  base  is  precipitated 
by  phosphotungstic  acid  and  by  silver  nitrate  in  the  presence  of  an 
excess  of  barium  hydroxide,  which  fact  is  of  importance  in  the  detection 
of  cytosine  (Kutscher).  The  double  bismuth-potassium  iodide  gives 
a  brick-red  precipitate.  Cytosine  gives  the  murexid  reaction  with 
chlorine-water  and  ammonia  (see  Chapter  XIV),  and  also  the  reaction 
described  by  Wheeler  and  Johnson  under  uracil.  In  regard  to 
preDaration  see  Kossel  and  Steudel  2  and  also  Kutscher.3 
HN— CO 

Uracil,  C4H4N202,  =  OC     CH      (2,  6  -  dioxypyrimidine) ,     was     first 

HN— CH 

obtained  by  Ascoli  and  Kossel  from  yeast  nucleic  acid  and  later  from 
various  complex  nucleic  acids,  perhaps  secondarily  from  the  cytosine  as  a 
cleavage  product.  The  synthetical  preparation  was  first  accomplished  by 
E.  Fischer  and  Roeder.4 

Uracil  crystallizes  in  needles  which  cluster  in  rosettes.  On  careful 
heating  it  sublimes  in  part  undecomposed,  but  develops  red  vapors  and 
decomposes  in  part.  It  is  readily  soluble  in  hot  water,  but  less  so  in  cold 
water,  and  nearly  insoluble  in  alcohol  and  in  ether.  Uracil  is  readily 
soluble  in  ammonia.  It  is  precipitated  by  mercuric  nitrate,  but  not  by 
phosphotungstic  acid.  It  is  precipitated  by  silver  nitrate  only  on  the 
careful  addition  of  ammonia  or  baryta-water.  Uracil  gives  the  Weidel 
reaction  and  the  following  reaction  described  by  Wheeler  and  John- 
son.5 The  uracil  solution  is  treated  with  bromine-water  until  it  is  per- 
manently cloudy  and  then  treated  with  baryta-water,  when  a  purple  or 
violet-colored  precipitate   appears  almost  immediately.     The  coloration 

1  Amer.  chem.  Journ.,  29. 

2  Zeitschr.  f.  physiol.  Chem.,  37  and  38. 

*  Ibid.,  38.  As  it  is  not  excluded,  but  rather  probable  according  to  Wheeler,  that 
besides  thymine  also  other  related  pyrimidine  bases  such  as  isocytosine,  6-amino 
pyrimidine  and  6-oxpyrimidine  can  be  formed  in  the  hydrolytic  cleavage  of  the  nucleic 
acids,  Wheeler  has  prepared  salts  and  compounds  of  these  bodies  and  described  them 
as  a  matter  of  comparison,  Journ.  of  biol.  Chem.,  3. 

4  Ascoli,  Zeitschr.  f.  physiol.  Chem.,  31;  Kossel  and  Steudel,  ibid.,  37;  Levene, 
HA'L,  3S,  39;  Levene  and  Mandel,  ibid.,  49;  E.  Fischer  and  Roeder,  Ber.  d.  d.  chem. 
Gesellsel,.,  34. 

5  Journ.  of  biol.  Chem.,  3. 


THYMINE.  195 

varies  with  the  dilution.  This  reaction  which,  as  remarked  above,  is  also 
given  by  cytosine,  depends  upon  the  fact  that  dibromoxyhydrouraeil 
us  first  formed,  and  from  this,  by  the  action  of  the  barium  hydroxide, 
first  isodialuric  and  then  dialuric  acid  is  produced,  both  of  which  give 
the  coloration.  In  regard  to  the  preparation  of  uracil  see  Kossel  and 
Steudel.1 

HN— CO 

I       I 
Thymine,   C5HGX2O2.  =  OC     C.CH3     (5-methyluracil).     This    body, 

HN— CH 

which  is  identical  with  nucleosin  obtained  by  Schmiedeberg  from  sal- 
mo-nucleic  acid,  was  first  prepared  by  Kossel  and  Neumann  from 
thymus-nucleic  acid,  and  then  by  other  investigators,  especially  Levene 
and  Mandel,  from  other  animal  nucleic  acids.  Fischer  and  Roeder  and 
later  Gerngross  2  have  prepared  it  synthetically. 

Thymine  crystallizes  in  small  leaves  grouped  in  stellar  or  dendriform 
clusters  or,  rarely,  in  short  needles  (Gulewitsch  3).  It  deflagrates  at 
318°  C.  and  melts  at  about  321°  and  sublimes.  It  is  soluble  with  diffi- 
culty in  cold  water,  mere  soluble  in  hot  water,  and  insoluble  in  alcohol. 
It  behaves  like  uracil  toward  ammonia  or  baryta-water  and  silver  nitrate. 
Thymine  is  precipitated  by  phosphotungstic  acid,  especially  when  impure. 
Bromine-water  is  decolorized  by  thymine,  producing  bromthymine. 
For  its  detection  we  make  use  of  the  sublimation,  the  behavior  toward 
silver  nitrate,  and  its  elementary  analysis. 

Meters  *  has  prepared  compounds  of  pyrimidine  bases  with  several  metals 
such  as  K,  Na,  Pb,  Hg  and  he  considers  it  incorrect  to  call  the  pyrimidine  bodies 
bases. 

In  regard  to  the  methods  of  preparation  see  Kossel  and  Neumann 
and  W.  Jones.0 

The  purine  and  pyrimidine  bodies  stand  in  close  chemical  and  phys- 
iological relation  to  each  other  and  for  this  reason  the  question  has 
been  repeatedly  raised  whether  the  pyrimidine  bases  might  not  be  formed, 
at  least  in  part,  from  the  purine  bases  by  the  action  of  acids.  Thus 
far  no  conclusive  investigations  have  been  made  supporting  this  view, 
while  on  the  contrary  the  investigations  of  Steudel  6  seem  to  contradict 
such  a  view. 

1  Zeitschr.  f.  physiol.  Chem.,  37. 

:  Schmiedeberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  37;  Kossel  and  Neumann,  Ber. 
d.  d.  chem.  Gesellsch.,  26  and  2";  Mandel  and  Levene,  Zeitschr.  f.  physiol.  Chem.,  46 
47,  49.  50;  E.  Fischer  and  Roeder,  ibid.,  34;  Gerngross,  ibid.,  38. 

3Zeitsch:.  f.  physiol.  Chem.,  27. 

4  Journ.  of  biol.  Chem.,  7. 

6  Kossel  and  Neumann,  1.  c,  and  W.  Jones,  Zeitschr.  f.  physiol.  Chem.,  29,  461. 

6  Zeitschr.  f.  physiol.  Chem.,  51  and  53  (against  Burian). 


CHAPTER  III. 
THE   CARBOHYDRATES. 

We  designate  by  this  name  bodies  which  are  especially  abundant 
in  the  plant  kingdom.  As  the  protein  bodies  form  the  chief  portion 
of  the  solids  in  animal  tissues,  so  the  carbohydrates  form  the  chief  por- 
tion of  the  dry  substance  of  the  plant  structure.  They  occur  in  the 
animal  kingdom  only  in  proportionately  small  quantities,  either  free  .or 
in  combination  with  more  complex  molecules,  forming  compound  pro- 
teins. Carbohydrates  are  of  extraordinarily  great  importance  as  food 
for  both  man  and  animals. 

The  carbohydrates  contain  only  carbon,  hydrogen,  and  oxygen.  The 
last  two  elements  occur,  as  a  rule,  in  the  same  proportion  as  they  do  in 
water,  namely,  2:1,  and  this  is  the  reason  why  the  name  carbohydrates 
has  been  given  to  them.  This  name  is  not  quite  pertinent,  if  strictly 
considered,  because  we  not  only  have  bodies,  such  as  acetic  acid  and 
lactic  acid,  which  are  not  carbohydrates  and  still  have  their  oxygen  and 
hydrogen  in  the  same  proportion  as  in  water,  but  we  also  have  a  sugar 
(the  methyl  pentoses,  C6H12O5)  which  has  these  two  elements  in  another 
proportion.  At  one  time  it  was  thought  possible  to  characterize  as 
carbohydrates  those  bodies  which  contained  6  atoms  of  carbon,  or  a 
multiple,  in  the  molecule,  but  this  is  not  considered  tenable  at  the  present 
time.  We  have  true  carbohydrates  containing  less  than  6,  and  also  those 
containing  7,  8,  and  9  carbon  atoms  in  the  molecule. 

The  carbohydrates  have  no  properties  or  characteristics  in  general 
which  differentiate  them  from  other  bodies;  on  the  contrary,  the  various 
carbohydrates  are  in  many  cases  very  different  in  their  external  prop- 
erties. Under  these  circumstances  it  is  very  difficult  to  give  a  positive 
definition  for  the  carbohydrates. 

From  a  chemical  standpoint  we  can  say  that  all  carbohydrates  are 
aldehyde  or  ketone  derivatives  of  polyhydric  alcohols.  The  simplest 
carbohydrates,  the  simple  sugars  or  monosaccharides,  are  either  alde- 
hyde or  ketone  derivatives  of  such  alcohols,  and  the  more  complex 
carbohydrates  seem  to  be  derived  from  these  by  the  formation  of  anhy- 
drides. It  is  a  fact  that  the  more  complex  carbohydrates  yield  two 
-or  even  more  molecules  of  the  simple  sugars  when  made  to  undergo 
hydrolytic  splitting. 

196 


MONOSACCHARIDES.  197 

Correspondingly  the  carbohydrates  can  he  divided  into  three  chief 
groups,  namely,  1.  Simple  sugars  or  monosaccharides,  2.  Complex  sugars 
or  disaccharides,  trisaccharides  and  crystalline  polysaccharides,  and 
3.  Non-crystalline  or  colloid  polysaccharides.  Of  these  groups  the  mono- 
saccharides, disaccharides  and  colloid  polysaccharides  are  of  special 
physiological  importance. 

Our  knowledge  of  the  carbohydrates  and  their  structural  relation- 
ships has  been  very  much  extended  by  the  pioneering  investigations  of 
Killiani  '  and  especially  those  of  E.  Fischer.2 

As  the  carbohydrates  occur  chiefly  in  the  plant  kingdom  it  is  naturally 
not  the  place  here  to  give  a  complete  discussion  of  the  numerous  carbo- 
hydrates known  up  to  the  present  time.  According  to  the  plan  of  this 
work  it  is  only  possible  to  give  a  short  review  of  those  carbohydrates 
which  occur  in  the  animal  kingdom  or  are  of  special  importance  as  food 
for  man  and  animals. 

1.  Monosaccharides. 

All  varieties  of  sugars  are  characterized  by  the  termination  "  ose," 
to  which  a  root  is  added  signifying  their  origin  or  other  relations.  Accord- 
ing to  the  number  of  carbon  atoms  contained  in  the  molecule  the  mono- 
saccharides are  divided  into,  trioses,  tetroses,  pentoses,  hexoses,  heptoses, 
and  so  on. 

All  monosaccharides  are  either  aldehydes  or  ketones  of  polyhydric 
alcohols.  The  former  are  termed  aldoses  and  the  latter  ketoses.  Ordinary 
glucose  is  an  aldose,  while  ordinary  fruit  sugar  (fructose)  is  a  ketose. 
The  difference  may  be  shown  by  the  structural  formula?  of  these  two 
varieties  of  sugar: 

Glucose  =  CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO; 
Fructose  =  CH2(OH).CH(OH).CH(OH).CH(OH).CO.CH2(OH). 

A  difference  is  also  observed  on  oxidation.  The  aldoses  can  be  con- 
verted into  oxyacids  having  the  same  quantity  of  carbon,  while  the  ketoses 
yield  acids  having  less  carbon.  On  mild  oxidation  the  aldoses  yield 
monobasic  oxyacids,  and  dibasic  acids  on  more  energetic  oxidation.  Thus 
ordinary  glucose  yields  gluconic  acid  in  the  first  case  and  saccharic 
acid  in  the  second. 


1  Ber.  d.  deutsch.  chem.  Gesellsch.,  18,  19,  and  20. 

2  See  E.  Fischer's  lecture,  Synthesen  in  der  Zuckergruppe,  Ber.  d.  deutsch.  chem. 
Gesellsch.,  23,  2114.  Excellent  works  on  carbohydrates  are  Tollen's  Kurzes  Hand- 
buch  der  Kohlehydrate,  Breslau,  2  (1895),  and  1,  2.  Auflage,  1898,  which  gives  a 
complete  review  of  the  literature,  and  E.  O.  v.  Lippmann,  Die  Chemie  der  Zucker- 
arten,  Braunschweig,  1904. 


198 


THE  CARBOHYDRATES. 


Gluconic  acid  =  CH2(OH).[CH(OH)]4.COOH; 
Saccharic  acid  =  COOH.[CH(OH)]4.COOH. 

The  monocarboxylic  acids  are  easily  transformed  into  their  anhydrides 
(lactones),  and  these  latter  are  of  special  interest  because,  as  shown  by 
Fischer,  they  can  be  changed  into  the  corresponding  aldehyde,  i.e., 
the  corresponding  aldose,  by  nascent  hydrogen. 

The  monosaccharides  are  converted  into  the  corresponding  poly- 
hydric  alcohol  by  nascent  hydrogen.  Thus  arabinose,  which  is  a 
pentose,  C5H10O5,  is  transformed  into  the  pentatomic  alcohol,  arabite, 
C5H12O5.  The  three  hexoses,  glucose,  mannose,  and  galactose, 
C6H12O6,  are  transformed  into  the  corresponding  three  hexites,  sorbite, 
mannite,  and  dulcite,  CeH^Oe-  The  ketoses,  on  the  contrary,  due 
to  their  constitution,  yield  a  mixture  of  two  alcohols  on  the  same  treat- 
ment. From  d-fructose,  for  example,  we  obtain  a  mixture  of  d-sorbite 
and  Z-mannite.  On  careful  oxidation  of  the  polyhydric  alcohols  the  cor- 
responding sugar  can  be  prepared. 

Numerous  isomers  occur  among  the  monosaccharides,  and  especially 
in  the  hexose  group.  In  certain  cases,  as,  for  instance,  in  glucose  and 
fructose,  we  are  dealing  with  a  different  constitution  (aldoses  and  ketoses), 
but  in  most  cases  we  have  stereoisomerism  due  to  the  presence  of  asym- 
metric carbon  atoms. 

As  the  monosaccharides  from  the  trioses  upward  contain  asymmetric 
carbon  atoms  they  occur  as  optically  active  bodies  in  an  1-,  dr  and  racemic 
form,  r  or  d-l  form,  which  is  a  mixture  or  a  combination  of  the  I-  and 
d-form  in  equal  parts.  As  the  number  of  asymmetric  carbon  atoms 
increases  so  does  the  number  of  possible  stereoisomeric  forms  enlarge. 
As  the  number  according  to  van't  Hoff  is  2n,  where  n  represents  the  num- 
ber of  asymmetric  carbon  atoms,  then  for  the  aldo-hexose,  which  con- 
tains 4  asymmetric  carbon  atoms,  24  =  16  stereo-chemically  different  forms 
can  exist.  In  fact,  of  these,  12  have  been  prepared  and  their  geometric 
structure  has  been  explained  and  for  which  Fischer  has  given  configura- 
tion formulae. 

As  these  relations  are  readily  conceived  we  will,  for  example,  give  only  the 
configuration  formula?  for  the  most  important  pentoses  and  hexoses  occurring 
in  the  animal  body. 


COH 

COH 

HOCH 

HCOH 

HCOH 

HOCH 

HCOH 

HOCH 

CH2OH 

CH2OH 

4-Arabmose 

f-Arabinose 

COH 
HOCH 

HCOH 
HOCH 

CH2OH 

d-Xylose 


COH 
HCOH 
HOCH 
HCOH 
CH2OH 

i-Xylose 


SPECIFIC  ROTATION. 


199 


1 

•    COH 

COH 

COH 

COH 

HCOH 

HOCH 

HCOH 

HOCH 

HCOH 

HOCH 

HOCH 

HCOH 

HCOH 

HOCH 

HCOH 

HOCH 

CHoOH 

CH2OH 

HCOH 

HOCH 

d-Ribose 

l-Riboee 

CH2OH 

d-Glucose 

CHoOH 

/-Glucose 

COH 

COH 

CH2OH 

CH2OH 

HCOH 

HOCH 

CO 

CO 

HOCH 

HCOH 

HOCH 

HCOH 

HOCH 

HCOH 

HCOH 

HOCH 

HCOH 

HOCH 

HCOH 

HOCH 

CH2OH 

d-Galactose 

CH2OH 

/-Galactose 

CH2OH 

d-Fructose 

CH2OH 

Z-Fructose 

We  designate  the  optical  activity  of  the  carbohydrates  with  the 
letter  I-  for  levogyrate,  d-  for  dextrogyrate,  and  r-  for  the  racemic. 
These  are  only  partly  indicative.  Thus  dextrorotatory  glucose  is 
designated  d-glucose,  levorotatory  Z-glucose,  but  Emil  Fischer  has 
used  these  signs  in  another  sense.  He  designates  by  these  signs  the 
mutual  relationship  of  the  various  kinds  of  sugars  instead  of  their 
optical  activity.  For  example,  he  does  not  designate  the  .levorotatory 
fructose  Z-fructose,  but  d-fructose,  showing  its  close  relation  in  stereometric 
structure  to  dextrorotatory  d-glucose.  This  designation  is  generally 
accepted,  and  the  above-mentioned  signs  only  show  the  optical  proper- 
ties in  certain  cases. 

Specific  rotation  means  the  rotation  in  degrees  produced  by  1  gm.  substance 
dissolved  in  1  cc.  liquid  placed  in  a  tube  1  dcm.  long.  The  reading  is  ordinarily 
made  at  20°  C.  and  with  the  monochromatic  sodium  light.  The  specific  rotation 
with  this  light  is  represented  by  («)D,  and  is  expressed  by  the  following  formula: 

(«)D  =  ±— r,  in  which  a  represents  the  reading  of  degrees,  1  the  length  of  the 

tube  in  decimeters,  and  p  the  weight  of  substance  in  1  cc.  of  the  liquid.    Inversely 
the  per  cent  P  of  substance  can  be  calculated,  when  the  specific  rotation  is  known, 

by  the  formula  P  = — r- ,  in  which  s  represents  the  known  specific  rotation. 

In  the  determination  of  the  change  in  specific  rotation  with  various  concen- 
trations we  must  know  also  the  amount  of  substance  in  grams  in  1  gram  of  the 
solution  (p)  and  the  specific  gravity  of  the  solution  (d)  at  20°.    The   rotation 

is  calculated  according  to  the  formula  (a)D  =  ± — j-r, 

A  freshly  prepared  solution  of  a  substance  often  shows  a  different  rotation 
from  one  that  has  been  allowed  to  stand  for  some  time  (multirotation).  The 
correct  values  which  are  found  on  allowing  the  solution  to  stand  for  a  sufficiently 
long  time  can  be  obtained  immediately  by  boiling  or  on  the  addition  of  very 
little  ammonia. 


200  THE  CARBOHYDRATES. 

In  order  to  explain  multirotation  Hudson  1  believes  that  the  aldoses  exist  in 
two  isomeric  forms  having  different  rotation,  which  on  being  dissolved  pass* 
into  each  other  by  means  of  a  reversible  reaction.  The  two  forms  can  be  derived 
because  a  lactone-like  binding  exists  between  the  end  carbon  atom  in  the  alde- 
hyde group  and  the  7-carbon  atom  according  to  the  formula 

CHo0HCHOH.CH.CHOH.CHOH.C(         .     In  this  way  the  end  carbon  becomes 

0 1 XoH 

asymmetric  and  according  to  whether  the  position  of  the  atoms  that  are  combined 
with  this  carbon  atom  are: 


C            H 

\   / 

C              or 

C           OH 

\  / 
C 

/  \ 
0           OH 

/   \ 
0           H 

-we  obtain  the  two  forms.  Tanret  2  has  obtained  two  isomeric  forms  of  glucose, 
galactose,  arabinose  and  lactose  which  pass  from  one  form  to  the  other.  The 
two  forms  of  glucose  correspond  according  to  E.  H.  Armstrong  3  to  the  syntheti- 
cally prepared  a-  and  /3-methyl  glucosides  prepared  by  E.  Fischer  (see  pages 
61-62). 

Like  the  ordinary  aldehydes  and  ketones,  the  sugars  may  be  made  to 
take  up  hydrocyanic  acid.  Cyanhydrins  are  thus  formed.  These  addi- 
tion products  are  of  special  interest  in  that  they  make  possible  the  arti- 
ficial preparation  of  sugars  rich  in  carbon  from  sugars  poor  in  carbon. 

As  an  example,  if  we  start  from  glucose  we  obtain  glucocyanhydrin 
on  the  addition  of  hydrocyanic  acid: 

CH2.(OH).[CH(OH)]4.COH+HCN  =  CH2(OH).[CH(OH)]4.CH(OH).CN. 

On  the  saponification  of  glucocyanhydrin  the  corresponding  oxyacid  is 
formed. 

CH2(OH).[CH(OH)]4.CH(OH).CN+2H20 

=  CH2(OH).[CH(OH)]4.CH(OH).COOH+NH3. 

By  the  action  of  nascent  hydrogen  on  the  lactone  of  this  acid  we  obtain 
glucoheptose,  C7H14O7  and  according  to  this  principle  the  construction 
of  sugars  up  to  nine  carbon  atoms  has  been  accomplished. 

The  monosaccharides  give  the  corresponding  oximes  with  hydroxyl- 
amine:  thus  glucose  yields  glucosoxime,  CH2(OH).|CH(OH)]4.CH:N.OH. 
These  compounds  are  of  importance  on  account  of  the  fact,  as  found  by 
Wohl,4  that  they  are  the  starting-point  in  the  formation  of  one  class 

Mourn.  Amer.  Chem.  Boo.,  31,  61,  955  (1909). 
2  Bull.  80C.  chim.,  13,  728  (1895),  15,  195,  349  (1896). 
'Journ.  Chem.  Soc,  83,  1305  (1903). 
*  Ber.  d.  d.  chem.  Gesellsch.,  26,  p.  730. 


DERIVATIVES.  201 

of  sugars  from  another  class,  namely,  the  preparation  of  sugars  poor  in 
carbon  from  those  rich  in  carbon,  for  example,  pentoses  from  hexoses 
(see  Wohl). 

According  to  Ruff,  by  the  action  of  hydrogen  peroxide  and  the  cata- 
lytic action  of  ferric  salts  upon  the  carbohydrate  monocarboxylic  acids 
the  carbon  chain  can  be  shortened  by  the  splitting  off  of  the  elements 
of  formic  acid,  and  with  the  formation  of  the  next  lower  aldose.  Nki  - 
berg1  has  accomplished  the  same  result  by  electrolysis,  and  by  this 
method  has  split  glucose  step  by  step  into  formaldehyde. 

By  the  action  of  alkalies,  even  in  small  amounts,  as  also  of  carbonates 
and  lead  hydroxide,  a  reciprocal  transformation  of  the  sugars,  such  as 
d-glucose,  d-fructose,  and  d-mannose,  may  take  place  (Lobry  de  Bruyn 
and  Alberda  van  Ekenstein2),  and  from  each  of  these  three  varieties 
of  sugar  the  two  others  are  produced  so  that  after  a  certain  time  the 
solution  contains  all  three  sugars. 

The  transformation  of  the  different  varieties  of  sugar  into  each  other 
also  occurs  in  the  animal  body.  Neuberg  and  Mayer3  have  shown  by 
experiments  on  rabbits  the  direct  partial  transformation  of  various 
mannoses  into  the  corresponding  glucoses.  Another  example  is,  it 
seems,  the  formation  of  galactose  (or  milk  sugar)  from  glucose  in  the 
mammary  gland. 

By  the  action  of  strong  alkali  the  sugars  are  decomposed  with  the 
formation  of  lactic  acid  and  many  other  products. 

With  ammonia  the  glucoses  may  form  compounds  which  have  been 
considered  as  osamines  by  Lobry  de  Bruyn,  but  to  differentiate  them 
from  the  true  osamines  have  been  called  osimines  by  E.  Fischer.4  The 
corresponding  osaminic  acid  can  be  obtained  from  such  an  osimine  by 
the  action  of  ammonia  and  hydrocyanic  acid,  and  from  the  hydrochloric- 
acid  lactone  of  this  acid  the  osamine  is  obtained  by  reduction  with  sodium 
amalgam.  In  this  manner  E.  Fischer  and  Leuchs  artificially  prepared 
first  d-arabinosimine  from  d-arabinose,  then  d-glucosaminic  acid  and 
finally  from  its  lactone  d-glucosamine,  which  occurs  in  the  animal 
body.  In  a  similar  manner  they 5  obtained  ^-glucosamine  from 
/-arabinose. 

Knoop  and  Windaus  6  have  obtained  large  amounts  of  methylimida- 


1  Ruff,  Ber.  d.  d.  chem.  Gesellsch.,  31  and  32;  Neuberg,  Biochem.  Zeitschr.,  7. 

2  Ber.  d.  d.  chem.  Gesellsch.,  28,  3078;  Bull.  soc.  chim.  de  Paris  (3),  15;  Chem. 
Centralbl.,  1896,  2,  and  1897,  2. 

3  Zeitschr.  f.  physiol.  Chem.,  37. 

4  Lobry  de  Bruyn,  Ber.  d.  d.  chem.  Gesellsch.,  28;  E.  Fischer,  ibid.,  35. 
6  Ibid.,  35,  p.  3787,  and  36,  24  (1903). 

8  Ibid.,  38,  and  Hofmeister's  Beitrage,  6. 


202  THE  CARBOHYDRATES. 

CH3 

I 
zol,  C — NHV        ,  from  glucose  by  the  action  of  ammonium-zinc  hydroxide 

CH— N^ 
at  ordinary  temperatures.  This  formation  can  be  conceived  as  follows: 
First  methyl  glyoxal  is  formed  from  the  sugar,  and  then  from  this,  or 
from  the  sugar,  formaldehyde  is  produced,  which  reacts  with  the  meth\  1 
glyoxal  with  the  formation  of  methylimidazole  according  to  the  following 
equation: 

CH3CO        NH3     Hx  H3C.C— NHX 

I        +         +     >CH=  ||  >CH+3H20 

COH    NH3     0*  CH— 1ST 

Methylglyoxal  Formaldehyde  Methylimidazole 

A  genetic  relationship  of  the  carbohydrates  to  histidine  and  the  purine 
bodies  is  thus  made  probable  by  the  imidazole  formation. 

As  the  sugars  are  derivatives  of  polyhydric  alcohols,  they  also  form 
esters,  among  which  the  benzoyl  ester  is  of  special  interest  because  it  is 
used  in  the  detection  and  isolation  of  the  sugars  and  also  of  other  car- 
bohydrates. The  nucleic  acids  probably  also  belong  to  the  acid  esters 
of  the  sugars,  and  thus  may  be  considered  as  complex  phosphoric  acid 
esters,  and  perhaps  the  chondroitin  sulphuric  acid  and  the  glucothionic 
acid  are  sulphuric  acid  esters.  The  nature  of  these  two  groups  of 
sulphuric  acid  esters  is  not  yet  thoroughly  understood. 

The  sugars  can  also  combine  with  other  bodies  and  with  each  other, 
forming  ether-like  combinations.  By  the  action  of  hydrochloric  acid 
as  catalyst,  as  shown  by  Fischer  and  collaborators,  the  sugars  split  off 
water  and  unite  with  "other  bodies,  producing  lactone-like  combinations, 
which  have  been  called  glucosides  (see  pages  61  and  200).  These  glucosides, 
which  are  generally  compounds  with  aromatic  groups,  occur  widely  dis- 
tributed in  the  vegetable  kingdom.  The  more  complex  carbohydrates 
may  be  considered,  according  to  Fischer,  as  glucosides  of  the  sugars. 
Thus  maltose,  for  example,  is  the  glucoside  and  lactose  the  galactoside 
of  glucose.  The  glucosides  can  be  split  into  their  components  by  chem- 
ical agents,  boiling  with  dilute  mineral  acids,  as  well  as  by  the  action 
of  enzymes.  The  complex  sugars  hereby  yield  simple  sugars  and  the  other 
glucosides  yield  compounds  which  belong  either  to  the  aromatic  or  the 
aliphatic  series  besides  the  sugar.  A  long-knoAvn  example  of  a  decom- 
position of  this  kind  is  the  splitting  of  amygdalin  by  the  enzyme  emulsin 
(see  page  60). 

With  phenylhydrazine  or  substituted  phenylhydrazines,  the  sugars 
first  yield  hydrazones  with  the  elimination  of  water,  and  then  on  the  fur- 
ther action  of  hydrazine  on  warming  in  an  acetic-acid  solution  we  obtain 
osazones. 


HYDRAZONES  AND  OSAZOXES.  203 

The  reaction  takes  place  with  the  aldoses  as  follows: 

<a)XH2(OH).[CH(OH)]3.CH(OH).CHO+H2X.XH.C6H5  = 

CH2(OH).[CH(OH)]3.CH(OH)CH:X.NH.C6H5+H20. 

Phenylglucosehydrazone 

(b)  CH,(OH)[CH(OH)]3.CH(OH).CH:X.NH.C6H5+H2N.NH.C8H5  = 

CH2(OH).[CH(OH)]3.C.CH:X.XH.C«H5 

N.XH.C6H6+H20+H2. 

Phenylglucosazone 

and  with  the  keteoses: 

CH,fOH)[CH(OH)]3CO.CH2(OH)+H2N.NH.C8H5  = 

CH2(OH)[CH(OH)]3C.CH2OH 

X.XH.C6H5+H20, 
and         CH2(OH)[CH(OH)];.C.CH,(OH) 

X.XH.C6H5+H2X.XH.C6H5  = 
CH2(OH)[CH(OH)]3.C.CH  :X.XH.C6H5  +H20+H2. 

X.XH.C6H3 

The  hydrogen  is  not  evolved,  but  acts  on  a  second  molecule  of  phenylhy- 
drazine  and  splits  it  into  aniline  and  ammonia : 

H2X.H.C6H3+H2=H2X.C6H5+XH3. 

As  seen  from  the  above  equations  the  aldoses  and  ketoses  yield  the 
same  osazones,  while  the  hydrazones  are  different. 

The  osazones,  which  are  more  important  than  the  hydrazones,  are 
generally  yellow  crystalline  compounds  which  differ  from  each  other  in 
melting-point,  solubility,  and  optical  properties,  and  hence  have  been  of 
great  importance  in  the  characterization  of  certain  sugars.  They  have 
also  become  of  extraordinarily  great  interest  in  the  study  of  the  carbo- 
hydrates for  other  reasons.  Thus  they  are  a  very  good  means  of  pre- 
cipitating sugars  from  solution  in  which  they  occur  mixed  with  other 
bodies,  and  they  are  of  the  greatest  importance  in  the  artificial  prepara- 
tion of  sugars.  On  cleavage,  by  the  brief  action  of  gentle  heat  and  fum- 
ing hydrochloric  acid  (for  disaccharides  still  better  with  benzaldehyde),1 
the  osazones  yield  so-called  osones,  which  on  reduction  yield  aldoses  or 
more  often  ketoses.  The  hydrazones  can  be  much  more  readily  retrans- 
formed  into  the  corresponding  sugar,  especially  by  decomposition  with 
benzaldehyde  (Herzfeld)  or  with  formaldehyde  (Ruff  and  Ollen- 
dorff2), whereby  the  sugar  is  replaced  by  the  aldehyde  used. 

An  important  property,  although  not  applicable  to  all  sugars,  is  their 
ability  to  undergo  fermentation,  especially  their  ability  to  undergo 
alcoholic  fermentation  with  alcohol-yeast.  We  must  state,  however, 
that  the  power  of  fermentation  with  pure  yeast  has  been  shown  only  for 

1  E.  Fischer  and  Armstrong,  Ber.  d.  d.  chem.  Gesellsch.,  35. 

2  Herzfeld,  ibid.,  2S;  Ruff  and  Ollendorff,  ibid.,  32. 


204  THE  CARBOHYDRATES. 

the  hexose  group,  and  in  fact  all  the  hexoses  do  not  ferment,  and  they 
do  not  all  ferment  with  the  same  readiness.  d-Glucose  and  d-mannose 
ferment  readily,  but  d-galactose  only  with  difficulty.  The  Worms  of 
the  above-mentioned  sugars  do  not  ferment,  and  from  the  racemic  forms 
of  these  sugars  the  optical  Z-antipode  can  be  prepared  by  the  fermenta- 
tion of  the  d-sugar.  Among  the  ketoses  the  d-fructose  ferments  while 
the  sorbose  does  not.  Among  the  sugars  containing  nine  atoms  of  car- 
bon, the  nonoses,  the  mannonose  ferments  while  the  glucononose  does 
not.  In  regard  to  the  fermentability  of  the  triose,  dioxyacetone,  see 
page  205.  The  different  behavior  of  the  various  sugars  with  yeast  stands 
in  fixed  relation  to  their  configuration,  and  is  not  only  of  great  importance 
for  the  behavior  of  the  sugar  in  lower  organisms,  but  also  for  their  behavior 
in  higher  developed  organisms.  Thus  the  investigations  of  Neuberg 
Wohlgemuth  x  upon  arabinose  and  of  Neuberg  and  Mayer  2  on  man- 
noses  have  shown  that  in  rabbits  the  Z-arabinose  and  the  d-mannose  are 
much  better  utilized  than  d-  and  r-arabinose  or  I-  and  r-mannose.  See 
also  pages  61-62. 

In  the  alcoholic  fermentation  the  sugar  is  decomposed  according 
to  the  general  equation  C6Hi206  =  2C2H60-f2C02.  The  exact  process 
is  not  clear,  and  seems  to  be  rather  complicated.  On  page  52  it  has 
been  mentioned  that  for  the  action  of  the  fermentation  enzymes  the 
presence  of  a  dialyzable  substance  present  in  the  press-juice  is  neces- 
sary (Harden  and  Young3).  On  the  other  hand  the  fermentation 
power  of  the  press-juice  can  also  be  considerably  increased  by  the  addi- 
tion of  secondary  sodium  phosphate.  The  phosphoric  acid  in  the  press- 
juice  after  fermentation  is  only  partly  precipitable  with  magnesia  mixture 
(Harden  and  Young).  The  most  favorable  action  of  boiled  press- 
juice  is  inhibited  by  lipase  (Buchner  and  Klatte).  According  to 
Harden  and  Young  we  must  consider  the  action  of  boiled  press-juice 
and  of  phosphate  in  that  first  a  hexose-phosphoric  acid  ester  is  produced 
with  the  simultaneous  formation  of  carbon  dioxide  and  alcohol,  according 
to  the  following  formula: 

2C6H12O6+2R2HPO4  =  2CO2+2C2H6O+C6H10O4(PO4R2)2+2H2O. 

The  hexose  phosphate  can  then  be  split  into  hexose  and  phosphate 
by  a  special  enzyme.  The  hexose  phosphoric  acid  has  been  isolated 
as  a  lead  salt  by  Young.  Glucose,  fructose  and  mannose  produce  in 
their   fermentation   the   same    hexose    phosphoric    acid.     According   to 

1  Zeitschr.  f.  physiol.  Chem.,  35. 
*1M&.,  37. 

3  Literature  in  Harden  and  Young,  Bioch.  Zeitschr.,  32,  173  (1911)  as  well  as 
Buchner  and  Klatte,  ibid.,  8,  520  (1908). 


ALCOHOLIC  FERMENTATION.  205 

Iwanoff  '  the  phosphoric  acid  combination  is  a  triose  phosphate  which 
is  fermented,  with  the  formation  of  carbon  dioxide,  alcohol  and  phos- 
phoric acid,  by  the  dead  and  not  by  the  living  yeast.  On  the  contrary 
Lebedew  finds  the  same  formula  as  Young  2  for  the  phosphoric  acid 
ester.  Iwanoff  as  well  as  Euler  and  their  collaborators  admit  that 
the  formation  of  phosphoric  acid  esters  is  brought  about  by  a  special 
enzyme.3  According  to  Iwanoff  and  to  Lebedew  the  sugar  is  first 
fermented  after  it  has  combined  with  the  phosphoric  acid.  It  seems, 
according  to  all  evidence,  that  phosphoric  acid  esters  of  carbohydrates 
are  formed  and  that  these  are  in  some  way  of  importance  for  the  accom- 
plishment of  the  fermentation.  It  is  not  probable  that  in  the  fermenta- 
tion the  hexose  does  not  directly  break  into  alcohol  and  CO2.  It  is 
generally  admitted  that  the  process  takes  place  through  intermediary 
steps.  Lactic  acid  is  considered  as  one  of  these,  although  in  fact,  this 
acid  is  not  fermented  with  the  formation  of  alcohol.  Recently  Buchner 
and  Meissenheimer4  have  proposed  dioxyacetone  (HOCH2.CO.CH2OH) 
as  a  probable  intermediary  step.  They  found  that  dioxyacetone  was 
very  readily  fermented  by  press-juice  in  the  presence  of  common  salt 
and  indeed  with  the  formation  of  alcohol  and  carbon  dioxide.  This  has 
been  substantiated  by  Lebedew.5  Harden  and  Young  disputed  the 
possibility  that  dioxyacetone  is  an  intermediary  step  in  the  alcoholic 
fermentation  of  sugar  because  it  is  more  slowly  fermented  than  the 
sugars.6 

Besides  ethyl  alcohol  and  carbon  dioxide  there  are  formed  in  the  fer- 
mentation of  sugar,  although  in  small  amounts,  several  higher  alcohols 
which  form  the  so-called  fusel  oil.  The  most  important  constituents 
of  fusel  oil  are  isoamylalcohol,  t/-amylalcohol,  isobutylalcohol  and  normal 
propylalcohol  in  varying  proportions.  The  formation  of  fusel  oil  was 
ascribed  for  a  long  time  to  the  action  of  bacteria  until  F.  Ehrlich  7 
found  that  the  higher  alcohols  could  be  produced  from  certain  amino- 
acids  by  the  living  activity  of  yeast.  From  an  amino-acid  probably 
the  corresponding  oxyacid  is  formed  first  by  the  splitting  off  of  ammonia, 


1Centralbl.  f.  Bakt.  24,  1  (1909). 
2Bioch.  Zeitschr.  36,  248  (1911). 

3  Euler  and  Kullberg,  Zeitschr.  f.  physiol.  Chem.,  74,  15  (1911);  80,  175  (1912); 
Bioch.  Zeitschr.,  37,  133  (1911). 

4  In  reference  to  the  intermediary  products  in  alcoholic  fermentation  see  Buchner 
and  Meissenheimer,  Ber.  d.  d.  chem.  Gesellsch.,  43,  1773  (1910)  which  also  contains 
the  literature. 

5Compt.  Rend.,  153,  136  (1911). 

6  Bioch.  Zeitschr.,  40,  458  (1912). 

7  Zeitschr.  f.  Ver.  d.  d.  Zuckerind,  55,  539  (1905)  also  Ber.  d.  d.  chem.  Gesellsch, 
40,  1027,  2538  (1907);  Bioch.  Zeitschr.  1,  8  (1906);  8,  438  (1908);  18,  391  (1909). 


206  THE  CARBOHYDRATES. 

and  then  from  this  by  loss  of  CO2  the  alcohol  is  derived.  The  ammonia 
is  assimilated  by  the  yeast.  If  the  amino-acid  is  in  the  racemic  form 
then  only  the  one  component  occurring  naturally  is  transformed  into 
alcohol  while  the  other  remains  in  great  part  unchanged.  In  this  manner 
leucine  is  converted  into  isoamylalcohol  according  to  the  following  equa- 
tion: 

hoco.ch(nh2).ch2.ch/    3+h2o= 

Leucine  XCH3 

/CH3 
NH3+C02+HOCH2.CH2.CH< 

Isoamylalcohol      ^P'TJo 

Other  examples  of  the  same  kind  is  the  formation  of  d-amylalcohol 
from  d-isoleucine  and  of  isobutylalcohol  from  a-amino-valeric  acid. 
The  formation  of  higher  alcohols  takes  place  with  yeast  poor  in  nitrogen 
and  in  the  presence  of  large  amounts  of  sugar.  In  an  analogous  man- 
ner, under  the  influence  of  yeast  in  the  presence  of  sugar  and  inor- 
ganic nutritive  salts,  from  tyrosine  tyrosol  (p-oxyphenylethyl  alcohol) 
HO.C6H4.CH2.CH2.OH  is  derived  and  from  tryptophane  we  get  tryptophol 
(/3-indoxylethyl  alcohol)1. 

C.CH2.CH2OH 
C6H4<f%CH 


NH 

Other  fermentation  processes  which  are  brought  about  by  yeast  but  without 
the  presence  of  sugar  have  been  studied  by  Neuberg  2  and  his  collaborators. 
Among  these  we  will  mention  the  decomposition  of  pyroracemic  acid  (pyruvic 
acid)  into  carbon  dioxide  and  acetaldehyde : 

HO.CO.CO.CH3  =C02+HOC.CH3. 

The  enzyme  active  in  this  fermentation  is  called  carboxylase.  If  the  pyro- 
racemic acid  exists  in  the  form  of  an  alkali  salt  then  the  cleavage  takes  place  ac- 
cording to  the  formula, 

2KO.CO.CO,CH3+H20=C02+2HOC.CH3+K2C03 

and  alkali  carbonate  is  formed  from  a  neutral  salt.     In  this  case  the  aldehyde 
is  condensed  by  the  alkali  to  aldol,  the  first  polymerization  product  of  acetaldehyde. 

The  previously  mentioned  (page  41)  lactic  acid  fermentation  of 
various  sugars  is  caused  by  the  action  of  different  varieties  of  bacteria. 
The  equation  represents  a  cleavage  of  one  hexose  molecule  into  two 
lactic  acid  molecules  CV,H120G  =  2HOCO.CH(OH).CH3.     Nothing  positive 

1  Ber.  d.  d.  chem.  Gesellsch.,  44,  139  (1910);  45,  883  (1912). 

s  Bioch.  Zeitschr.,  31,  170  (1910);  32,  323;  36,  60,  68,  76  (1911);  47,  405,  413  (1912). 


PENTOSES.  207 

is  known  as  to  how  this  cleavage  occurs.  According  to  Buchner  and 
Meissenheimer1  the  fermentation  with  the  enzymes  contained  in  the 
bacteria  produces  chiefly  the  racemic,  inactive  form  of  the  acid.  This 
also  occurs  as  a  rule  by  the  action  of  living  bacteria.  In  reference  to  the 
formation  of  lactic  acid  within  the  organism  see  Chapter  X. 

The  monosaccharides  are  colorless  and  odorless  bodies,  neutral  in 
reaction,  with  a  sweet  taste,  readily  soluble  in  water,  generally  soluble 
with  difficulty  in  absolute  alcohol,  and  insoluble  in  ether.  Some  of  them 
crystallize  well  in  the  pure  state.  They  are  strong  reducing  substances. 
They  reduce  metallic  silver  from  ammoniacal  silver  solutions  and  they 
also  reduce  other  metallic  oxides  such  as  copper,  bismuth  and  mercury 
oxides,  on  heating  in  alkaline  solution.  This  behavior  is  of  great 
importance  in  the  detection  and  quantitative  estimation  of  the  sugars. 

The  simple  varieties  of  sugar  occur  in  part  in  nature  as  such,  already 
formed,  which  is  the  case  with  both  of  the  very  important  sugars,  glucose 
and  fructose.  They  also  occur  in  great  abundance  in  nature  as  more 
complex  carbohydrates  (di-  and  polysaccharides);  also  as  ester-like 
combinations  with  different  substances,  as  so-called  glucosides. 

Among  the  groups  of  monosaccharides  known  at  the  present  time, 
those  containing  less  than  five  and  more  than  six  carbon  atoms  in  the 
molecule  have  no  great  importance  in  biochemistry,  although  they  are 
of  high  scientific  interest.  Of  the  two  groups  the  hexoses  are  the  more 
abundant  and  are  cf  special  interest.  The  pentoses  are  becoming  cf 
greater  importance,  net  only  for  the  chemistry  of  plants,  but  also  for 
the  chemical  processes  in  the  animal  body. 

4 

Pentoses  (C5H10O5). 

As  a  rule  the  pentoses  do  not  occur  as  such  in  nature.  They  are 
obtained  from  animal  tissues,  organs  and  fluids  as  cleavage  products 
of  the  nucleic  acids,  or  nucleoproteins.  The  pentoses  are  chiefly  obtained 
from  the  plant  kingdom,  where  nucleic  acids  also  occur,  by  the  hydro- 
lytic  cleavage  with  dilute  mineral  acids,  of  more  complex  carbohydrates, 
the  so-called  pentosans.  The  pentosans  exist  very  widely  distributed 
in  the  plant  kingdom,  and  are  of  especially  great  importance  in  the  build- 
ing up  of  certain  plant  constituents.  Methyl  pentosans  and  methyl 
pentoses  also  occur  in  the  plants,  and  of  these,  the  methyl  pentose,  rham- 
nose,  which  occurs  in  several  glucosides,  must  be  specially  mentioned. 

The  pentoses  were  first  found  in  the  animal  kingdom  by  Salkowski 
and  Jastrowitz  in  the  urine  of  a  person  addicted  to  the  morphine  habit, 

1  Ann.  d.  Chem.  u.  Pharm.,  349,  125  (1906). 


208  THE  CARBOHYDRATES. 

and  later  by  Salkowski  and  others  in  human  urine.  Small  quantities 
of  pentoses  have  been  detected  by  Kulz  and  Vogel  x  in  the  urine  of 
diabetics,  as  also  in  dogs  with  pancreas  diabetes  or  phlorhizin  diabetes. 
Pentose  has  also  been  found  by  Hammarsten  among  the  cleavage 
products  of  a  nucleoprotein  obtained  from  the  pancreas,  or  from  the 
corresponding  guanylic  acid,  and  seems  also,  according  to  the  observa- 
tions of  Blumenthal,  to  be  a  constituent  of  nucleoproteins  of  various 
organs,  such  as  the  thymus,  thyroid,  brain,  spleen,  and  liver.  Their 
occurrence  in  the  nucleic  acids  has  been  previously  discussed.  In  regard 
to  the  quantity  of  pentoses  found  in  the  various  organs,  we  must  refer 
to  the  works  of  Grund  and  of  Bendix  and  Ebstein  and  Mancini.2 

The  pentosans  (Stone,  Slowtzoff)  as  well  as  the  pentoses  are  of  the 
greatest  importance  as  foods  for  herbivorous  animals.  In  regard  to  the 
value  of  the  pentoses,  the  researches  of  Salkowski,  Cremer,  Neuberg, 
and  Wohlgemuth  3  upon  rabbits  and  hens  show  that  these  animals 
can  utilize  the  pentoses.  The  question  whether  the  pentoses  are  active 
as  glycogen-formers  is  still  an  open  one  (see  Chapter  VII).  The  pen- 
toses seem  to  be  absorbed  by  human  beings  and  in  part  utilized,  but 
they  pass  in  part  into  the  urine  even  when  small  quantities  are  taken.4 

The  natural  pentoses  are  reducing  aldoses,  and  as  a  rule  do  not  belong 
to  the  sugars  fermentable  by  yeast.  Still,  the  observations  of  Sal- 
kowski, Bendlx,  Schone  and  Tollens  seem  to  indicate  that  the  pen- 
toses are  fermentable.5  They  are  readily  decomposed  by  putrefaction 
bacteria.  With  phenylhydrazine  and  acetic  acid  they  give  crystalline 
osazones  which  are  soluble  in  hot  water,  and  whose  melting-points  and 
optical  behavior  are  important  for  the  detection  of  the  pentoses.  On 
heating  with  hydrochloric  acid  they  yield  furfurol,  but  no  levulinic  acid. 
In  this  reaction  furfuran  is  formed  from  the  pentose  molecule,  and  then 


1  Salkowski  and  Jastrowitz,  Centralbl.  f.  d.  med.  Wissensch.,  1892,  337  and  593; 
Salkowski,  Berl.  klin.  Wochenscbr.,  1895;  Bial,  Zeitschr.  f.  klin.  Med.,  39;  Bial  and 
Blumenthal,  Deutsch.  med.  Wochenschr.,  1901,  No.  2;  Kulz  and  Vogel,  Zeitschr.  f. 
Biologie,  32. 

2  Hammarsten  Zeitschr.  f.  physiol.  Chem.,  19;  also  Salkowski,  Berl.  klin.  Wochen- 
schr., 1895;  Blumenthal,  Zeitschr.  f.  klin.  Med.,  34;  Grund,  Zeitschr.  f.  physiol.  Chem. 
35;  Bendix  and  Ebstein,  Zeitschr.  f.  allgemein.  Physiol.,  2;  Mancini,  Chem.  Centralbl., 
1906. 

3  Stone,  Amer.  Chem.  Journ.,  14;  Slowtzoff,  Zeitschr.  f.  physiol.  Chem.,  34;  Sal- 
kowski, ibid.,  32;  Cremer,  Zeitschr.  f.  Biologie,  29  and  42;  Neuberg  and  Wohlgemuth, 
Zeitschr.  f.  physiol.  Chem.,  35. 

4  See  Ebstein,  Virchow's  Arch.,  129;  Tollens,  Ber.  d.  deutsch.  chem.  Gesellsch.,  29, 
1208;  Cremer,  1.  c;  Lindemann  and  May,  Deutsch.  Arch.  f.  klin.  Med.,  56;  Salkowski, 
Zeitschr.  f.  physiol.  Chem.,  30. 

Salkowski,  Zeitschr.  f.  physiol.  Chem.,  30;  Bendix,  see  Chem.  Centralbl.,  1900,  1; 
Sr-lione  and  Tollens,  ibid.,  1901,  1. 


PENTOSE  REACTIONS.  209 

HC— CH 

II      II 
from  this  its  aldehyde,  the  furfurol  HC     C.CHO.     The  furfurol   pass- 

O 
ing  over,  on  distilling  with  hydrochloric  acid,  can  be  detected  by  the  aid 
of  aniline-acetate  or  xylidine  acetate  paper,  which  is  colored  a  beautiful 
red  by  furfurol.  In  the  quantitative  estimation  we  can  use  the  method 
suggested  by  Tollens,  which  consists  in  converting  the  furfurol  in  the 
distillate  into  phloroglucide  by  means  of  phloroglucin  and  weighing  (see 
Tollens  and  Krober,  Grund,  Bendix  and  Ebstein),  or  according  to 
Jolles  l  by  bisulphite  and  retitrating  with  iodine  solution.  In  using 
these  methods  it  must  be  borne  in  mind  that  glucuronic-acid  compounds 
also  yield  furfurol  under  the  same  conditions.  The  two  following  pentose 
reactions,  as  suggested  by  Tollens,  are  especially  applicable. 

The  orcin-hydrochloric  acid  test.  Mix  with  the  solution  or  the  substance 
introduced  into  water  an  equal  volume  of  concentrated  hydrochloric  acid,  add 
some  orcin  in  substance,  and  heat.  In  the  presence  of  pentoses  the  solution 
becomes  reddish-blue,  then  bluish-green,  and  on  spectroscopic  examination  an 
absorption-band  is  observed  between  C  and  D.  If  it  is  cooled  and  shaken  with 
amyl  alcohol,  a  bluish-green  solution  which  shows  the  same  band  is  obtained. 

The  phloroglucin-hydrochloric  acid  test.  This  test  is  performed  in  the 
same  manner  as  the  above,  using  phloroglucin  instead  of  orcin.  The  solution 
becomes  cherry-red  on  heating  and  then  becomes  cloudy  and  hence  a  shaking  out 
with  amyl  alcohol  is  very  necessary.  The  red  amyl-alcohol  solution  shows  an 
absorption-band  between  D  and  E.  The  orcin  test  is  better  for  several  reasons 
than  the  phloroglucin  test  (Salkowski  and  Neuberg  2).  In  regard  to  the  use 
of  these  tests  in  urine  examination  see  Chapter  XIV. 

Many  modifications  of  these  tests  have  been  suggested.  Brat  3  performs 
the  crcin  reaction  by  the  addition  of  NaCl  and  heating  to  only  90-95°.  Bial  4 
uses  a  hydrochloric  acid  containing  ferric  chloride  for  the  orcin  test  and  claims 
to  get  a  greater  delicacy.  On  too  strong  and  too  long  heating  (l§-2  minutes), 
when  using  this  modification,  a  confusion  with  sugars  of  the  six  carbon  series  may 
occur  (Bial,  van  Leersum).5  According  to  R.  Adler  and  0.  Adler  the  phlo- 
roglucin and  orcin  tests  can  be  made  with  glacial  acetic  acid  and  a  few  drops 
hydrochloric  acid  instead  of  with  the  hydrochloric  acid  alone.  These  investigators 
also  use  a  mixture  of  equal  volumes  of  aniline  and  glacial  acetic  acid  as  a  reagent 
for  pentoses.  On  the  addition  of  a  little  pentose  to  the  boiling  mixture  a  beautiful 
red  color  of  furfurol-aniline  acetate  is  obtained.  A.  Neumann  6  performs  the 
orcin  test  with  glacial  acetic  acid  and  adds  concentrated  sulphuric  acid  drop 
by  drop.  On  following  the  exact  instructions  not  only  do  the  pentoses  give 
this  reaction,  but  also  glucuronic  acid,  glucose,  and  fructose   give   characteristic 

1  Bendix  and  Ebstein,  1.  c,  which  contains  the  literature;  Jolles,  Ber.  d.  d.  chem. 
Gesellsch.,  39  and  Zeitschr.  f.  anal.  Chem.,  46. 

2  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  27;  Neuberg,  ibid.,  31. 

3  Zeitschr.  f.  klin.  Med.,  47. 

4  Deutsch.  med.  Wochenschr.,  1902  and  1903,  and  Zeitschr.  f.  klin.  Med.,  50. 

5  Bial,  Zeitschr.  f.  klin.  Med.,  50;  van  Leersum,  Hofmeister's  Beitrage,  5. 

6  R.  and  O,  Adler,  Pfliiger's  Arch.,  106;  A.  Neumann,  Berl.  klin.  Wochenschr., 
1904. 


210  THE  CARBOHYDRATES. 

colored  solutions  with  special  absorption-bands  which  can  be  made  use  of  in 
identifying  the  various  sugars.  Fr.  Sachs  has  tested  Bial's  test  and  has  given 
special  precautions  to  prevent  confusion  with  glucuronic  acid.  Jolles  *  pre- 
cipitates (from  urine)  the  pentoses  as  osazones,  distills  the  precipitate  with 
hydrochloric  acid,  and  tests  the  distillate  with  Bial's  reagent. 

In  performing  the  above  two  tests  for  pentose  it  must  be  borne  in  mind  that 
glucuronic  acid  gives  the  same  reactions  and  also  that  the  colors  alone  are  not 
sufficient.  The  spectroscopic  examination  must  therefore  never  be  omitted. 
Both  tests  are  to  be  considered  as  tests  of  detection  rather  than  definite  pentose 
reactions,  and  therefore  for  a  positive  detection  of  pentoses  we  must  prepare  also 
the  osazones  or  other  compounds. 

Arabinoses.  The  pentose  isolated  by  Neuberg  from  human  urine 
is  r-arabinose.  It  can  be  isolated  from  the  urine  as  the  diphenylhydra- 
zone,  from  which  the  arabinose  can  be  separated  by  splitting  with  for- 
maldehyde. The  inactive  r-arabinose  seems  to  be  the  pentose  regularly 
occurring  in  pentosuria  and  thus  far,  in  only  one  case,  has  /-arabinose 
been  found.  /-Arabinose  is  said  to  pass  into  the  urine  after  partaking  of 
certain  fruits,  such  as  plums,  in  large  amounts  (C.  Barszczewski  2). 

The  r-arabinose  is  crystalline,  has  a  sweetish  taste,  and  melts  at 
163-164°  C.  Its  diphenylhydrazone,  which,  according  to  Neuberg 
and  Wohlgemuth,3  can  be  used  in  its  quantitative  estimation,  melts 
at  206°  C,  is  insoluble  in  cold  water  and  alcohol,  but  readily  soluble 
in  pyridine.     The  osazone  melts  at  166-168°  C. 

The  dextrorotatory  /-arabinose  is  obtained  by  boiling  gum  arabic  or 
cherry  gum  with  dilute  sulphuric  acid.  The  d-arabinose  has  been  pre- 
pared synthetically.  The  phenylosazone  of  /-arabinose  melts  at  160°. 
The  /-arabinose  which  crystallizes  in  plates  or  prisms  melts  at  about 
164°.     The  specific  rotation  is  (a)D  =  +104.5°. 

Xyloses.  The  /-xylose  occurs  extensively  in  the  plant  kingdom  and 
is  prepared  from  wood-gum  by  the  action  of  dilute  acid.  Xylose  is 
crystalline,  melts  at  150-153°  C,  dissolves  very  readily  in  water  but 
with  difficulty  in  alcohol,  is  faintly  dextrorotatory,  (a)D= +18.1°,  and 
gives  a  phenylosazone  which  melts  at  155-158°  C,  and  according  to 
Tollens  and  Muther  a  diphenylhydrazone  which  melts  at  107-108°. 
According  to  Bertrand  xylose  can  be  transformed  into  xylonic  acid, 
CH2(OH)[CH(OH)]3COOH,  by  bromine-water  and  the  brom-cadmium 
compound  or  the  brucine  salt  (Neuberg)  of  this  acid  is  well  suited  for 
the  detection  and  isolation  of  /-xylose.  On  oxidation  with  nitric  acid 
the  optically  inactive  trioxyglutaric  acid,  with  a  melting-point  of  152°  C. 
is  obtained. 


1  Fr.  Sachs,  Biochem.  Zeitschr.,  1  and  2;  Jolles,  ibid.,  2,  Centralbl.  f.    inn.  Med., 
1907.  and  Zeitschr.  f.  anal.  Chem.,  46. 

2  Neuberg,  Ber.  d.  d.  chem.  Gesellsch.,  33;  Barszczewski,  Maly's  Jahrsb.,  27,  733. 
•Zeitschr.  f.  physiol.  Chem.,  35. 


HEXOSES:  211 

According  to  Neitberg  and  to  Rewald1  the  pentose  obtained  from  a 
pancreas  nucleoprotein  and  the  pentose  isolated  by  Neuberg  and  Bkahn 
from  inosinic  acid  is  identical  with  /-xylose. 

Ribose.  This  pentose  has  been  prepared  synthetically  by  E.  Fischer. 
The  phenylhydrazone  melts  at  154-155°  C,  the  p-bromphenylhydr:iz<>n<- 
at  164-165°  C.  The  osazone  is  identical  with  arabinosazone.  On  oxida- 
tion it  yields  an  optically  inactive  trioxyglutaric  acid,  which  melts  at  170- 
171°  C.  d-ribose  is",  according  to  Levene  and  Jacobs,  the  pentose  of 
inosinic  acid,  guanylic  acid  and  yeast  nucleic  acid.  According  to  these 
workers  the  pentose  exists  in  these  nucleic  acids  in  a  glucoside-like  com- 
bination with  the  purine  bases,  as  so-called  nucleosides.  It  must  be 
remarked  that  Neuberg  adheres  to  his  claim  that  /-xylose  exists  at 
least  in  the  pancreas.2 

Hexoses  (C0H12O6). 

The  most  important  and  best-known  simple  sugars  belong  to  this 
group,  and  most  of  the  other  bodies  which  have  been  considered  as  car- 
bohydrates in  the  past  are  anhydrides  of  this  group.  Certain  hexoses, 
such  as  glucose  and  fructose,  either  occur  in  nature  already  formed 
or  are  produced  by  the  hydrolytic  splitting  of  other  more  complicated 
carbohydrates  or  glucosides.  Others,  such  as  mannose  or  galactose, 
are  formed  by  the  hydrolytic  cleavage  of  other  natural  products,  while 
some,  on  the  contrary,  such  as  gulcse,  talose,  and  others,  are  obtained 
only  by  artificial  means. 

All  hexoses,  as  also  their  anhydrides,  yield  levulinic  acid,  C5H8O3, 
besides  formic  acid  and  humus  substances  on  boiling  with  dilute  min- 
eral acids.  Oxymethyl  furfurol,  CqHqO^,  occurs  here  as  an  intermediary 
step  and  this  then  quantitatively  decomposes  into  levulinic  acid  and 
formic  acid.3  Some  of  the  hexoses,  as  above  stated,  are  fermentable 
with  yeast. 

Some  hexoses  are  aldoses,  while  others  are  ketoses.  Belonging  to  the 
first  group  we  have  mannose,  glucose,  and  galactose,  and  to  the  other 
fructose,  and  also  sorbinose. 

The  most  important  syntheses  of  the  carbohydrates  have  been  made 
by  E.  Fischer  and  his  pupils,  chiefly  within  the  members  of  the  hexose 
group.      A  short  summary  of  the  syntheses  of  hexoses  will  be  given. 

1  Tollens  and  Miither,  Ber.  d.  d.  chem.  Gesellsch.,  37;  Bertrand,  Bull.  soc.  chim. 
(3),  5;  Neuberg,  Ber.  d.  d.  chem.  Gesellsch.,  35;  Neuberg  and  Brahn,  Biochem.  Zeitchr., 
5;  Rewald,  Ber.  d.  d.  chem.  Gesellsch.,  42  (1909). 

2  E.  Fischer,  Ber.  d.  d.  chem.  Gesellsch,  24,  Levene  and  Jacobs,  ibid,  42,  2102,  2469 
2474,  3247  (1909);  43,  3147  (1910);  Neuberg,  ibid.,  42,  2806  (1909);  43,  3501  (1910). 

1  Kiermeyer,  Chem.  Zeitung.,  1895;  v.  Ekenstein  and  Blanksma,  Ber.  d.  d.  chem. 
Gesellsch.,  43,  2355  (1910). 


212  THE  CARBOHYDRATES. 

The  first  artificial  preparation  of  a  sugar  was  made  by  Butlerow.  On 
treating  trioxymethylene,  a  polymer  of  formaldehyde,  with  lime-water  he  obtained 
a  faintly  sweetish  syrup  called  methylenitan.  Loew  l  later  obtained  a  mixture 
of  serveral  sugars,  from  which  he  isolated  a  fermentable  sugar,  called  methose, 
by  condensation  of  formaldehyde  in  the  presence  of  bases.  The  most  important 
and  comprehensive  syntheses  of  sugar  have  been  performed  by  E.  Fischer  2 

The  starting-point  of  these  syntheses  is  a-acrose,  which  occurs  as  a  condensa- 
tion product  of  formaldehyde.  The  name  a-acrose  has  been  given  to  this  body 
because  it  is  obtained  from  acrolein  bromide  by  the  action  of  bases  (Fischer). 
It  is  also  obtained  admixed  with  fi-acrose  on  the  oxidation  of  glycerin  with 
bromine  in  the  presence  of  sodium  carbonate  and  treating  the  resulting  mixture 
with  alkali.  On  the  oxidation  with  bromine  a  mixture  of  glyceric  aldehyde, 
CH2OH.CH(OH).CHO,  and  dioxyacetone,  CH2(OH).CO.CH2OH,  is  obtained. 
These  two  bodies  may  be  considered  as  true  sugars,  namely,  glyceroses  or  trioses. 
It  seems  as  if  a  condensation  to  hexoses  takes  place  on  treatment  with  alkalies. 

a-acrose  may  be  isolated  from  the  above  mixture  and  obtained  pure  by  first 
converting  it  into  osazone  and  then  retransforming  this  into  the  sugar,  a-acrose 
seems  to  be  identical  with  r-fructose.  With  yeast  one-half,  the  levogyrate 
rf-fructose  ferments,  while  the  dextrogyrate  Z-fructose  remains.  The  r-  and 
Z-fructose  may  be  prepared  in  this  way. 

On  the  reduction  of  a-acrose  we  obtain  a-acrite,  which  is  identical  with  r- 
mannite.  On  oxidation  of  r-mannite  we  obtain  r-mannose,  from  which  only 
Z-mannose  remains  on  fermentation.  On  further  oxidation  of  r-mannose  it 
yields  r-mannonic  acid.  The  two  active  mannonic  acids  may  be  separated  from 
each  other  by  the  fractional  crystallization  of  their  strychnine  or  morphine  salts. 
The  two  corresponding  mannoses  may  be  obtained  from  these  two  acids,  d-  and 
Z-mannonic  acids,  by  reduction. 

rf-fructose  can  be  obtained  from  cZ-mannose  with  the  osazone  as  an  inter- 
mediary step  and  it  remains  now  to  speak  of  .the  formation  of  glucoses.  The 
d-  and  Z-mannonic  acids  are  partly  converted  into  d-  and  Z-gluconic  acids  on  heat- 
ing with  quinoline,  and  d-  or  Z-glucose  is  obtained  on  the  reduction  of  these  acids; 
Z-glucose  is  best  prepared  from  Z-arabinose  by  means  of  the  cyanhydrin  reaction, 
using  Z-gluconic  acid  as  the  intermediate  step.  The  combination  of  Z-  and  d- 
gluconic  acids,  forming  r-gluconic  acid,  yields  r-glucose  on  reduction. 

The  artificial  preparation  of  sugars  by  means  of  the  condensation  of  formalde- 
hyde has  received  special  interest  because,  according  to  Baeyer's  assimilation 
hypothesis,  in  plants  formaldehyde  is  first  formed  by  the  reduction  of  carbon 
dioxide,  and  the  sugars  are  produced  by  the  condensation  of  this  formaldelvyde. 
Bokorny  3  has  shown,  by  special  experiments  on  algse  Spirogyra,  that  formalde- 
hyde sodium  sulphite  was  split  by  the  living  algse  cells.  The  formaldehyde  set 
free  is  immediately  condensed  to  carbohydrate  and  precipitated  as  starch.4 

Among  the  hexoses  known  at  the  present  time  only  glucose,  fructose, 
and  galactose  are  really  of  physiological-chemical  interest;  therefore  of 
the  other  hexoses  only  mannose  will  be  incidentally  mentioned. 

rf-Glucose  (grape  sugar)  also  called  dextrose  and  diabetic  sugar — 
occurs  abundantly  in  the  grape,  and  also,  often  accompanied  with  fructose 

1  Butlerow,  Ann.  d.  Chem.  u.  Pharm.,  102;  Compt.  rend.,  53;  O.  Loew,  Journ.  f. 
prakt.  Chem.  (N.  F.),  33,  and  Ber.  d.  deutsch.  chem.  Gesellsch.,  20,  21,  22. 

2  Ber.  d.  d.  chem.  Gesellsch.,  21,  and  1.  c,  p.  197. 

3  Biolog.  Centralbl.  12,  pp.  321  and  481. 

*  In  regard  to  the  syntheses  of  sugar  see  also  W.  Lob  and  Pulvermacher  Bioch. 
Zeitschr,  23,  10  (1909),  26,  231  (1910). 


GLUCOSE.  213 

(levulose),  in  honey,  sweet  fruits,  seeds,  roots,  etc.  It  occurs  in  the 
human  and  animal  intestinal  tract  during  digestion,  also  in  small  quan- 
tities in  the  blood  and  lymph,  and  as  traces  in  other  animal  fluids  and 
tissues.  It  occurs  only  as  traces  in  urine  under  normal  conditions, 
while  in  diabetes  the  quantity  is  very  large.  It  is  formed  in  the  hydro- 
lytic  cleavage  of  starch,  dextrin,  and  other  compound  carbohydrates, 
as  also  in  the  splitting  of  glucosides.  The  question  whether  glucose 
can  be  formed  in  the  body  from  proteins  or  from  fats  is  disputed  and  will 
be  discussed  in  a  following  chapter  (VII). 

Properties  of  Glucose.  Glucose  crystallizes  sometimes  with  1  mole- 
cule of  water  of  crystallization  in  warty  masses  consisting  of  small  leaves 
or  plates,  and  sometimes  when  free  from  water  in  fine  needles  or  prisms. 
The  sugar  containing  water  of  crystallization  melts  even  below  100°  C. 
and  loses  its  water  of  crystallization  at  110°  C.  The  anhydrous  sugar 
melts  at  146°  C,  and  is  converted  into  glucosan,  CeHioOs,  at  170°  C. 
with  the  elimination  of  water.  On  strongly  heating  it  is  converted  into 
caramel  and  then  decomposes. 

Glucose  is  readily  soluble  in  water.  This  solution,  which  is  not  as 
sweet  as  a  cane-sugar  solution  of  the  same  strength,  is  dextrogyrate  and 
shows  strong  birotation.  The  specific  rotation  is  dependent  upon  the 
concentration  of  the  solution,  as  it  increases  with  an  increase  in  the  con- 
centration. A  10  per  cent  solution  of  anhydrous  glucose  can  be  taken  as 
+52.5°  at  20°  C.1  Glucose  dissolves  sparingly  in  cold,  but  more  freely 
in  boiling  alcohol.  One  hundred  parts  alcohol  of  sp.  gr.  0.837  dissolves 
1.95  parts  anhydrous  glucose  at  17.5°  C.  and  27.7  parts  at  the  boiling 
temperature  (Anthon2).     Glucose  is  insoluble  in  ether. 

If  an  alcoholic  caustic-potash  solution  is  added  to  an  alcoholic  solu- 
tion of  glucose,  an  amorphous  precipitate  of  insoluble  sugar-potash 
compound  is  formed.  On  warming  this  compound  it  decomposes  easily 
with  the  formation  of  a  yellow  or  brownish  color,  which  is  the  basis  of 
Moore's  test.     Glucose  also  forms  compounds  with  lime  and  baryta. 

Moore's  Test.  If  a  glucose  solution  is  treated  with  about  one 
quarter  of  its  volume  of  caustic  potash  or  soda  and  warmed,  the  solution 
becomes  first  yellow,  then  orange,  yellowish-brown,  and  lastly  dark 
brown.  It  has  at  the  same  time  a  faint  odor  of  caramel,  and  this  odor 
is  more  pronounced  on  acidifiying.3 

Glucose  forms  several  crystallizable  combinations  with  NaCl  of 
which   the   easiest   to   obtain   is    (CeHioOG^-NaCl-fH^O,   which    forms 

1  For  further  information  see  Tollens'  Handbuch  der  Kohlehydrate,  2.  AuflL,  44. 

2  Cited  from  Tollens'  Handbuch. 

3  In  regard  to  the  products  formed  in  this  reaction,  see  Framm,  Pfluger's  Arch.,  64; 
Neff,  Annal.  d.  Chem.  u.  Pharm.,  357;  Buchner  and  Meisenheimer,  Ber.  d.  d.  chem. 
Gesellsch.,  39;  Meisenheimer,  ibid.,  41. 


214  THE  CARBOHYDRATES. 

large  colorless  six-sided  double  pyramids  or  rhomboids  with  13.52  per 
cent  NaCl. 

Glucose  in  neutral  or  very  faintly  acid  (organic  acid)  solution  under- 
goes alcoholic  fermentation  with  beer-yeast:  CeHi206=:2C2H5.0H+2C02. 
In  the  presence  of  acid  milk  or  cheese  the  glucose  undergoes  lactic-acid 
fermentation,  especially  in  the  presence  of  a  base  such  as  ZnO  or  CaC03. 
The  lactic  acid  may  then  further  undergo  butyric-acid  fermentation: 
2C3H603  =  C4H802+2C02+4H. 

Glucose  reduces  several  metallic  oxides,  such  as  copper,  bismuth, 
and  mercuric  oxide,  in  alkaline  solutions,  and  the  most  important 
reactions  for  sugar  are  based  on  this  fact.1 

Trommer's  test  is  based  on  the  property  that  glucose  possesses  of 
reducing  cupric  hydroxide  in  alkaline  solution  into  cuprous  oxide.  Treat 
the  glucose  solution  with  about  \- \  vol.  caustic  soda  and  then  carefully 
add  a  dilute  copper-sulphate  solution.  The  cupric  hydroxide  is  thereby 
dissolved,  forming  a  beautiful  blue  solution,  and  the  addition  of  copper 
sulphate  is  continued  until  a  very  small  amount  of  hydroxide  remains 
undissolved  in  the  liquid.  This  is  now  warmed,  and  a  yellow  hydrated 
suboxide  or  red  suboxide  separates  even  below  the  boiling  temperature. 
If  too  little  copper  salt  has  been  added,  the  test  will  be  yellowish-brown 
in  color,  as  in  Moore's  test;  but  if  an  excess  of  copper  salt  has  been  added, 
the  excess  of  hydroxide  is  converted  on  boiling  into  a  dark-brown  hydrate 
which  interferes  with  the  test.  To  prevent  these  difficulties  the  so- 
called  Fehling's  solution  may  be  employed.  This  solution  is  obtained 
by  mixing  just  before  use  equal  volumes  of  an  alkaline  solution  of  Rochelle 
salt  and  a  copper-sulphate' solution  (173  grams  Rochelle  salt  and  about 
50-60  grams  NaOH  per  liter  and  34.65  grams  crystalline  copper  sulphate 
per  liter).  This  solution  is  not  reduced  or  noticeably  changed  by  boiling. 
The  tartrate  holds  the  excess  of  cupric  hydroxide  in  solution,  and  an  excess 
of  the  reagent  does  not  interfere  in  the  performance  of  the  test.  In 
the  presence  of  sugar  this  solution  is  reduced. 

According  to  Benedict  2  this  test  is  more  delicate  if  sodium  carbonate  is 
used  instead  of  sodium  hydroxide  in  the  preparation  of  Fehling's  solution. 

B6ttger-Alm£n's  test  is  based  on  the  property  glucose  possesses 
of  reducing  bismuth  oxide  in  alkaline  solution.  The  reagent  best  adapted 
for  this  purpose  is  obtained,  according  to  Nylander's  3  modification  of 
Almex's  original  test,  by  dissolving  4  grams  of  Rochelle  salt  in  100  parts 
of  10  per  cent  caustic-soda  solution  and  adding  2  grams  of  bismuth 
subnitrate  and  digesting  on  the  water-bath  until  as  much  of  the  bismuth 

1  In  regard  to  the  products  produced  see  Neff,  Annal.  d.  Chem.  u.  Pharm.,  357. 

2  Journ.  of  biol.  Chem.,  3. 

3  Zeitschr.  f.  physiol.  Chem.,  8. 


GLUCOSE.  215 

salt  is  dissolved  as  possible.  If  a  glucose  solution  \s  treated  with  about 
tV  vol.,  or  with  a  larger  quantity  of  the  solution  when  large  quantities 
of  sugar  are  present,  and  boiled  for  a  few  minutes,  the  solution  becomes 
first  yellow,  then  yellowish-brown,  and  finally  nearly  black,  and  after  a 
time  a  black  deposit  of  bismuth  (?)  settles. 

The  property  that  glucose  has  of  reducing  an  alkaline  solution  of 
mercury  on  boiling  is  the  basis  of  Knapp's  reaction  with  alkaline  mercuric 
cyanide,  and  of  Sachsse's  reaction  witli  an  alkaline  potassium-mercuric 
iodide  solution. 

On  heating  with  phenylhydrazine  acetate  a  glucose  solution 
gives  a  precipitate  consisting  of  fine  yellow  crystalline  needles  which  are 
almost  insoluble  in  water,  but  soluble  in  boiling  alcohol,  and  which  separate 
again,  on  treating  the  alcoholic  solution  with  water.  The  crystalline 
precipitate  consists  of  phenylglucosazone  (see  page  203).  This  com- 
pound melts  when  pure  at  205°  C.  It  must  be  borne  in  mind  that 
the  melting-point  of  this  and  other  osazones  is  somewhat  variable,  depend- 
ing upon  the  rapidity  of  the  heating,  the  diameter  of  the  tube  and  the 
thickness  of  the  sides  of  the  tube.1  The  osazone  dissolves  readily  in 
pyridine  (0.25  gram  in  1  gram),  and  precipitates  again  from  this  solu- 
tion as  crystals  on  the  addition  of  benzene,  ligroin,  or  ether.  According 
to  Neuberg  2  this  behavior  can  be  used  in  the  purification  of  the  osazone. 
The  diphenylhydrazone  and  the  methyl  phenylhydrazone  are  also  of 
interest. 

Glucose  is  not  precipitated  by  a  lead-acetate  solution,  but  is  almost 
completely  precipitated  by  a  solution  of  ammoniacal  basic  lead  acetate. 
On  warming,  the  precipitate  becomes  flesh-color  or  rose-red  (Rubner's 
reaction  3) . 

If  a  watery  solution  of  glucose  is  treated  with  benzoylchloride  and 
an  excess  of  caustic  soda,  and  shaken  until  the  odor  of  benzoylchloride 
has  disappeared,  a  precipitate  of  benzoic-acid  ester  of  glucose  will  be 
produced  which  is  insoluble  in  water  or  alkali  (B aumann  4) . 

If  \-\  cc.  of  a  dilute  watery  solution  of  glucose  is  treated  with  a  few 
drops  of  a  10  per  cent  alcoholic  solution  (free  from  acetone)  of  a-nayhthol , 
on  the  slow  addition  of  1-2  cc.  of  concentrated  sulphuric  acid  a  beautiful 
reddish-violet  ring  forms  at  the  juncture  of  the  liquids,  or  on  shaking,  the 
entire    mixture    becomes    a    beautiful    reddish-violet  color  (Molisch5). 

1  See  E.  Fischer,  Ber.  d.  d.  ehem.  Gesellsch.,  41. 

2  Ber.  d.  d.  chim.  Gesellsch.,  32,  3384. 

3  Zeitschr.  f.  Biologie,  20. 

4  Ber.  d.  deutsch.  chem.  Gesellsch.,  19;  also  Kueny,  Zeitschr.  f.  physiol.  Chem.,  14, 
and  Skraup,  ^Vien.  Sitzungsber.,  98  (1888). 

5  Molisch,  Monatshefte  f .  Chem.,  7,  and  Centralbl.  f.  d.  med.  Wissensch.,  1887, 
pp.  34  and  49. 


216  THE  CARBOHYDRATES. 

This  reaction  depends,  according  to  Ville  and  Derrien,  as  well  as  to 
V.  Ekenstein  and  Blanksma  1  upon  the  formation  of  oxymethylfurfurol 
which  reacts  with  the  a-naphthol.  As  oxymethylfurfurol  is  formed  from 
all  hexoses,  hence  Molisch's  reaction  is  a  general  reaction  for  hexoses. 

Diazobenzenesulphonic  acid  gives  with  a  glucose  solution  made  alkaline 
with  a  fixed  alkali  a  red  color,  which  after  10-15  minutes  gradually  changes  to 
violet.  Orthonitrophenylpropiolic  acid  yields  indigo  when  boiled  with  a 
small  quantity  of  glucose  and  sodium  carbonate,  and  this  is  converted  into 
indigo-white  by  an  excess  of  sugar.  An  alkaline  solution  of  glucose  is  colored 
deep  red  on  being  warmed  with  a  dilute  solution  of  picric  acid.  The  behavior 
of  glucose  toward  certain  pentose  reactions  has  been  given  on  page  209. 

A  more  complete  description  as  to  the  performance  of  these  several 
tests  will  be  given  in  detail  in  a  subsequent  chapter  (on  the  urine). 

Glucose  is  prepared,  pure,  by  inverting  cane-sugar  by  the  follow- 
ing simple  method  of  Soxhlet  and  Tollens,  which  is  a  modification  of 
Schwarz's2  method: 

Treat  12  liters  90  per  cent  alcohol  with  480  cc.  fuming  hydrochloric 
acid  and  warm  to  45-50°  C;  gradually  add  4  kilos  of  powdered  cane- 
sugar,  and  allow  to  cool  after  two  hours,  when  all  the  sugar  will  have 
dissolved  and  been  inverted.  To  incite  crystallization,  some  crystals  of 
anhydrous  glucose  are  added,  and  after  several  days  the  crystals  are 
sucked  dry  by  the  air-pump,  washed  with  dilute  alcohol  to  remove 
hydrochloric  acid,  and  crystallized  from  alcohol  or  methyl  alcohol. 
According  to  Tollens  it  is  best  to  dissolve  the  sugar  in  one-half  its 
weight  of  water  on  the  water-bath  and  then  add  double  this  volume  of 
90-95  per  cent  alcohol. 

In  detecting  glucose  in  animal  fluids  or  extracts  of  tissues  we  may 
make  use  of  the  above-mentioned  reduction  tests,  the  optieal  deter- 
mination, fermentation,  and  phenylhydrazine  tests.  For  the  quantitative 
estimation  the  reader  is  referred  to  the  chapter  on  the  urine.  Those 
liquids  containing  proteins  must  first  have  these  removed  by  coagulation 
with  heat  and  addition  of  acetic  acid,  or  by  precipitation  with  alcohoL 
or  metallic  salts,  before  testing  for  glucose.  In  regard  to  the  difficulties 
of  operating  with  blood  and  serous  fluids  we  refer  the  student  to  larger 
works. 

Mannoses.  d-Mannose,  also  called  seminose,  is  obtained  with  d-fructose  on 
the  careful  oxidation  of  d-mannite.  It  is  also  obtained  on  the  hydrolysis  of 
natural  carbohydrates,  such  as  salep  slime  and  reserve  cellulose  (especially 
from  the  shavings  of  the  ivory-nut).  It  is  dextrorotatory,  readily  ferments, 
with  beer-yeast,  gives  a  hydrazone  not  readily  soluble  in  water,  and  an  osazone 
which  is  identical  with  that  from  d-glucose. 

d-Galactose  (not  to  be  mistaken  for  lactose  or  milk-sugar)  is  obtained  on 
the  hydrolytic  cleavage  of  milk-sugar,  and  by  the  hydrolysis  of  many  other 

1  Bull.  soc.  chim.  (4),  5,  895  (1909);  Ber.  d.  d.  ohem.  Gesellsch.,  43,  2358  (1910). 

2  Tollens,  Handbuch  der  Kohlehydrate,  2.  Aufl.  I,  39. 


FRUCTOSE.  217 

carbohydrates,  especially  varieties  of  gums  and  mucilaginous  bodies. 
It  is  also  obtained  on  heating  cerebrin,  a  nitrogenized  glucoside  prepared 
from  the  brain,  with  dilute  mineral  acids. 

It  crystallizes  in  needles  or  leaves  which  melt  at  1G8°  C.  It  is  some- 
what less  soluble  in  water  than  glucose.  It  is  dextrogyrate,  and  according 
to  Neuberg  l  has  a  rotation  (a)D=+81°.  With  ordinary  yeast  galac- 
tose is  slowly,  but  nevertheless  completely,  fermented.  It  is  fermented 
by  a  great  variety  of  yeasts  (E.  Fischer  and  Thierfelder),  but  not  by 
Saccharomyces  apiculatus,2  which  is  of  importance  in  physiological- 
chemical  investigations.  Galactose  reduces  Fehling's  solution  to  a 
less  extent  than  glucose,  and  10  cc.  of  this  solution  are  reduced,  accord- 
ing to  Soxhlet,  by  0.0511  gram  galactose  in  1  per  cent  solution.  Its 
phenylosazone  melts  according  to  Neuberg  at  196-197°  C,  and  is  soluble 
with  difficulty  in  hot  water,  but  with  relative  ease  in  hot  alcohol.  Its 
solution  in  glacial  acetic  acid  is  optically  inactive.  In  the  test  with 
hydrochloric  acid  and  phloroglucin  galactose  gives  a  color  similar  to  that 
of  the  pentoses,  but  the  solution  does  not  give  the  absorption  spectrum. 
On  oxidation  it  first  yields  galactonic  acid  and  then  mucic  acid,  and 
these  serve  in  the  detection  of  galactose. 

d-Fructose  (levulose)  also  fruit-sugar,  occurs,  as  above  stated,  mixed 
with  glucose,  extensively  distributed  in  the  vegetable  kingdom  and 
also  in  honey.  It  is  formed  in  the  hydrolytic  cleavage  of  cane-sugar 
and  several  other  carbohydrates,  but  it  is  very  readily  obtained  by  the 
hydrolytic  splitting  of  inulin.  In  extraordinary  cases  of  diabetes  mellitus 
we  find  fructose  in  the  urine.  Neuberg  and  Strauss3  have  detected 
fructose  with  positiveness  in  human  blood-serum,  and  exudates  in  cer- 
tain cases. 

Fructose  crystallizes  with  comparative  difficulty  in  coarse  crusts 
or  warts  or  in  fine  needles.  C.  Morner  4  has  obtained  crystals  2-3  mm. 
long  which  belonged  to  the  rhombic  system,  and  neither  melted  nor  lost 
in  weight  on  heating  to  100°  C.  The  melting-point  is  110°  C.  Fructose 
is  readily  soluble  in  water,  but  almost  insoluble  in  cold  absolute  alcohol, 
though  rather  readily  in  boiling  alcohol.  Its  aqueous  solution  is  levogy- 
rate.  C.  Morner  found  the  rotation  for  a  10  and  20  per  cent  solution 
was  (a)D=— 93°  and  —94.1°  respectively.  Fructose  ferments  with 
yeast,  and  gives  the  same  reduction  tests  as  glucose,  and  also  the  same 
osazone.  It  gives  a  compound  with  lime  which  is  less  soluble  than  the 
corresponding  glucose  compound.  Fructose  is  not  precipitated  by 
sugar  of  lead  or  basic  lead  acetate. 

1  See  C.  Oppenheimer,  Handb.  d.  Biochem.  1,  p.  197. 

2  See  F.  Voit,  Zeitschr.  f.  Biol.,  28  and  29. 

3  Zeitschr.  f.  physiol.  Chem.,  36,  which  also  contains  the  older  literature. 

*  Svensk.  Farmac.  Tidskr,  No.  6,  1907.     See  also  Maly's  Jahresb.,  37,  p.  95. 


218  THE  CARBOHYDRATES. 

Fructose  does  not  reduce  copper  to  the  same  extent  as  glucose. 
Under  similar  conditions  the  reduction  relationship  is  100  :  92.08. 

In  detecting  fructose  and  those  varieties  of  sugar  which  yield  fructose  on 
cleavage  we  make  use  of  the  following  reaction,  suggested  by  Seliwanoff  which 
consists  in  heating  with  hydrochloric  acid  and  resorcinol.  This  depends  upon 
the  formation  of  oxymethylfurfurol  and  is  therefore  obtained  by  all  hexoses. 
As  the  ketoses  give  about  20  per  cent  oxymethylfurfurol  and  the  aldoses  only 
1  per  cent  the  reaction  is  more  readily  obtained  with  the  ketohexoses  than  with 
the  aldohexoses  (v.  Ekenstein  and  Blanksma,  page  216).  To  a  few  cubic 
centimeters  of  fuming  hydrochloric  acid  add  an  equal  volume  of  water  and  a  small 
quantity  of  the  sugar  solution  or  of  the  solid  substance  and  a  few  crystals  of 
resorcinol,  and  apply  heat.  The  liquid  becomes  a  beautiful  red,  and  gradually 
a  substance  precipitates  which  is  red  in  color  and  soluble  in  alcohol.  According 
to  Ofnek  1  the  mixture  must  not  contain  more  than  12  per  cent  HC1,  and  the 
boiling  must  not  be  continued  longer  than  twenty  seconds,  if  it  is  boiled  for 
a  longer  time  and  with  more  hydrochloric  acid  this  reaction  is  also  given  with 
the  aldoses.  R.  and  0.  Adler  2  perform  the  test  with  glacial  acetic  acid  and  a 
drop  of  hydrochloric  acid  and  some  resorcinol,  in  which  case  a  reaction  with 
aldoses  is  not  obtained.  Seliwanoff' s  reaction,  according  to  Rosin,3  may 
be  made  more  delicate  by  a  combination  with  the  spectroscopic  examination. 
In  regard  to  its  use  in  urine  examinations  see  Chapter  XIV. 

The  naphtho-resorcinol  reaction  as  suggested  by  B.  Tollens  and  Rorive  4 
can  be  carried  out  as  follows:  A  few  particles  of  the  sugar  and  about  the  same 
quantity  of  naphthoresorcinol  are  treated  with  about  10  cc.  of  a  mixture  of  equal 
volumes  of  water  and  concentrated  hydrochloric  acid  of  sp.  gr.  1.19.  This  is 
slowly  heated  to  boiling  over  a  low  flame,  and  is  continued  for  1-3  minutes. 
The  fluid  becomes  more  purple  or  violet  than  with  Seliwanoff's  resorcin  test. 
The  spectroscopic  examination  shows  a  faint  band  in  the  green. 

According  to  Neuberg,5  methylphenlhydrazine  is  an  excellent  substance 
to  use  for  the  separation  and  detection  of  fructose,  as  it  gives  a  characteristic 
fructose  methylphenylosazone.  This  osazone  when  recrystallized  from  alcohol 
melts  at  153°.  It  shows  a  dextrorotation  of  1°  40'  when  0.2  gram  of  the  osazone 
is  dissolved  in  4  cc.  pyridine  and  6  cc.  absolute  alcohol. 

Ofner  has  made  objections  to  the  use  of  methylphenylhydrazine  in  the  detec- 
tion of  fructose.  He  has  obtained  the  osazone  from  glucose  and  methylphenylhy- 
drazine, although  the  osazone  is  formed  much  more  quickly  with  fructose  than 
with  glucose.  Only  when  the  separation  of  the  osazone  crystals  with  methyl- 
phen}rlhydrazine  after  the  addition  of  acetic  acid  takes  place  within  five  hours 
at  ordinary  temperatures  is  the  presence  of  fructose  positively  proven  (Ofner  6). 

The  use  of  secondary  asymmetric  hydrazines  as  a  general  reagent  for  ketoses 
and  as  a  means  of  separation  from  aldoses  is  objected  to  by  Ofner. 

(/-Sorbinose  (sorbin)  is  a  ketose  obtained  from  the  juice  of  the  berry  of  the 
mountain  ash  under  certain  conditions.  It  is  crystalline  and  levogyrate,  and 
is  converted  into  c/-sorbite  by  reduction. 


1  Monatshefte  f.  Chem.,  25. 

2  See  footnote  6,  p.  209. 

3  Ber.  d.  d.  chem.  Gesellsch.,  38. 

*  Ibid.,  41,  p.  1783  and  Tollens,  ibid.,  41,  p.  1788.     See  also  Mandel  and  Neuberg, 
Biochem.  Zeitschr.,  13. 

1  Ibid.,  35;  also  Neuberg  and  Strauss,  ibid.,  36. 
6  Ibid.,  37,  and  Zeitschr.  f.  physiol.  Chem.,  45. 


GLUCOSAMINE.  219 

Appendix   to  the   Monosaccharides, 
a.  Amino-sugars. 

The  most  important  amino-sugar  is  the  already  mentioned  glucosamine. 

CH2OH 

(/-Glucosamine    (chitosamine),    C6H13NO5,  =  /,„ "  TTJ    ,   whose    synthet- 

Cn.1N.ri2 

COH 
ical  preparation  has  been  given  on  page  201  was  first  prepared  by 
Ledderhose  J  from  chitin  by  the  action  of  concentrated  hydrochloric 
acid.  Recently  it  has  been  obtained  as  a  cleavage  product  of  several 
mucin  substances  and  proteins  (see  pages  84  and  168).  Glucosamine  is, 
as  E.  Fischer  and  Leuchs  2  have  shown,  a  derivative  of  glucose  or  of 
d-mannose  (probably  glucose),  and  is  an  a-amino-sugar. 

The  free  base,  which  can  crystallize  in  needles,  is  readily  soluble  in 
water  giving  an  alkaline  reaction,  and  quickly  decomposes.  The  charac- 
teristic hydrochloride  forms  colorless  crystals  which  are  stable  in  the  air 
and  readily  soluble  in  water,  soluble  with  difficulty  in  alcohol,  and  insoluble 
in  ether.  The  solution  is  dextrorotatory,  (a)D  =  +70.15°  to  74.64°,  at  vari- 
ous concentrations.3  Glucosamine  has  a  reducing  action  similar  to  that  of 
glucose,  and  gives  the  same  osazone,  but  is  not  fermentable.  With 
benzoyl-chloride  and  caustic  soda  it  gives  a  crystalline  ester.  In  alkaline 
solution  it  gives  with  phenylisocyanate  a  compound  which  can  be  con- 
verted into  its  anhydride  by  acetic  acid,  and  is  used  in  the  separation  and 
detection  of  glucosamine  (Steudel).4  On  oxidation  with  nitric  acid  it 
yields  norisosaccharic  acid,  whose  lead  salt  can  be  separated,  and  whose 
salts  with  cinchonine  or  quinine  are  soluble  with  difficulty  in  water  and  can 
also  be  used  very  successfully  in  the  detection  of  glucosamine  (Neuberg  and 
Wolff5).  On  oxidation  with  bromine,  chitaminic  acid  (rf-glucosaminic 
acid)  is  produced,  and  this  is  converted  into  chitaric  acid,  CgHioOg, 
by  nitrous  acid.  On  treatment  with  nitrous  acid  glucosamine  yields 
a  non-fermentable  sugar  called  chitose. 

Ehrlich  6  has  suggested  a  test  which  does  not  respond  with  the  free  glucos- 
amine, but  with  the  mucins  and  other  protein  bodies  containing  an  acetylated 
glucosamine.     It  consists  in  warming  the  substance,  which  has  been  previously 

1  Zeitschr.  f.  physiol.  Chem.,  2  and  4. 

2  Ber.  d.  d.  chem.  Gesellsch.,  36. 

3  See  Hoppe-Seyler-Thierfelder's  Handbuch,  8,  Aufl.;  Sundvik,  Zeitschr.  f.  physiol 
Chem.,  34. 

*  Zeitschr.  f.  physiol.  Chem.,  34. 
6  Ber.  d.  d.  chem.  Gesellsch.,  34. 
p  Mediz.  Woche,  1901,  No.  15;  see  Langstein,  Ergebnisse  der  Physiol.,  I,  Abt.  1,  88. 


220  THE  CAKBOHYDRATES. 

treated  with  alkali,  with  a  hydrochloric-acid  solution  of  dimethylaminobenzalde- 
hyde,  when  a  beautiful  red  color  is  obtained. 

Glucosamine  is  best  prepared  from  decalcified  lobster-shells  by  treat- 
ing with  hot  concentrated  hydrochloric  acid.1  In  regard  to  its  prepara- 
tion from  protein  substances  we  must  refer  to  the  works  cited  on  page 
84,  footnote  5. 

Albamine  (diglucosamine),  (CeHnC^N^+H^O,  is  the  name  given  by  S.  Fran- 
kel  -  to  a  body  which  he  isolated  from  the  products  of  the  hydrolysis  of  ovalbumin 
with  baryta,  as  well  as  in  its  digestion.  Albamine  is  amorphous,  dextrogyrate, 
and  reduces  after  boiling  with  acids.  As  hydrolytic  cleavage  product  it  yields 
^-glucosamine. 

Galactosamine  is  claimed  to  have  been  found  by  Schulz  and  Ditthorn  in 
a  glycoprotein  of  the  spawn  of  the  frog.  This  claim  is  not  generally  accepted, 
v.  Ekexstein  and  Blanksma  3  obtained  galactose  on  the  hydrolysis  of  the  slimy 
envelope  of  frog  eggs. 

According  to  Offer,4  pentosamine  occurs  in  the  liver  of  the  horse.  Accord- 
ing to  Offer,  the  pentose  derivative,  which  he  calls  dipentosamine  (CsHyOs.NH^o-l- 
H20  and  a  second,  perhaps  a  diacetyl-pentosamine  2(CH3CO)CioHi8N207  (?), 
also  occur  in  the  liver.  The  first  gives  pentose  reactions  and  reduces  Fehling's 
solution  after  boiling  with  acid.  The  only  amino-sugar  positively  detected  in 
the  animal  organs  is  glucosamine. 

The  amino-sugars,  as  intermediary  bodies  between  the  carbohydrates 
and  oxyamino-acids,  are  of  great  physiological  interest,  and  this  interest 
has  become  still  more  important  since  Netjberg  was  first  able  to  pre- 
pare the  corresponding  amino-aldehyde  from  glycocoll  and  then  also 
from  other  amino-acids.  From  the  ethyl  ester  of  glycocoll  in  acid  solu- 
tion Neuberg  5  obtained  the  amino-acetaldehyde.  NH2.CH2.CHO,  by 
treatment  with  sodium  amalgam.  This  aldehyde  is  very  unstable  and 
has  a  tendency  to  condensation  with  ring  formation,  and  Neuberg 
obtained  therefrom  by  oxidation  with  corrosive  sublimate  and  caustic 
soda,  pyrazine  according  to  the  equation: 


NH2 

1 

N 

CH2+CHO 

y\ 

1           1 

HC      CH 

CHO   CH24-0  = 

=      |       ||     +3H20 

1 

HC      CH 

NH2 

\/ 

N 

1  See  Hoppe-Seyler-Thierfelder's  Handbuch,  8.  Aufl. 

2  Monatsh.  f.  Chem.,  19. 

-'•hulz  and  Ditthorn,  Zeitsohr.  f.  physiol.  Chem.,  29;  v.  Ekenstein  and  Blanksma, 
Chem.  Centralis,  1907,  2,  p.  1001. 
4  Hofmeister's  Beitrage,  8. 
6  Ber.  d.  d.  chem.  Gesellsch.,  41. 


GLUCURONIC  ACIDS.  221 

On  account  of  this  tendency  to  ring-formation  the  amino-acetalde- 
hyde  as  well  as  the  amino-aldehydes  as  a  group,  stand,  according  to  Neu- 
berg,  in  close  relationship  to  many  ring  systems,  such*  as  imidazole, 
piperazine,    pyrazine,    pyridine   and   others,    and   also   to   the   alkaloids. 

The  amino-sugars,  like  the  amino-aldehydes,  can  also  unite,  form- 
ing ring  compounds,  and  this  seems  to  be  the  case  on  the  decomposi- 
tion of  free  glucosamine  in  aqueous  solution,  which  occurs  with  access 
cf  air  (Lobry  de  Bruyn).  As  found  by  Stolte  l  2,5-ditetraoxybutyl 
pyrazine  (=  fructosazine)  is  hereby  produced  according  to  the  following 
equation: 

NH2  N 

/  y\ 

O.1H9C4.CH+CHO  O4H9C4.C      CH 

+0=  I       ||  +3H20 

CHO  CH.C4H9O4  HC      C.C4H9O4 

/  V 

NH2  N 

Fructosazine 

The  2,5-ditetraoxybutyl  pyrazine,  which  Stolte  obtained  by  Lobry 
de  Bruyn's2  method  from  fructose  in  methyl  alcohol  solution  and 
ammonia,  and  which  he  calls  fructosazine,  can  be  oxidized  outside  of  the 
body  into  2,5-pyrazine  dicarboxylic  acid. 

The  same  acid  can  be  formed  in  the  animal  body  (rabbits),  although  not 
constantly,  after  introducing  fructosazine.  It  also  passes  into  the  urine  of 
rabbits  after  intravenous  injection  of  d-fructose  and  glycocoll  (Spiro),  a  behavior 
which  Spiro  claims  indicates  that  carbohydrates  in  metabolism  react  with  the 
cleavage  products  of  proteins.  Stolte's  experiments  to  decide  the  question 
whether  in  the  animal  body  the  glucosamine  in  its  decomposition  passes  into 
fructosazine  did  not  at  first  yield  conclusive  results.  His  more  recent  investiga- 
tions 3  show  on  the  contrary  that  in  rabbits  2-oxymethylpyrazine-5-carboxylic 
acid  is  formed  as  an  oxidation  product,  and  this  can  be  oxidized  outside  of  the 
body  into  pyrazine-2,  5-dicarboxyIic  acid. 

b.  Glucuronic  Acids. 

The  glucuronic  acids  occurring  in  the  animal  body  either  physiolog- 
ically or  pathologically,  are  conjugated  acids  which  will  be  described  in 
detail  in  a  subsequent  chapter  (XIV).  We  will  here  describe  only  the 
d-glucuronic  acid  in  connection  with  the  carbohydrates. 

CHO 
d-Glucuronic  acid  (glycuronic  acid),  C6Hio07  =  (CH.OH)4,  is  a  deriva- 

COOH 
tive  of  glucose,  and  has  been  synthetically  prepared  by  E.  Fischer  and 

1  Hofmeister's  Beitriige,  11. 

2  Cited  by  Stolte,  Hofmeister's  Beitrage,  11. 

3  Spiro,  Hofmeister's  Beitriige,  10,  p.  283;  Stolte,  Biochem.  Zeitschr.,  12. 


222  THE  CARBOHYDRATES. 

Piloty  l  by  the  reduction  cf  the  lactone  of  saccharic  acid.  On  oxidation 
with  bromine  it  forms  saccharic  acid,  and  on  reduction  it  yields  gulonic- 
acid  lactone.  -Salkowski  and  Neuberg  2  have  obtained  i-xylcse  from 
glucuronic  acid  by  splitting  off  CO2  by  means  of  putrefaction  bacteria. 

Glucuronic  acid  has  not  been  found  in  the  free  state  in  the  animal 
body.  It  occurs  to  a  slight  extent  in  normal  urine  as  a  conjugated  acid 
(Mayer  and  Neuberg).  It  occurs  to  a  much  greater  extent  in  urine  as 
conjugated  acid  after  the  ingestion  of  certain  aromatic  and  also  aliphatic 
substances,  especially  camphor  and  chloral  hydrate.  It  was  obtained 
first  by  Schmiedeberg  and  Meyer  from  camphoglucuronic  acid,  and 
then  by  v.  Mering  3  from  urochloralic  acid  by  cleavage  with  dilute  acids. 
According  to  P.  Mayer,4  on  the  oxidation  of  glucose  a  partial  forma- 
tion of  glucuronic  acid  and  oxalic  acid  takes  place,  and  therefore,  according 
to  him,  an  increased  elimination  of  conjugated  glucuronic  acids  shows 
in  certain  cases  an  incomplete  oxidation  of  glucose.  Conjugated  glucu- 
ronic acids  may  also  occur  in  the  blood  (P.  Mayer,  Lepine  and  Boulud  5), 
in  the  feces,  and  in  the  bile.6  Neuberg  and  Neumann  7  have  prepared 
certain  conjugated  glucuronic  acids  (see  Chapter  XIV)  synthetically, 
among  them  being  euxanthic  acid.  The  most  abundant  source  of  glucu- 
ronic acid  is  the  artist's  pigment  "  Jaune  indien,"  which  contains  the 
magnesium  salt  cf  euxanthic  acid  (euxanthon-glucuronic  acid). 

Glucuronic  acid  is  not  crystalline,  but  is  only  obtainable  as  a  syrup. 
It  dissolves  in  alcohol  and  is  readily  soluble  in  water.  If  the  aqueous 
solution  is  boiled  for  an  hour  the  acid  is  partly  (20  per  cent)  converted 
into  the  crystalline  lactone,  glucurone,  CaHsOo,  which  is  soluble  in  water 
and  insoluble  in  alcohol,  and  which  has  a  melting-point  of  175-178°  C. 
The  alkali  salts  of  the  acid  are  crystalline.  If  a  concentrated  solution 
of  the  acid  is  saturated  with  barium  hydroxide  the  basic  barium  salt  is 
obtained  as  a  precipitate.  The  neutral  lead  salt  is  soluble  in  water, 
while  the  basic  salt  is  insoluble.  The  readily  crystallizable  cinchonine 
salt  can  be  used  in  isolating  glucuronic  acid  (Neuberg8).  Glucuronic 
acid  is  dextrorotatory,  while  the  conjugated  acids  are  levorotatory; 
they  behave  like  glucose  with  the  reduction  tests,  and  do  not  ferment 


1  Ber.  d.  d.  chem.  Gesellsch.,  24. 

J  Zeitschr.  f.  physiol.  Chem.,  36. 

3  Mayer  and  Neuberg,  Zeitschr.  f.  physiol.  Chem.,  29;  Schmiedeberg  u.  Meyer, 
ibid. ,3;  v.  Mering,  ibid,  6. 

*  Zeitschr.  f.  klin.  Med.,  47.     See  Chapter  XIV. 

5  Mayer,  Zeitschr.  f.  physiol.  Chem.,  32;  Lepine  and  Boulud,  Compt.  rend..,  133, 
134,  138. 

8  See  Bial,  Hofmeister's  Beitrage,  2,  and  v.  Leersum,  ibid.,  3. 

7  Zeitschr.  f.  physiol.  Chem.,  44. 

8  Ber.  d.  d.  chem.  Gesellsch.,  33. 


DISACCHAKIDES.  223 

with  yeast.  With  the  phcnylhydrazine  test  it  gives  crystalline  emu- 
pounds  which  are  not  sufficiently  characteristic  (Thierfelder,  P. 
Mayer  l).  By  the  action  of  3  mol.  phenylhydrazine  and  the  necessary 
amount  of  acetic  acid  upon  1  mol.  glucuronic  acid  at  40°  for  a  few  days, 
Neuber<;  and  Neimann  obtained  the  glucuronic-acid  osazone,  which 
was  very  similar  to  glucosazone  and  melted  at  200-205°.  With  p-brom- 
phenylhydrazine  hydrochloride  and  sodium  acetate,  glucuronic  acid  gives 
p-bromphenylhydrazine  glucuronate,  which  is  characterized  by  its  insolu- 
bility in  absolute  alcohol  and  by  a  very  prominent  levorotatory  action. 
This  compound  is  very  well  suited  for  the  detection  of  glucuronic  acid.2 
Dissolved  in  a  mixture  of  alcohol  and  pyridine  (0.2  gram  substance  in  4  cc. 
pyridine  and  6  cc.  alcohol)  the  rotation  is  7°  25',  which  corresponds  to 
(a)o=— 309°.  On  distillation  with  hydrochloric  acid,  glucuronic  acid 
yields  furfurol  and  also  carbon  dioxide,  and  on  this  behavior  Tollens 
and  Lefevre  3  have  based  their  quantitative  method  for  the  estimation 
of  glucuronic  acid. 

They  give  the  pentose  reactions  with  phloroglucin  or  orcin  and  hydrochloric 
acid,  and  also  a  good  reaction  with  naphthoresorcinol  and  hydrochloric  acid 
(see  page  218).  The  product  produced  herewith  is  soluble  in  ether  with  a  blue, 
bluish-violet  or  reddish-violet  color,  and  the  solution  shows  an  absorption  band 
somewhat  to  the  right  and  on  the  D-line.  According  to  Maxdel  and  Xeuberg  4 
this  reaction  is  not  characteristic  of  glucuronic  acid,  as  many  aldehyde  and  ketone 
acids  give  the  same  reaction;  still,  it  is  important  in  the  differentiation  of  the 
pentoses. 

Glucuronic  acid  is  best  prepared  from  euxanthic  acid,  which  decom- 
poses on  heating  it  with  water  to  120°  C.  for  several  hours.  The  nitrate 
from  the  euxanthon  is  concentrated  at  40°  C,  when  the  anhydride 
gradually  crystallizes  out.  On  boiling  the  mother-liquor  for  some  time 
and  evaporating  further,  the  crystals  of  the  lactone  are  obtained.  In 
regard  to  the  quantitative  estimation  of  glucuronic  acid  we  must  refer 
the  reader  to  the  works  of  Tollens  and  his  collaborators  and  of  Neuberg 
and  Neimann.5 

2.  Disaccharides. 

Some  of  the  varieties  of  sugar  belonging  to  this  group  occur  ready 
formed  in  nature.  Thus  we  have  saccharose  and  lactose.  Some,  on  the 
contrary,  such  as  maltose  and  isomaltose,  are  produced  by  the  partial 

1  Thierfelder,  Zeitschr.  f.  physiol.  Chem.,  11,  13,  15;  P.  Mayer,  ibid.,  29. 

2  See  Neuberg,  Ber  d.  d.  chem.  Gesellsch.,  32;  and  Mayer  and  Neuberg,  Zeitschr. 
f.  physiol.  Chem.,  29. 

3  Ber.  d.  d.  chem.  Gesellsch.,  40. 

4  Bioch.  Zeitschr.  13. 

6  Tollens,  Zeitschr.  f.  physiol.  Chem.,  41,  which  cites  also  the  older  work;  Neuberg 
and  Neimann,  ibid.,  44;  Neuberg,  ibid.,  45. 


224  THE  CARBOHYDRATES. 

hydrolytic  cleavage  of  complex  carbohydrates.     Isomaltose  is  also  obtained 
from  glucose  by  reversion  (see  page  225). 

The  disaccharides  or  hexobioses  are  to  be  considered  as  glucosides, 
each  of  which  is  derived  from  two  monosaccharides  with  the  exit  of  1 
molecule  of  water.  Corresponding  to  this,  their  general  formula  is 
C12H22O11.  On  hydrolytic  cleavage  and  the  addition  of  water  they 
yield  2  molecules  of  hexoses,  either  2  molecules  of  the  same  hexose  or 
one  each  of  two  different  hexoses.     Thus 

Saccharose -j- H2O  =  glucose + fructose ; 
Maltose      -f-  H2O  =  glucose + glucose ; 
Lactose      +H2O  =  glucose + galactose. 

The  configuration  of  the  disaccharides  has  not  been  positively  determined. 

The  fructose  turns  the  polarized  ray  more  to  the  left  than  the  glucose 
does  to  the  right;  hence  the  mixture  of  hexoses  obtained  on  the  cleavage 
of  saccharose  has  an  opposite  rotation  to  the  saccharose  itself.  On 
this  account  the  mixture  is  called  invert-sugar,  and  the  hydrolytic 
splitting  is  designated  as  inversion.  This  term,  "inversion,"  is  not  only 
used  for  the  splitting  of  saccharose,  but  is  also  used  for  the  hydrolytic 
cleavage  of  compound  sugars  into  monosaccharides.  The  reverse  reaction, 
whereby  monosaccharides  are  condensed  into  complex  carbohydrates,  is 
called  reversion. 

We  subdivide  the  disaccharides  into  two  groups,  first,  the  group  to 
which  saccharose  belongs,  where  the  members  do  not  have  the  property 
of  reducing  certain  metallic  oxides;  and  the  second  group,  to  which  the 
two  maltoses  and  lactose  belong,  the  members  acting  like  monosaccharides 
in  regard  to  the  ordinary  reduction  tests.  The  members  of  the  latter 
group  have  the  character  of  aldehyde  alcohols,  and  in  milk-sugar  the 
aldehyde  characteristics  are  connected  with  the  glucose  fraction. 

Saccharose,  or  cane-sugar,  occurs  extensively  distributed  in  the 
plant  kingdom.  It  occurs  to  the  greatest  extent  in  the  stalk  of  the  sugar- 
millet  and  sugar-cane,  the  roots  of  the  sugar-beet,  the  trunks  of  certain 
varieties  of  palms  and  maples,  in  carrots,  etc.  Cane-sugar  is  of  extraor- 
dinary great  importance  as  a  food  and  condiment. 

Saccharose  forms  large,  colorless  monoclinic  crystals.  On  heating  it 
melts  in  the  neighborhood  of  160°  C,  and  on  heating  more  strongly  it 
turns  brown,  forming  so-called  caramel.  It  dissolves  very  readily  in 
water,  and  according  to  Herzfeld,1  100  parts  of  saturated  saccharose 
solution  contain  67  parts  of  sugar  at  20°  C.  It  dissolves  with  difficulty 
in  strong  alcohol.  Cane-sugar  is  strongly  dextrorotatory.  The  specific 
rotation   is   only   slightly   modified   by   concentration,    but   is   markedly 

1  See  Tollens'  Handbuch  der  Kohlehydrate,  2.  Aufl.  1,  154. 


MALTOSE.  225 

changed  by  the  presence  of  other  inactive  substances.  The  specific 
rotation  is  (a)D= +66.5°. 

Saccharose  acts  indifferently  toward  Moore's  test  and  to  the  ordinary 
reduction  tests.  On  continuous  boiling  it  may  reduce  an  alkaline  copper 
solution,  perhaps  on  account  of  a  partial  inversion.  It  does  not  ferment 
directly,  but  only  after  inversion,  which  can  be  brought  about  by  an 
enzyme  (invertin)  contained  in  the  yeast.  An  inversion  of  cane-sugar  also 
takes  place  in  the  intestinal  canal.  Cane-sugar  does  not  combine  with 
hydrazines.  Concentrated  sulphuric  acid  blackens  cane-sugar  very 
quickly  even  at  the  ordinary  temperature,  and  anhydrous  oxalic  acid 
does  the  same  on  warming  on  the  water-bath.  Various  products  are 
obtained  on  the  oxidation  of  cane-sugar,  dependent  upon  the  variety  of 
oxidizing  agent  and  also  upon  the  intensity  of  the  action.  Saccharic  acid 
and  oxalic  acid  are  the  most  important  products. 

The  reader  is  referred  to  complete  text-books  on  chemistry  for'  the 
preparation  and  quantitative  estimation  of  cane-sugar. 

Maltose  (malt-sugar)  is  formed  in  the  hydrolytic  cleavage  of  starch 
by  malt  diastase,  saliva,  or  pancreatic  juice.  It  is  obtained  from  glyco- 
gen under  the  same  conditions  (see  Chapter  VII).  Maltose  is  also  pro- 
duced transitorily  in  the  action  of  sulphuric  acid  on  starch.  Maltose 
forms  the  fermentable  sugar  of  the  potato  or  grain  mash,  and  also  of  the 
beerwort. 

Maltose  crystallizes  with  one  molecule  water  of  crystallization  in  fine 
white  needles.  It  is  readily  soluble  in  water,  rather  easily  in  alcohol, 
but  insoluble  in  ether.  Its  solutions  are  dextrorotatory;  and  the  specific 
rotation  is  variable,  depending  upon  the  concentration  and  temperature, 
but  is  considerably  stronger  than  glucose,1  and  is  generally  given  as 
(o0d=+137  to  138°.  Maltose  ferments  readily  and  completely  with 
yeast,  and  acts  like  glucose  in  regard  to  the  reduction  tests.  It  yields 
phenylmaltosazone  on  warming  with  phenylhydrazine  for  1^  hours. 
This  phenylmaltosazone  melts  at  205°  C,  and  is  more  soluble  in  hot 
water  than  the  glucosazone.  Maltose  differs  from  glucose  chiefly  in  the 
following:  It  does  not  dissolve  as  readily  in  alcohol,  has  a  stronger  dex- 
trorotatory power,  and  has  a  feebler  reducing  action  on  Fehling's  solu- 
tion; 10  cc.  Fehling's  solution  are,  according  to  Soxhlet,2  reduced 
by  77.8  milligrams  anhydrous  maltose  in  approximately  1  per  cent  solution. 

Isomaltose.  This  variety  of  sugar,  as  has  been  shown  by  Fischer,3 
is  produced,  as  are  dextrin-like  products,  by  reversion,  and  by  the  action 
of  fuming  hydrochloric  acid  on  glucose.     A  re-formation  of  isomaltose 


1  See  Hoppe-Seyler-Thierfelder's  Handbuch,  S.  Aufl. 

2  Cited  from  Tollens'  Handbuoh.  der  Kohlehydrate,  2.  Aufl.  1,  154. 
5  Ber.  d.  deutsch.  ehem.  Gesellsch.,  23  and  28. 


226  THE   CARBOHYDRATES. 

and  other  sugars  from  glucose  can  also  Le  Lrought  about  by  means  of 
yeast  maltase  (Hill  and  Emmerling,  see  page  58).  It  is  also  formed, 
besides  ordinary  maltose,  in  the  action  of  diastase  on  starch  paste,  and 
occurs  in  beer  and  in  commercial  starch-sugar.  It  is  produced,  with 
maltose,  by  the  action  of  saliva  or  pancreatic  juice  (Kulz  and  Vogel) 
or  blood-serum  (Rohmann  *)  on  starch.  The  formation  of  isomaltose 
in  the  hydrolysis  of  starch  has  been  denied  by  many  investigators  because 
they  considered  isomaltose  only  as  contaminated  maltose.2 

Isomaltose  dissolves  very  readily  in  water,  has  a  pronounced  sweetish 
taste,  and  does  not  ferment,  or,  according  to  some,  only  very  slowly. 
It  is  dextrorotatory,  and  has  very  nearly  the  same  power  of  rotation  as 
maltose.  Isomaltose  is  characterized  by  its  osazone.  This  forms  fine 
yellow  needles,  which  begin  to  form  drops  at  140°  C.  and  melt  at  150- 
153°  C.  These  are  rather  easily  soluble  in  hot  water  and  dissolve  in  hot 
absolute  alcohol  much  more  readily  than  the  maltosazone.  Isomaltose 
reduces  copper  as  well  as  bismuth  solutions. 

Lactose  (milk-sugar).  As  this  sugar  occurs  exclusively  in  the  animal 
world,  in  the  milk  of  human  beings  and  animals,  it  will  be  treated  in  a 
following  chapter  (on  milk). 

3.  Colloid  Polysaccharides. 

If  we  exclude  the  not  well  known  trisaccharides  and  the  tetrasaccharide 
stachyose  this  group  includes  a  great  number  of  very  complex  carbo- 
hydrates which  occur  only  in  the  amorphous  condition,  or  at  least  not 
as  crystals  in  the  ordinary  sense.  Unlike  the  bodies  belonging  to  the 
other  groups,  these  have  no  sweet  taste.  Some  are  soluble  in  water, 
while  others  swell  up  therein,  especially  in  warm  water,  and  finally 
some  are  neither  dissolved  nor  visibly  changed.  Polysaccharides  are 
ultimately    converted    into    monosaccharides    by    hydrolytic    cleavage. 

The  polysaccharides  are  ordinarily  divided  into  the  following  groups: 
starches  with  the  dextrins,  plant  gums  and  mucilages,  and  the  celluloses. 

Starch  Group. 

Starch,  amylum  (CeHic-C^x.  This  substance  occurs  in  the  plant 
kingdom  very  extensively  distributed  in  the  different  parts  of  the  plant, 
especially  as  reserve  food  in  the  seed,  roots,  tubers,  and  trunks. 

Starch  is  a  white,  odorless,  and  tasteless  powder,  consisting  of  small 

1  Kulz  and  Vogel,  Zeitschr.  f.  Biologie,  31;    Rohmann,  Centralbl.  f.  d.  med.  Wis- 
eh.,  1893,  849. 

2  Brown  and  Morris,  Journ.  of  Chera.  Soc,  1895;  Chem.  News,  72.  See  also  Ost 
Ulrich,  and  Jalowetz,  Ref.  in  Ber.  d.  deutsch.  chem.  Gesellsch.,  28;  Ling  and  Baker, 
Journ.  of  Chem.  Soc,  1895;  Pottevin,  Chem.  Centralbl.,  1899,  II,  1023. 


STARCH.  227 

granules  which  have  ;i  stratified  structure  and  different  shape  and  size 
in  different  plants.  Starch  is  considered  insoluble  in  cold  water.  The 
grains  swell  up  in  warm  water  and  hurst,  yielding  a  paste. 

According  to  the  ordinary  opinion  the  starch  granules  consist  of  two 
different  substances,  starch  granulose  and  starch  cellulose  (v. 
Nageli),  the  first  of  which  turns  blue  with  iodine  and  forms  the  chief 
part  of  the  granule.  According  to  Maquenne  and  Roux 1  this  is 
not  the  fact.  According  to  them  the  starch  granule  consists  of  two 
constituents,  of  which  the  .first,  amylase,  forms  the  chief  mass  (80-85 
per  cent)  and  the  other,  amijlopectin,  forms  only  15-20  per  cent  of  the 
granule.  Amylopectin  is  not  identical  with  v.  Nageli's  starch  cellulose, 
and  the  above  investigators  consider  starch  cellulose  as  only  an  insoluble 
form  of  amylase.  The  amy  lose  can  occur  in  two  forms;  one,  which  is 
soluble,  is  colored  blue  by  iodine  and  is  immediately  transformed  into 
sugar  by  malt,  the  other  is  a  solid  substance,  which  is  not  colored 
with  iodine  and  resists  the  action  of  malt  infusion.  One  modification 
can  be  transformed  into  the  other. 

In  the  paste,  besides  amylopectin,  we  also  have  soluble  amylose,  and 
this  can,  by  a  process  called  retrogradation  by  Maquenne  and  Roux,  be 
transformed  into  the  solid  modification,  "  artificial  starch."  This 
solid  form  occurs  in  the  starch  granule,  and  is  identical  with  v.  Nageli's 
starch  cellulose.  As  the  starch  granules  are  directly  colored  blue  by 
iodine  they  must,  besides  this,  also  contain  soluble  amylose.  If  the  author 
understands  the  above  investigators  correctly  the  starch  granules  con- 
tain three  constituents,  namely,  soluble  amylose,  which  is  colored  blue 
by  iodine  (  =  starch  granulose),  insoluble  amylose,  which  is  not  colored 
by  iodine  (  =  starch  cellulose),  and  amylopectin. 

In  the  formation  of  paste  the  amount  of  amylose  is  not  the  essential 
but  rather  the  quantity  of  amylopectin.  The  amylopectin  is  a  slime- 
like substance,  insoluble  in  boiling  water  and  dilute  alkalies,  only  becom- 
ing pasty  therein,  and  not  colored  blue  by  iodine.  Accordingly  the 
paste  is  a  solution  cf  amylose  made  thick  by  amylopectin.  The  amylo- 
pectin, unlike  the  amylose,  is  only  slowly  transformed  into  sugar  with 
dextrin  formation.  Starch  is  insoluble  in  alcohol  and  ether.  On  heat- 
ing starch  with  water  alone,  or  heating  with  glycerin  to  190°  C,  or  on 
treating  the  starch  grains  with  6  parts  dilute  hydrochloric  acid  of  sp.  gr. 
1.06  at  ordinary  temperature  for  six  to  eight  weeks,2  it  is  converted  into 
soluble    starch     (amylodextrin,    amidulin).     Soluble    starch    is    also 


*v.  Nageli,  Botan.  Mitteil.,  1863;  Maquenne  and  Roux,  Compt.  rend.,  138,  140, 
142,  146,  and  Bull.  Soc.  chim.  de  Paris  (3),  33  and  35. 

2  See  Tollens'  Handb.,  191.  In  regard  to  other  methods,  see  Wr6blewsky,  Ber.  d. 
deutsch.  chem.,  Gesellsch.,  30;  Syniewski,  ibid. 


228  THE  CARBOHYDRATES. 

formed  as  an  intermediate  step  in  the  conversion  of  starch  into  sugar 
by  dilute  acids  or  diastatic  enzymes.  Soluble  starch  may  be  precipitated 
from  very  dilute  solutions  by  baryta-water.1 

Starch  granules  swell  up  and  form  a  pasty  mass  in  caustic  potash  or 
soda.  This  mass  gives  neither  Moore's  nor  Trommer's  test.  Starch 
paste  does  not  ferment  with  yeast.  The  most  characteristic  test  for  starch 
is  the  blue  coloration  produced  by  iodine  in  the  presence  of  hydriodic 
acid  or  alkali  iodides.2  This  blue  coloration  disappears  on  the  addition  of 
alcohol  or  alkalies,  and  also  on  warming,  but  reappears  again  on  cooling. 

On  boiling  with  dilute  acids  starch  is  converted  into  glucose.  In 
the  conversion  by  means  of  diastatic  enzymes  we  have,  as  a  rule,  besides 
dextrin,  maltose,  and  isomaltose,  onl}-  very  little  glucose.  We  are 
considerably  in  the  dark  as  to  the  kind  and  number  of  intermediate 
products  produced  in  this  process  (see  Dextrins) . 

Starch  may  be  detected  by  means  of  the  microscope  and  by  the 
iodine  reaction.  Starch  is  quantitatively  estimated,  according  to  Sachsse's 
method,3  by  converting  it  into  glucose  by  hydrochloric  acid  and  then 
determining  the  glucose  by  the  ordinary  methods. 

Inulin  (C6Hio05)x+H20,  occurs  in  the  underground  parts  of  many 
Composite,  especially  in  the  roots  of  the  Inula  helenium,  the  tubers 
of  the  Dahlia,  the  varieties  of  Helianthus,  etc.  It  is  ordinarily  obtained 
from  the  tubers  of  the  Dahlia. 

Inulin  forms  a  white  powder  similar  to  starch,  consisting  of  spheroid 
cr}rstals  which  are  readily  soluble  in  warm  water  without  forming  a  paste. 
It  separates  slowly  on  cooling,  but  more  rapidly  on  freezing.  Its  solu- 
tions are  levogyrate  and  are  precipitated  by  alcohol,  and  are  colored 
only  yellow  with  iodine.  Inulin  is  converted  into  the  levogyrate  mono- 
saccharide d-fructose  on  boiling  with  dilute  sulphuric  acid.  Diastatic 
enzymes  of  higher  animals  have  no,  or  only  a  ver}r  slight,  action  on  inulin.4 

According  to  Dean  5  inulin  occurs  in  combination  with  other  substances, 
levulins,  which  are  more  soluble  and  have  less  rotation.  He  suggests  that  we 
limit  the  name  inulin  to  that  carbohydrate  (or  mixture  of  carbohydrates),  which 
is  readily  precipitable  by  60  per  cent  alcohol  and  shows  a  specific  rotation  of 
(«)D=-3Sto40°. 

Lichenin  (moss-starch)  occurs  in  many  lichens,  especially  in  Iceland  moss. 
It  is  not  soluble  in  cold  water,  but  swells  up  into  a  jelly.  It  is  soluble  in  hot 
water,  forming  a  jelly  on  allowing  the  concentrated  solution  to  cool.  It  is  colored 
yellow  by  iodine  and  yields  glucose  on  boiling  with  dilute  acids.  Lichenin  is 
not  changed  by  diastatic  enzymes  such  as  ptyalin  or  amylopsin  (Nilson  6). 

1  In  regard  to  the  compounds  of  soluble  starch  and  dextrins  with  barium  hydroxide, 
Bee  Biilow,  Pfliiger's  Arch.,  62. 

2  See  Mylius,  Ber.  d.  deutsch.  chem.  Gesellsch.,  20,  and  Zeitsch.  f.  physioL  Chem.,  11. 

3  Tollens'  Handb.,  2.  Aufl.,  1,  187. 
*  To] lens'  Handbuch,  208. 

6  Amer.  Chem.  Journ.,  32. 
c  Upsala  Lakaref.  Forh.,  28. 


DEXTRINS.  229 

Glycogen.  This  carbohydrate,  which  stands  to  a  certain  extent 
between  starch  and  dextrin,  is  principally  found  in  the  animal  kingdom, 
hence  it  will  be  considered  in  a  subsequent  chapter  (on  the  liver). 

Dextrins  and  Gums. 

The  dextrins  stand  in  close  relation  to  the  starches,  and  are  formed 
therefrom  as  intermediate  products  by  the  action  of  acids  or  diastatic 
enzymes.  They  yield  as  final  products  only  hexoses,  indeed  only  glu- 
cose, on  complete  hydrolysis.  The  vegetable  gums,  the  vegetable 
mucilages  and  the  pectin  bodies,  which  all  stand  close  to  the  hemicellu- 
loses,  yield,  on  the  contrary,  abundance  of  pentose  and,  among  the  hex- 
oses. galactose  is  very  often  found. 

Dextrin  (starch-gum,  British  gum),  is  produced  on  heating  starch  to 
200-210°  C.,  or  by  heating  starch,  which  has  previously  been  moistened 
with  water  containing  a  little  nitric  acid,  to  100-110°  C.  Dextrins  are 
also  produced  by  the  action  of  dilute  acids  and  diastatic  enzymes  on 
starch.  There  have  been  numerous  investigations  as  to  the  steps 
involved  in  the  last-mentioned  process,  but  they  have  led  to  conflicting 
views.  One  of  these,  which  used  to  be  generally  accepted,  is  as  follows: 
The  first  product,  which  gives  a  blue  color  with  iodine,  is  soluble  starch 
or  amylodextrin,  which  on  further  hydrolytic  cleavage  yields  sugar  and 
erythrodextrin,  which  is  colored  red  by  iodine.  On  further  cleavage  of 
this  erythrodextrin  more  sugar  and  a  dextrin,  achroodextrin,  which  is 
not  colored  by  iodine,  is  formed.  From  this  achroodextrin  after  suc- 
cessive splittings  Ave  have  sugar  and  dextrins  of  lower  molecular  weights 
formed,  until  finally  we  have  sugar  and  a  dextrin,  maltodextrin,  which 
refuses  to  split  further,  as  final  products.  The  views  are  rather  contra- 
dictory in  regard  to  the  number  of  dextrins  which  occur  as  intermediate 
steps.  The  sugar  formed  is  maltose  (or  in  first  place  isomaltose),  and 
only  very  little  glucose  is  produced.  Another  view  is  that  first  several 
dextrins  are  formed  consecutively  in  the  successive  splittings,  by  hydra- 
tion, and  then  finally  the  sugar  is  formed  by  the  splitting  of  the  last 
dextrin.  According  to  Moreau,  in  the  first  stages  of  saccharification 
amylodextrin,  erythrodextrin,  achroodextrin  and  sugar  are  formed  sim- 
ultaneously. Other  investigators,  especially  Syniewski,  have  recently 
suggested  other  views  on  the  subject.1 

This  question  has  taken  another  direction  by  the  investigations  c£ 

1  In  regard  to  the  various  views  on  the  theories  of  the  saccharification  of  starch, 
see  Musculus  and  Gruber,  Zeitschr.  f.  physiol.  Chem.,  2;  Lintner  and  Dull,  Ber.  d.  d. 
chem.  Gesellsch.,  26  and  28;  Brown  and  Heron,  Journ.  of  Chem.  Soc,  1S79;  Brown 
and  Morris,  ibid.,  1885  and  1S89;  Moreau,  Biochem.  Centralbl.,  3,  648;  Syniewski, 
Annal.  d.  Chem.  u.  Pliarm.,  309,  and  Chem.  Centralbl.,  1902,  2. 


230  THE   CARBOHYDRATES. 

Maquenne,  mentioned  above.  According  to  him  the  amylose  passes 
directly  into  maltose  without  the  formation  of  dextrin  by  the  action  of 
malt  infusion.  The  dextrins  produced  are  only  formed  from  the  amylo- 
pectin,  which  does  not  undergo  saccharification  with  freshly  prepared 
malt  infusions,  but  only  with  older  or  especially  active  infusions.  This 
also  explains  why  in  the  older  investigations  the  saccharification  was 
only  about  80  per  cent  while  Maquenne  has  been  able  to  completely 
convert  the  starch  into  sugar  by  enzymotic  action. 

The  various  dextrins  are  very  hard  to  isolate  as  chemical  individuals 
and  to  separate  from  each  other.  Young  i  has  tried  their  separation 
by  means  of  neutral  salts,  especially  ammonium  sulphate,  and  Moreau 
by  the  aid  of  a  baryta-alcohol  method.  We  cannot  enter  into  the  dif- 
ferences as  to  the  dextrins  so  separated,  and  only  the  characteristic 
properties  and  reactions  will  be  given  for  the  dextrins  in  general. 

The  dextrins  appear  as  amorphous,  white  or  yellowish-white  powders 
which  are  readily  soluble  in  water.  Their  concentrated  solutions  are 
viscid  and  sticky,  like  gum  solutions.  The  dextrins  are  dextrogyrate. 
They  are  insoluble  or  nearly  so  in  alcohol,  and  insoluble  in  ether.  Watery 
solutions  of  dextrins  are  not  precipitated  by  basic  lead  acetate.  Dex- 
trins dissolve  cupric  hydroxide  in  alkaline  liquids,  forming  a  beautiful 
blue  solution,  which,  as  is  generally  admitted,  is  reduced  by  pure  dex- 
trins. According  to  Moreau  pure  dextrin  has  no  reducing  action.  The 
dextrins  are  not  directly  fermentable. 

Schardinger  has  discovered  a  bacillus  which  forms  acetone  from 
starch  and  which  is  especially  useful  for  the  perparation  of  crystalline 
cleavage  products  from  starch.  He  obtained  two  crystalline  substances, 
dextrin  a  and  f3,  which  are  not  fermentable  by  yeast  and  on  hydrolysis 
with  acid  yield  glucose.  For  the  a-dextrin  Pringsheim  and  Langhans 
have  determined  the  formula  (CeHinOs^  while  Biltz  and  Truthe  2 
found  the  formula  (CgHioCs^g  for  the  /3-dextrin. 

The  vegetable  gums  are  soluble  in  water,  forming  solutions  which  are  viscid 
but  may  be  filtered.  We  designate,  on  the  contrary,  as  vegetable  mucilages 
those  varieties  of  gum  which  do  not  or  only  partly  dissolve  in  water,  and  which 
swell  up  therein  to  a  greater  or  less  extent.  The  natural  varieties  of  gum  and 
mucilage,  to  which  belong  several  generally  known  and  important  substances, 
such  as  gum  arabic,  wood-gum,  cherry-gum,  salep,  and  quince  mucilage,  and 
probably  also  the  little-studied  pectin  substances,  will  not  be  treated  in  detail, 
because  of  their  unimportance  from  a  physiological  standpoint. 

1  Journ.  of  Physiol.,  22,  which  contains  the  older  researches  of  Nasse,  Kriiger 
Neumeister,  Pohl,  and  Halliburton.     Moreau,  1.  c. 

-  Schardinger,  Centralbl.  f.  Bak.  u.  Parasitenkunde,  II,  22,  98  (1909);  29,  118  (1911); 
Pringsheim  and  Langhans,  Ber.  d.  d.  Chem.  Gesellsch.,  45,  2533  (1912);  Biltz  and 
Truthe,  ibid.,  46,  1377  (1913). 


CELLULOSE.  231 

The  Cellulose  Group  (C6Hi0O5)x. 

Cellulose  is  that  carbohydrate,  or  perhaps  more  correctly,  mixture 
of  carbohydrates,  which  forms  the  chief  constituent  of  the  walls  of  the 
plant-cells.  This  is  true  for  at  least  the  walls  of  the  young  cells,  while 
in  the  walls  of  the  older  cells  the  cellulose  is  extensively  incrusted  with  a 
substance  called  lignin,  and  with  many  other  cellulose  derivatives  and 
compounds. 

The  true  celluloses  are  characterized  by  their  great  insolubility.  They 
are  insoluble  in  cold  or  hot  water,  alcohol,  ether,  dilute  acids,  and  alkalies. 
We  have  only  one  specific  solvent  for  cellulose,  and  that  is,  an  ammo- 
niacal  solution  of  copper  oxide  called  Schweitzer's  reagent.  The 
cellulose  may  be  precipitated  from  this  solvent  by  the  addition  of  acids, 
and  obtained  as  an  amorphous  powder  after  washing  with  water. 

Cellulose  is  converted  into  a  substance,  so-called  amyloid,  which 
gives  a  blue  coloration  with  iodine,  by  the  action  of  concentrated  sul- 
phuric acid.  With  oxidizing  agents  (nitric  acid,  etc.)  oxycelluloses  are 
produced.  By  the  action  of  strong  nitric  acid  or  a  mixture  of  nitric 
acid  and  concentrated  sulphuric  acid,  celluloses  are  converted  into  nitric- 
acid  esters  or  nitrocelluloses,  which  are  highly  explosive  and  have  found 
great  practical  use. 

The  ordinary  celluloses  when  treated  at  the  ordinary  temperature 
with  strong  sulphuric  acid  and  then  boiled  for  some  time  after  diluting 
with  water  are  coverted  into  glucose.  In  this  case  it  must  be  observed, 
according  to  Maqtjenne,  that  it  is  not  maltose  that  is  produced  as  an 
intermediate  step,  but  another  disaccharide,   called  cellose  or  cellobiose. 

The  cellulose,  at  least  in  part,  undergoes  decomposition  in  the  intestinal 
tract  of  man  and  animals.  A  closer  discussion  of  the  nutritive  value 
of  cellulose  will  be  given  in  a  future  chapter  (on  digestion).  The  great 
importance  of  the  carbohydrates  in  the  animal  economy  and  to  animal 
metabolism  will  also  be  given  in  the  following  chapters. 

Hemicelluloses  are,  according  to  E.  Schulze,1  thoseconstituents  of  the  cell- 
wall  related  to  cellulose  which  differ  from  the  ordinary  cellulose  by  dissolving 
on  heating  with  strongly  diluted  mineral  acids,  such  as  1.25  per  cent  sulphuric 
acid,  and  of  yielding  arabinose,  xylose,  galactose,  and  mannose  instead  of  glucose. 
Those  hemicelluloses  which  serve  partly  as  reserve  food  and  partly  as  support- 
substance,  are  very  widely  distributed  in  the  plant  kingdom.  It  must  be  recalled 
that  according  to  Bierry  and  Giaja  2  the  digestive  organs  of  different  inverte- 
brates (Helix,  Astacus,  Maja.  Hommarus)  contain  enzymes  which  have  an 
energetic  splitting  action  upon  such  polysaccharides  as  well  as  on  the  natural 
celluloses. 


1  E.  Schulze,  Zeitschr.  f.  physiol.  Chem.,  16  and  19,  with  Castro,  ibid.,  36. 
2Bioch.  Zeitschr.,  40,  370  (1912). 


CHAPTER  IV. 
ANIMAL  FATS  AND  PHOSPHATIDES. 

1.  Neutral  Fats  and  Fatty  Acids. 

The  fats  form  the  third  chief  group  of  the  organic  food  of  man  and 
animals.  They  occur  very  widely  distributed  in  the  animal  and  plant 
kingdoms.  Fat  occurs  in  all  organs  and  tissues  of  the  animal  organism, 
though  the  quantity  may  be  so  variable  that  a  tabular  exhibit  of  the 
amount  of  fat  in  different  organs  is  of  little  interest.  The  marrow  con- 
tains the  largest  quantity,  having  over  96  per  cent.  The  three  most 
important  deposits  of  fat  in  the  animal  organism  are  the  intermuscular 
connective  tissue,  the  fatty  tissue  in  the  abdominal  cavity,  and  the 
subcutaneous  connective  tissues.  In  plants,  the  seeds  and  fruit  and  in 
certain  instances  also  the  roots,  are  rich  in  fat.  Fat  also  occurs  deposited, 
during  the  winter's  rest,  in  the  trunks  of  trees. 

The  fats  consist  almost  entirely  of  so-called  neutral  fats,  with  only 
very  small  quantities  of  fatty  acids.  The  neutral  fats  are  esters  of  the 
triatomic  alcohol,  glycerin,  with  monobasic  fatty  acids.  These  esters 
are  triglycerides;  that  is,  the  hydrogen  atoms  of  the  three  hydroxyl 
groups  of  the  glycerin  are  replaced  by  the  fatty-acid  radicals,  and  their 
general  formula  is  therefore,  C3H5.O3.R3-  The  animal  fats  consist 
chiefly 'of  esters  of  the  three  fatty  acids,  stearic,  palmitic,  and  oleic  acids. 
In  certain  fats,  especially  in  milk-fat,  glycerides  of  fatty  acids  such  as 
butyric,  caproic,  caprylic,  and  capric  acids  also  occur  in  considerable 
amounts.  Besides  the  above-mentioned  ordinary  fatty  acids,  stearic, 
palmitic,  and  oleic  acids,  we  also  find  in  human  and  animal  fat,  exclusive 
of  certain  fatty  acids  only  little  studied,  the  following  non-volatile  fatty 
acids,  as  glycerides,  namely,  lauric  acid,  C12H24O2,  myristic  acid,  C14H28O2, 
and  arachidic  acid,  C20H40O2.  Of  the  unsaturated  fatty  acids,  besides 
oleic  acid,  we  probably  also  have  in  small  quantities  glycerides  of  acids 
of  the  linolic  acid  series  C„H2a-402  and  of  the  linolenic  acid  series, 
C„H2O-G-02.  In  this  case  the  question  can  be  raised  whether  or  not 
tin -c  acids  are  not  derived  from  the  phosphatides  mixed  with  the  fats, 
in  the  plant  kingdom  triglycerides  of  other  fatty  acids,  such  as  lauric 
acid,    myristic    acid,    linoleic   acid,    erucic   acid,    etc.,    sometimes   occur 

232 


NEUTRAL  FATS.  233 

abundantly.  Besides  these,  oxyacids  and  high  molecular  alcohols  have 
been  found  in  many  plant  fats.  The  extent  to  which  traces  of  I 
oxyacids  occur  in  the  animal  kingdom  has  not  been  thoroughly  inves- 
tigated, but  the  occurrence  of  monoxystearic  acid  seems  to  have  been 
proved.1  The  occurrence  of  high  molecular  alcohols,  although  ordinarily 
only  in  small  amounts,  has  on  the  contrary  been  positively  shown  in 
animal  fat. 

The  animal  fats  are  of  the  greatest  interest  and  consist  of  a  mixture  of 
varying  quantities  of  tristearin,  tripalmitin,  and  triolein,  having 
an  average  elementary  composition  of  C  76.5,  H  12.0,  and  O  11.5  per 
cent.  It  must  be  remarked  that  in  animal  fat  (mutton  and  beef  tallow) 
as  well  as  in  plant  fat  (olive-oil)  mixed  triglycerides,  such  as  dipalmityl- 
olein,  distearyl-palmitin  and  distearyl-olein,  occur,  and  that  these  mixed 
glycerides  may  also  be  prepared  synthetically.2 

Fats  from  different  species  of  animals,  and  even  from  different  parts 
of  the  same  animal,  have  an  essentially  different  consistency,  depending 
upon  the  relative  amounts  of  the  different  individual  fats  present.  In 
solid  fats — as  tallow — tristearin  and  tripalmitin  are  in  excess,  while 
the  less  solid  fats  are  characterized  by  a  greater  abundance  of  triolein. 
This  last-mentioned  fat  is  found  in  greater  quantities  proportionally 
in  cold-blooded  animals,  and  this  accounts  for  the  fact  that  the  fat  of 
these  animals  remains  fluid  at  temperatures  at  which  the  fat  of  warm- 
blooded animals  solidifies.  Human  fat  from  different  organs  and  tissues 
contains,  in  full  numbers,  67-85  per  cent  triolein.3  The  melting-point 
of  different  fats  depends  upon  the  composition  of  the  mixtures,  and  it 
not  only  varies  for  fat  from  different  tissues  of  the  same  animal,  but  also 
for  the  fat  from  the  same  tissues  in  various  kinds  of  animals.4 

Neutral  fats  are  colorless  or  yellowish,  and,  when  perfectly  pure, 
ordorless  and  tasteless.  They  are  lighter  than  water,  on  which  they 
float  when  in  a  molten  condition.  They  are  insoluble  in  water,  dissolve 
in  boiling  alcohol,  but  separate  on  cooling — often  in  crystals.  They  are 
easily  soluble  in  ether,  benzene,  chloroform,  carbon  disulphide  and  petro- 
leum ether.  The  fluid  neutral  fats  give  an  emulsion  when  shaken  with 
a  solution  of  gum  or  albumin.     With  water  alone  they  give  an  emulsion 


1  Erben,  Zeitschr.  f.  physiol.  Chem.,  30;  Bernert,  Arch,  f .  exp.  Path.  u.  Pharm.,  40. 
2Guth,  Zeitschr.  f.  Biologie,  44;    W.  Hansen,  Arch.  f.  Hygiene,  42;    Holde  and 
Stange,  Ber.  d.  d.  chem.  Gesellsch.,  34;  Kreis  and  Hafner,  ibid.,  36. 

3  See  Knopfelmacher,  "Untersuch.  iiber  das  Fett  im  Sauglingsalter,"  etc.,  Jarhbuch 
f.  Kinderheilkunde  (X.  F.),  45,  which  also  contains  the  older  literature;  Jaeckle, 
Zeitschr.  f.  physiol.  Chem.,  36. 

4  According  to  Gilkin  (Ber.  d.  d.  chem.  Gesellsch.,  41)  the  fat  from  bone-marrow 
and  also  other  fats  of  animal  and  plant  origin  contain  iron,  which  cannot  be  removed 
by  water  containing  hydrochloric  acid. 


234  ANIMAL  FATS  AND  PHOSPHATIDES. 

only  after  vigorous  and  prolonged  shaking,  but  the  emulsion  is  not  per- 
sistent. The  presence  of  some  soap  causes  a  very  fine  and  permanent 
emulsion  to  form  easily.  Fat  produces  spots  on  paper  which  do  not 
disappear;  it  is  not  volatile;  it  boils  at  about  300°  C.  with  partial  decom- 
position, and  burns  with  a  luminous  and  smoky  flame.  The  fatty  acids 
have  most  of  the  above-mentioned  properties  in  common  with  the  neutral 
fats,  but  differ  from  them  in  being  soluble  in  alcohol-ether,  in  having 
an  acid  reaction,  and  by  not  giving  the  acrolein  test.  The  neutral  fats 
generate  a  strong  irritating  vapor  of  acrolein,  due  to  the  decomposition 
of  glycerin,  C3H5(OH)3— 2H20  =  C2H3.CHO,  when  heated  alone,  or 
more  easily  when  heated  with  potassium  bisulphate  or  with  other  dehy- 
drating substances. 

The  neutral  fats  may  be  split  by  the  addition  of  the  constituents  of 
water  according  to  the  following  equation: 

C3H5(OR)34-3H20  =  CVH5(OH)34-3HOR. 

This  splitting  may  be  produced  by  the  pancreatic  enzjine  and  other 
enzymes  occurring  in  the  animal  and  vegetable  kingdoms, 'for  example, 
the  castor  lipase.  The  reverse  action,  namely,  the  synthesis  of  fatty  acid 
esters,  can  be  brought  about  by  enzymes,  such  as  pancreatic  lipase  (see 
page  60).  The  cleavage  of  the  neutral  fats  can  also  be  accomplished 
by  superheated  steam  or  by  dilute  acids.  We  most  frequently  decompose 
the  neutral  fats  by  boiling  them  with  not  too  concentrated  caustic  alkali, 
or.  still  better  (in  biochemical  researches),  with  an  alcoholic  potash  solu- 
tion or  with  sodium  alcoholate.  By  this  procedure,  which  is  called  sapon- 
ification, the  alkali  salts  of  the  fatty  acids  (soaps)  are  formed.  If  the 
saponification  is  made  with  lead  oxide,  then  lead  plaster,  the  lead  salt  of 
the  fatty  acids  is  produced.  By  saponification  is  to  be  understood  not 
only  the  cleavage  of  neutral  fats  by  alkalies,  but  also  the  splitting  of  neutral 
fats  into  fatty  acids  and  glycerin  in  general. 

On  keeping  fats  for  a  long  time  in  contact  with  air  they  undergo  a 
change,  becoming  yellow  in  color  and  acid  in  reaction,  and  they  develop 
an  unpleasant  odor  and  taste,  becoming  rancid.  In  this  change  a  part 
of  the  fat  is  split  into  fatty  acids  and  glycerin,  and  then  an  oxidation 
of  the  free  fatty  acids  takes  place,  producing  volatile  bodies  of  an 
unpleasant  odor. 

The  three  most  important  fats  of  the  animal  kingdom  are  stearin, 

valmitin,  and  olein. 

CH2.O.Ci8H350 

Stearin,  or  tristearin,  CsyHnoOe^CH.O.CisHssO,  occurs  especially  in 

CH2.O.CisH350 

the  solid  varieties  of  tallow  but  also  in  the  vegetable  fats.     Stearic  acid, 

C18H:jf;02,  is  found  in  the  free  state  in  decomposed  pus,  in  the  expectora- 


PALM  IT  IN.  235 

tions  in  gangrene  of  the  lungs,  and  in  cheesy  tuberculous  masses.  It 
occurs  as  lime  soap  in  excrement  and  adipocere,  and  in  this  last  product 
also  as  an  ammonium  soap.  It  also  exists  as  alkali  soap  in  the  blood, 
bile,  transudations  and  pus,  and  in  the  urine  to  a  slight  extent. 

Stearin  is  the  hardest  and  most  insoluble  of  the  three  ordinary  neutral 
fats.  It  is  nearly  insoluble  in  cold  alcohol,  and  soluble  with  great  dif- 
ficulty in  cold  ether  (225  parts).  It  separates  from  warm  alcohol  on 
cooling  as  rectangular,  and  less  frequently  as  rhombic  plates.  The  opinions 
regarding  the  melting-point  are  somewhat  varied.  Pure  stearin,  ac- 
cording to  Heintz,1  melts  transitorily  at  55°  and  permanently  at  71.5°. 
The  stearin  from  the  fatty  tissues  (not  pure)  melts  at  63°  C. 
CH3 

Stearic  acid,  (CH2)i6,  crystallizes  (on  cooling  from  boiling  alcohol)  in 
COOH 
large,  shining,  long  rhombic  scales  or  plates.     It  is  less  soluble  than  the 
other  fatty  acids  and  melts  at  68.2°  C.2     Its  barium  salt  contains  19.49 
per  cent  barium,  and  its  silver  salt  contains  27.59  per  cent  silver. 

CH2.O.C16H3iO 

Palmitin,  or  tripalmitin,  C5iH9s06,  =  CH.O.Ci6H310.     Of  the  two  solid 

CH2.O.Ci6H3iO 
varieties  of  fats,  palmitin  is  the  one  which  occurs  in  predominant  quan- 
tities in  human  fat  (Langer3).  Palmitin  is  present  in  all  animal  fats 
and  in  several  kinds  of  vegetable  fat.  A  mixture  of  stearin  and  palmitin 
■was  formerly  called  margarin.  As  to  the  occurrence  of  palmitic  acid, 
C16H32O2,  about  the  same  remarks  apply  as  to  stearic  acid.  The  mixture 
of  these  two  acids  has  been  called  margaric  acid,  and  this  mixture  occurs 
— often  as  very  long,  thin,  crystalline  plates — in  old  pus,  in  expectora- 
tions from  gangrene  of  the  lungs,  etc. 

Palmitin  crystallizes,  on  cooling  from  a  warm  saturated  solution  in 
ether  or  alcohol,  in  starry  rosettes  of  fine  needles.  The  mixture  of  pal- 
mitin and  stearin,  called  margarin,  crystallizes,  on  cooling  from  a  solu- 
tion, as  balls  or  round  masses  which  consist  of  short  or  long,  thin  plates 
or  needles  which  often  appear  like  blades  of  grass.  Palmitin,  like  stearin, 
has  a  variable  melting-  and  solidifying-point,  depending  upon  the  way  it 
has  been  previously  treated.  The  melting-point  is  often  given  as  62°  C, 
but  some  investigators 4  claim  that  it  melts  at  50.5°  C,  solidifies  on 
further  heating,  and  melts  again  at  66.5°  C. 


1  Annal.  d.  Chem.  u.  Pharm.,  92. 

2  According  to  Carlinfanti  and  Levi-Malvano,  Chem.  Centralbl.,  1910. 

3  Monatshefte  f.  Chem.,  2;  see  also  Jaeckle,  Zeitschr.  f.  physiol.  Chem.,  36. 
*  R.  Benedikt,  Analyse  der  Fette,  3.  Aufl.,  1897,  p.  44. 


236  ANIMAL  FATS  AND  PHOSPHATIDES. 

CH3 
Palmitic  acid,  (CHo)i4,  crystallizes  from  an  alcoholic  solution  in  tufts 
COOH 
of  fine  needles.     It  melts  at  61°  C.;1   still  the  admixture  with  stearic  acid, 
essentially  changes  them  elting-  and  solidifying-points  according  to  th 
relative  amounts  of  the  two  acids.      Palmitic   acid  is  somewhat  more 
soluble  in  cold  alcohol  than  stearic  acid;   but  they  have  about  the  same 
solubility  in  boiling  alcohol,  ether,  chloroform,  and  benzene.     Its  barium 
salt  contains  21.17  per  cent  barium,  and  silver  salt  contains  29.72  per 
cent  silver. 

CH2.O.C18H33O 
Olein,  or  triolein,  C57Hi04O6,  =  CH.O.Ci8H33O,  is  present  in  all  animal 

CH2.O.C18H33O 
fats,   and  in  greater  quantities  in  vegetable  fats.     It  is  a  solvent  for 
stearin  and  palmitin.     The  oleic  acid  (elaic  acid),  C18H34O2,  as  soaps, 
probably  has  about  the  same  occurrence  as  the  other  fatty  acids. 

Olein  is,  at  ordinary  temperatures,  a  nearly  colorless  oil  of  a  specific 
gravity  of  0.914,  without  odor  or  marked  taste,  and  solidifies  in  crystalline 
needles  at  -6°  C.  It  becomes  rancid  quickly  if  exposed  to  the  air.  It 
dissolves  with  difficulty  in  cold  alcohol,  but  more  easily  in  warm  alcohol 
or  in  ether.  It  is  converted  into  its  isomer,  elaidin,  by  nitrous  acid. 
CH3 

(CH2)7 
r*vx 
Oleic  acid,  =,„,  is  an  unsaturated  acid  of  the  series  CnH„-202,  and 

(CH2)7 
COOH 
correspondingly  takes  up  two  halogen  atoms,  i.e.,  iodine,  at  the  double 
bondage,  a  factor  which  is  the  basis  of  v.  Hubl's  method  for  determining 
the  iodine  equivalent.  On  taking  up  hydrogen,  which  can  be  accomplished 
by  heating  with  hydroiodic  acid  and  amorphous  phosphorus,  it  is  trans- 
formed into  the  corresponding  saturated  acid,  namely,  stearic  acid.  On 
oxidation  the  double  bonds  are  satisfied  by  2HO  groups,  and  dioxystearic 
acid,  CH3(CH2)7CHOH.CHOH(CH2)7COOH,  is  formed.  Oleic  acid 
readily  undergoes  oxidation  in  the  air  with  the  formation  of  acid  products, 
and  the  occurrence  of  monoxystearic  acid,  found  in  animal  fats  in  certain 
instances,  can  be  explained  by  this  oxidation.  Oleic  acid  on  heating 
yields,  besides  volatile  fatty  acids,  sebacic  acid,  C10H18O4,  which  melts 
at  127°C;  and  with  nitrous  acid  it  is  transformed  into  its  isomer,  solid 
elaidic  acid,  which  melts  at  45°  C. 

Oleic  acid  forms  at  ordinary  temperature  a  colorless,  tasteless,  and 

1Carlinfanti  and  Levi-Malvano.  Chem.  Centralbl.  1910. 


OLEIC  ACID.  237 

odorless  oily  liquid  which  solidifies  in  crystals  at  about  4°  C,  which 
latter  melt  at  14°  C.  Oleic  acid  is  insoluble  in  water,  but  dissolves 
in  alcohol,  ether,  chloroform  and  petroleum  ether.  With  concentrated 
sulphuric  acid  and  some  cane-sugar  it  gives  a  beautiful  red  or  reddish- 
violet  liquid  whose  color  is  similar  to  that  produced  in  Pettenkofer's 
test  for  bile-acids.  If  a  solution  of  oleic  acid  in  glacial  acetic  acid  is  treated 
with  a  little  chromic  acid  (in  glacial  acetic  acid)  and  then  with  concen- 
trated sulphuric  acid,  the  green  solution  gradually  becomes  violet  or 
cherry-red,  and  shows  two  characteristic  absorption  bands  in  the  green, 
one  a  broad  band  near  the  blue  and  a  second  but  fainter  band  near  the 
yellow  (Lifschutz).1  The  barium  salt  of  oleic  acid  contains  19. G5  per 
cent  barium  and  the  silver  salt  27.73  per  cent  silver. 

If  the  watery  solution  of  the  alkali  compounds  of  oleic  acid  is  pre- 
cipitated with  lead  acetate,  a  white,  tough,  sticky  mass  of  lead  oleate  is 
obtained,  which  is  not  soluble  in  water  and  only  slightly  in  alcohol,  but  is 
soluble  in  ether.  This  salt  is  more  easily  soluble  in  benzene  than  the  lead 
salts  of  stearic  and  palmitic  acids,  and  this  behavior  of  the  lead  salts 
toward  ether  and  benzene  is  made  use  of  in  separating  oleic  acid  from 
the  other  fatty  acids. 

An  acid  related  to  oleic  acid,  doeglic  acid,  which  is  solid  at  4°  C,  liquid  at 
16°  C,  and  soluble  in  alcohol,  is  found  in  the  blubber  of  the  Balcena  rostrata. 
According  to  Bull  this  acid  is  probably  only  a  mixture  of  oleic  acid  and  another 
acid — gadoleic  acid,  C^oH.-sOo,  having  a  melting-point  of  +24.5°  C,  and  occurring 
in  cod-liver  oil,  herring  oil  and  in  whale  blubber.  In  addition  to  this  acid  Bull 
found  in  cod-liver  oil,  besides  myristic,  palmitic,  oleic  and  erucic  acids,  another 
acid,  having  the  formula  CieEUCK.  According  to  Ellmer  2  the  most  abundant 
acid  (80-90  per  cent)  in  cod-liver  oil  is  therapinic  acid,  Ci8H2802  which  is  changed 
into  stearic  acid  by  reduction  and  jecoleic  acid,  which  seems  to  be  identical  with 
Bull's  gadoleic  acid.  Kurbatoff  has  demonstrated  the  presence  of  linoleic 
acid  in  the  fat  of  the  silurus,  sturgeon,  seal,  and  certain  other  animals.  Drying 
fats  have  also  been  found  by  Amthor  and  Zink  3  in  hares,  wild  rabbits,  wild 
boar,  and  mountain-cock. 

To  detect  the  presence  of  fat  in  an  animal  fluid  or  tissue  the  fat  must 
first  be  shaken  out  or  extracted  with  ether.  After  the  evaporation  of 
the  ether  the  residue  is  tested  for  fat  and  fatty  acids.  The  neutral 
fats  are  differentiated  from  the  fatty  acids  by  the  acrolein  test,  and  the 
fatty  acids  by  the  fact  that  their  solution  in  a  mixture  of  alcohol  and 
ether  has  an  acid  reaction.  In  separating  the  fats  from  cholesterin 
and  other  non-saponifiable  substances,  as  well  as  for  the  determination 
of  the  kind  of  the  various  fatty  bodies,  they  are  saponified  with  caustic 
alkali,  alcoholic  potash,  or  with  sodium  alcoholate.  In  regard  to  these 
operations,  as  well  as  the  further  investigation  and  the  separation  of  the 


1  Zeitschr.  f.  physiol.  Chem.,  56. 

2  Bull,  Ber.  d.  d.  chem.,  Gesellsch.,  39;  Ellmer,  Bioch.  Zeitschr.,  9. 

3  Kurbatoff,  Maly's  Jahresb.,  22;  Amthor  and  Zink,  Zeitschr.  f.  anal.  Chem.,  36. 


238  ANIMAL  FATS  AND  PHOSPHATIDES. 

various  fatty  acids  from  each  other,  we   must  refer  to  more   complete 
hand-books. 

In  addition  to  the  methods  already  suggested  there  are  other  chemical  methods 
which  are  important  in  investigating  fats.  Besides  ascertaining  the  melting- 
and  congealing-point  we  also  determine  the  following:  1.  The  acid  equivalent, 
which  is  a  measure  of  the  amount  of  fatty  acids  in  a  fat,  is  determined  by  titrat- 
ing the  fat  dissolved  in  alcohol-ether  with  N/10  alcoholic  caustic  potash,  using 
phenolphthalein  as  indicator.  2.  The  saponification  equivalent,  which  gives 
the  milligrams  of  caustic  potash  uniting  with  the  fatty  acids  in  the  saponification 
of  1  gram  fat  with  N/2  alcoholic  caustic  potash.  3.  Reichert-Meissl's  equivalent, 
which  gives  the  quantity  of  volatile  fatty  acids  contained  in  a  given  amount 
of  neutral  fat  (5  grams).  The  fat  is  saponified,  then  acidified  with  mineral  acid, 
and  distilled,  whereby  the  volatile  fatty  acids  pass  over;  the  distillate  is  then 
titrated  with  alkali.  4.  Iodine  equivalent  is  the  quantity  of  iodine  absorbed  by 
a  certain  amount  of  the  fat  by  addition.  It  is  chiefly  a  measure  of  the  quantity 
of  unsaturated  fatty  acids,  principally  oelic  acid  or  olein,  in  the  fat.  Other  bodies, 
such  as  cholesterin,  may  also  absorb  iodine  or  halogens.  The  iodine  equiva- 
lent is  generally  determined  according  to  the  method  suggested  by  v.  Hubl. 
5.  The  acetyl  equivalent  measures  the  quantity  of  those  constituents  of  fats  which 
contain  OH  groups,  and'  is  found  by  converting  these  bodies  (oxyfatty  acids, 
alcohols  and  others)  into  the  corresponding  acetyl  ester  by  boiling  them  with 
acetic  acid  anhydride. 

In  the  quantitative  estimation  of  fats,  the  finely  divided  dried  tissues 
or  the  finely  divided  residue  from  an  evaporated  fluid  is  extracted  with 
ether,  alcohol-ether,  benzene,  or  any  other  proper  extraction  medium. 
The  lecithin  (phosphatides)  and  other  bodies  are  dissolved  by  the  various 
extraction  media,  hence  the  results  for  fats  are  too  high.  The  most 
exact  method  for  the  estimation  of  fat  seems  to  be  the  method  sug- 
gested by  Ktjmagawa  and  Suto,1  who  give  a  complete  review  of  the 
literature  of  the  subject. 

The  fats  are  poor  in  oxygen,  but  rich  in  carbon  and  hydrogen.  They 
therefore  represent  a  large  amount  of  chemical  energy,  and  yield  correspond- 
ingly large  quantities  of  heat  on  combustion.  They  take  first  rank 
among  the  foods  in  this  regard,  and  are  therefore  of  very  great  impor- 
tance in  animal  life.  We  will  speak  more  in  detail  of  this  significance, 
also  of  fat  formation  and  of  the  behavior  of  the  fats  in  the  body,  in  the 
following  chapters. 

Cholesterin  and  isocholesterin  ester,  which  will  be  discussed  in  a  sub- 
sequent chapter,  as  well  as  the  following  bodies,  are  closely  related  to 
the  fats. 

Spermaceti.  In  the  living  spermaceti  or  white  whale  there  is  found,  in  a  large 
cavity  in  the  skull,  an  oily  liquid  called  spermaceti,  which  on  cooling,  after  death, 
separates  into  a  solid  crystalline  part  ordinarily  called  spermaceti,  and  into  a 
liquid,  spermaceti-oil.  This  last  is  separated  by  pressure.  Spermaceti  is  also 
found  in  other  whales  and  in  certain  species  of  dolphin. 

The  purified,  solid  spermaceti,  which  is  called  cetin,  is  a  mixture  of  esters  of 
fatty  acids.     The  chief  constituent  is  the  cetyl-palmitic  ester  mixed  with  small 

1  Biochem.  Zeitschr.,  8.     See  also  y.  Schimidzu,  ibid.,  28. 


PHOSPHATIDES.  230 

quantities  of  compound  esters  of  Iauric,  myristic,  and  stearic  acid-;  with  radicals 
of  the  alcohols,  lethal.  CuH«.OH,  methal,  CmH».OH,  and  stethal,  CuHit.OH. 
Cetin  is  a  snow-white  mass  shining  like  mother-of-pearl,  crystallizing  in  plates, 
brittle,  fatty  to  the  touch,  and  which  has  a  varying  melting-point  of  30  to  50°  C., 
depending  upon  its  purity.  Cetin  is  insoluble  in  water,  hut  dissolves  easily 
in  cold  ether  or  volatile  and  fatly  oils.  It  dissolves  in  boiling  alcohol,  but  crys- 
tallizes on  cooling.  It  is  saponified  with  difficulty  by  a  solution  of  caustic  potash 
in  water,  but  with  an  alcoholic  solution  it  saponifies  readily,  and  the  above-men- 
tioned  alcohols  are  set  free. 

CH3 

Ethal  or  cetyl  alcohol,  Ci6H310.  =  (CHj)H,  which  occurs  in  smaller  quantities 

CH..OH 
in  beeswax   and  was  found  by  Ludwig  and  v.  Zeyxek  in  the  fat  from  dermoid 
Cysts — though  this  is  denied  by  Ameseder,1 — forms  white,  transparent,  odorless, 
and  tasteless  crystals  which  are  insoluble  in  water  but  dissolve  easily  in  alcohol 
and  ether.     Ethal  melts  at  4!ho°  C. 

Spermaceti-oil  yields  on  saponification  valeric  acid,  small  amounts  of  solid 
fatty  acids,  and  PHY6ETOLEIC  ACID.  This  acid,  which  has.  like  hypogffiic  acid, 
the  composition  CuH&Os,  occurs  also,  as  found  by  Ljubarsky,2  in  considerable 
amounts  in  the  fat  of  the  seal.  It  forms  colorless  and  odorless  needle-shaped 
crystals  which  easily  dissolve  in  alcohol  and  ether  and  melt  at  34°  C. 

Beeswax  may  be  treated  here  as  concluding  the  subject  of  fats.  It  con- 
tains three  chief  constituents:  (1)  cerotic  acid,  CseHo-.Oi,3  which  occurs  as  cetyl 
ether  in  Chinese  wax  and  as  free  acid  in  ordinarv  wax.  It  dissolves  in  boiling 
alcohol  and  separates  as  crystals  on  cooling.  The  cooled  alcoholic  extract  of 
wax  contains  (2)  ceroleix,  which  is  probably  a  mixture  of  several  bodies,  and 
(3)  mvricix,  which  forms  the  chief  constituent  of  that  part  of  wax  which  is 
insoluble  in  warm  or  cold  alcohol.  Myricin  consists  chiefly  of  palmitic-acid 
ester  of  nielissyl  (myricyl)  alcohol,  C3oH6i.OH.  This  alcohol  is  a  silky,  shining, 
crystalline  body  melting  at  So0  C.  Dunham  *  has  found  camaubic  acid,  C^J^sO-- 
in  a  phosphatide  from  the  ox  kidney. 

2.  Phosphatides. 

In  close  relaticn  to  the  fats  stands  a  group  of  esters  containing 
nitrogen,  phosphoric  acid  and  fatty  acid  radicals.  The  representative 
of  this  group  longest  known  is  lecithin.  This  latter  is  an  ester  combina- 
tion of  a  nitrogenous  base,  choline,  with  a  fatty  acid-glycerophosphoric 
acid,  and  Thudichum  5  has  shown  that  a  large  number  of  more  or  less 
analogous  bodies  occur  in  the  animal  body,  especially  in  the  brain. 
All  of  these  bodies  have  received  the  name  phosphatides. 

Those  phosphatides  which  contain  only  one  phosphoric  acid  radical 
in  the  molecule  are  called  monophosphatides;  those  with  two  such  radicals 
di phosphatides.     The  monophosphatides  may  contain  one,  two  or  more 


1  Ludwig  and  v.  Zeynek,  Zeitschr.  f.  physiol.  Chem.  23;  Ameseder,    ibid.,  52. 
;  Journ.  f.  prakt.  Chem.  (N.  F.),  57. 

1  See  Henriques,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30,  1415. 
4  Journ.  of  biol.  Chem.,  4. 

s  J.  L.  W.  Thudichum,  Die  chemische  Konstitution  des  Gehirns  des  Menschen, 
etc.,  Tubingen,  1901. 


240  ANIMAL  FATS  AND  PHOSPHATIDES. 

atoms  of  nitrogen  in  the  molecule,  and  hence  we  differentiate  between 
monamido-  (P :  N  =  1 :  1) ,  diamido-  (P :  N  =  1 :  2) ,  triamido-  (P :  N  =  1 :  3) 
monophosphatides,  etc. 

So  also  may  the  diphosphatides  contain  1,  2  or  3  atoms  of  nitrogen 
for  every  2  atoms  of  phosphorus  (mono-  di-  or  triaminodiphosphatides) . 
Phosphatides  with  4  or  more  atoms  of  nitrogen  for  every  atom  of  phos- 
phorus are  also  claimed  to  occur,  but  these  statements  seem  to  be  uncer- 
tain. On  the  other  hand,  according  to  Thudichum,  non-nitrogenous 
phosphatides  occur  in  the  brain;  but  if  such  be  true  these  bodies  must 
not,  for  the  present  at  least,  be  classified  as  phosphatides. 

The  phosphatides  thus  far  investigated  seem  to  be  chiefly  ester  com- 
binations between  nitrogenous  bases  and  fatty  acid-glycerophosphoric 
acid.  According  to  Thudichum  phosphatides  exist  which  contain  no 
glycerin  group  and  the  carnaubon  obtained  by  Dunham  *  from  beef 
kidneys  seems  to  be  such  a  phosphatide.  The  fatty  acids  occurring  in 
the  phosphatides  may  be  of  different  kinds.  It  seems  that  at  least 
one  oleic  acid  radical,  or  another  still  less  saturated  fatty  acid,  occurs 
in  most  of  the  phosphatides;  still  we  know  of  phosphatides  that  con- 
tain only  saturated  fatty  acids.  On  this  account  the  phosphatides  may. 
be  divided  into  saturated  and  unsaturated  phosphatides.  The  unsaturated 
add  iodine,  take  up  oxygen  from  the  air  and  are  auto-oxidizable  and 
are  changed  readily.  They  also  give  a  beautiful  reaction  with  Petten- 
kofer's  bile-acid  test. 

Choline  has  generally  been  obtained  as  a  basic  constituent  of  the 
phosphatides.  Still  other  not  sufficiently  studied  bases,  have  been  found 
in  the  plant  as  well  as  animal  phosphatides  and  according  to  Trier  2 
aminoeihyl  alcohol  is  a  probable  generally  distributed  component  of  the 
lecithins  (phosphatides). 

The  phosphatides  are  very  widely  distributed  in  the  plant  as  well  as 
the  animal  kingdom  and  they  must  undoubtedly  exist  as  primary  cell 
constituents.  We  differentiate  between  such  cell  constituents  which  seem 
to  be  absolutely  necessary  for  the  life  of  the  cells,  and  those  which  are 
stored  up  as  reserve  material,  or  are  products  of  metabolism.  The  first, 
which  seem  to  occur  in  all  developing  cells,  have  been  called  'primary 
by  Kossel3  while  he  calls  the  others  secondary.  The  question  as  to 
the  division  of  the  known  cell  constituents  into  the  primary  or  secondary 
groups  in  the  above  sense,  cannot  be  answered  positively  in  many  cases. 
In  the  primary  group  besides  water  and  mineral  bodies  we  include  pro- 
teins of  various  kinds,  nucleic  acids  and  the  so-called  lipoids  (see  below) 
to  which  the  phosphatides  belong. 

lZeitschr.  f.  physiol.  Chem.,  64,  303  (1910). 
2  Ibid.,  73,  76  and  80. 
8  Verhandl.  d.  physiol.  Gesellsch.  zu  Berlin,  1890-91. 


PHOSPHATIDES.  241 

Attention  has  been  called  in  Chapter  I  to  the  importance  of  the  phos- 
phatides (lipoids)  for  the  limiting  layer  of  the  cells  as  well  as  for  the 
osmotic  processes  and  for  the  metabolism  of  the  cells.  The  unsat- 
urated, readily  oxidizable  phosphatides  also  play  a  possible  role  as  oxygen 
carriers  and  the  phosphatides  are  undoubtedly  of  great  importance  as 
constituents  of  the  food-stuffs.  There  is  also  no  doubt  that  they  are 
very  important  for  development  and  growth.  It  has  been  found  that 
the  amount  of  phosphatides  is  especially  abundant  in  the  new-born, 
and  that  these  latter,  to  a  certain  extent,  bring  into  the  world  a  store  of 
phosphatides  and  this  store  diminishes  during  growth.1 

The  phosphatides  seem  to  be  closely  related  to  one  another;  they 
influence  the  solubility  and  precipitation  properties  of  one  another,  and 
are  generally  precipitated  as  mixtures  which  are  extremely  difficult 
to  separate  into  individual  constituents.  They  are  also  amorphous, 
and  readily  oxidized,  and  it  is  easy  to  understand  why  their  preparation 
in  a  pure  state  is  so  extremely  difficult.  Under  these  circumstances 
we  have  no  sufficient  guarantee  as  to  their  chemical  individuality,  and 
the  description  of  their  properties  and  composition  must  be  accepted 
with  some  reservation. 

The  phosphatides  are  generally  amorphous,  colorless  or  yellowish; 
they  melt,  on  warming,  and  burn.  As  a  rule  they  are  insoluble  in  water 
and  swell  up  therein  forming  colloidal  solutions,  which  are  precipitated  by 
certain  salts.  The  phosphatides  as  above  stated,  belong  to  the  lipoids 
and  it  is  for  this  reason  that  each  phosphatide  is  dissolved  by  at  least  one 
of  the  solvents  for  fats  (alcohol,  ether,  benzene,  petroleum  ether,  etc.). 
The  lipoid  group  cannot  be  otherwise  characterized.  Originally  we 
included  in  this  group,  bodies  similar  to  fat  or  in  certain  respects  related 
to  the  fats  such  as  phosphatides,  cholesterin  and  cerebrosides,  but  later 
the  conception  has  been  developed  and  now  we  consider  as  lipoids 
those  bodies  that  are  soluble  in  ether  or  equivalent.  Under  these 
circumstances,  as  the  diverse  known  and  unknown  bodies,  such  as  lactic 
acid,  phenols,  alkaloids  and  extractive  bodies  of  various  kinds  may 
belong  to  the  lipoid  group  there  does  not  seem  to  be  any  sense  in  speaking 
of  a  special  lipoid  group,  and  especially  from  a  chemical  standpoint  it 
would  be  better  to  drop  the  name  entirely. 

The  various  phosphatides  show  a  different  behavior  toward  the 
solvents  for  lipoids,  namely  some  are  soluble  in  ether  while  others 
are  insoluble  therein,  etc.,  and  these  differences  are  important  for  their 


1  In  regard  to  the  quantity  and  importance  of  the  phosphatides  (the  lecithins) 
see  Siwertzow,  Bioch.  Centralbl.,  2,  310;  Glikin,  Bioch.  Zeitschr.,  4  and  7;  Nerking, 
ibid.,  10;  Stoklasa,  Ber.  d.  d.  chem.  Gesellsch.,  29,  Wien.  Sitz.  Ber.,  104;  Zeitschr. 
f.  physiol.  Chem.  25;  Danilewsky,  Compt.  Rend.,  121  and  123  and  \Y.  Koch.,  Zeitschr. 
f.  physiol.  Chem.,  37;  Kyes,  ibid.,  41  and  Berl.  klin.  Wochenschr.,  1904. 


242  ANIMAL  FATS  AND  PHOSPHATIDES. 

preparation.  They  are  generally  all  precipitated  from  their  solution 
by  acetone  although  not  completely,  and  this  behavior  is  also  of  especial 
importance  in  their  preparation.  The  phosphatides  are  also  nearly  all 
precipitated  by  metallic  salts,  especially  by  platinum  chloride  and  cad- 
mium chloride,  and  this  method  is  also  often  used  in  their  preparation. 
The  usefulness  of  this  method  has  been  questioned  at  least  for  certain 
phosphatides,   since  Erlandsen  *   showed  that  a  decomposition  occurs. 

Erlandsen  has  also  found  that  when  finely  divided  heart-muscle,  dried  in 
the  air,  is  completely  extracted  with  ether  and  then  with  alcohol,  the  first  extract 
contains  the  monophosphatides,  and  the  alcohol  extract  contains  the  diamino 
phosphatides  which  were  not  free  in  the  tissues,  but  existed  in  the  combined  state. 
Whether  this  observation  is  of  general  importance  in  the  preparation  of  pure 
phosphatides  remains  to  be  seen. 

As  the  phosphatides  among  themselves  are  rather  difficult  to  char- 
acterize and  as  there  is  a  question  whether  a  pure  phosphatide  has  thus  far 
been  prepared  it  seems  of  little  interest  to  give  a  review  here  of  the  division 
of  the  isolated  phosphatides  among  the  different  groups.  In  this  chapter 
we  will  only  discuss  the  three  most  studied  phosphatides,  namely,  lecithin, 
cephalin  and  cuorin;  the  others  will  be  treated  of  in  the  respective  chapters. 
Lecithins.  In  correspondence  with  the  generally  accepted  view 
lecithin  is  a  monoaminomonophosphatide,  which'  forms  an  ester  com- 
pound of  glycerophosphoric  acid  substituted  by  two  fatty-acid  radicals 
with  a  base  called  choline,2  hence  there  must  exist  several  groups  of 
lecithins.  According  to  the  kind  of  fatty  acid  contained  in  the  lecithin 
molecule  it  is  possible  to  have  various  lecithins,  such  as  stearyl-,  palmityl-, 
and  oleyl-lecithins.  According  to  Thudichum  3  every  true  lecithin  always 
contains  at  least  one  oleic-acid  radical.  According  to  the  investigations 
of  Henriques  and  Hansen,  Cousin  and  Erlandsen,4  there  is  no  ques- 
tion that  the  so-called  lecithin  of  the  egg-yolk  and  muscles  must  contain 
a  fatty  acid,  still  less  saturated  than  oleic  acid.  All  lecithins  are  mon- 
aminophosphatides,  according  to  the  following  type: 

CH2 — 0 — fatty-acid  radical. 

CH  — 0 — fatty-acid  radical. 

CH2— (X 

hcApo 

/C2H4— O/ 
N£-(CH8)3 
MDH 

1  Zeitschr.  f.  physiol.  Chem.,  51. 

2  Strecker,  Annal.  d.  Chem.  u.  Pharm.,  148;   Hundeshagen,  Journ.  f.  prakt.  Chem. 
X.    V.),   28;    Gilson,  Zeitschr.  f.  physiol.  Chem.,   12.     A  different  view  is  held  by 

Malengreau  and  Pridgent,  ibid.,  77. 

*  Thudichum,  Die  chemische  ^Constitution  des  Gehirns  des  Menschen,  etc.,  Tubingen, 
IfiOl. 

4  Henriques  and  Hansen,  Skand.  Arch.  f.  Physiol.,  14  (1903);  Cousin,  Compt. 
Piend.,  137;  Erlandsen,  Zeitschr.  f.  physiol.  Chem.,  51. 


LECITHINS.  243 

The  various  lecithins  stand  close  to  each  other  in  regard  to  constitu- 
tion. The  amount  of  phosphorus  varies  between  3.7-3.97  per  cent  and 
the  amount  of  nitrogen  between  1.7-1.9  per  cent.  The  so-called 
di-stearyl-lecithin  studied  by  Hoppe-Seyler  and  Diacoxow,1  which 
probably  has  a  different  structure,  has  the  formula  C44H90XPO9.  Erlaxd- 
sex  gives  the  formula  C^HsoNPOj  for  the  lecithin  isolated  by  him  from 
the  heart  muscles. 

On  saponification  with  alkalies  or  baryta-water,  lecithin  yields  fatty 
acids,  glycerophosphoric  acid,  and  choline.  It  is  remarkable  that  in 
the  cleavage  of  lecithins  a  smaller  amount  of  nitrogen  than  corresponds 
to  the  choline  is  obtained.  Mac  Lean2  who  has  especially  investigated 
this  could  not  re-obtain  the  total  nitrogen  in  the  lecithins  as  choline  but 
only  a  part  thereof — from  heart  muscle  lecithin,  42  per  cent,  and  from  egg- 
yolk  lecithin,  G5  per  cent.  He  is  therefore  of  the  opinion  that  the  choline 
group  is  not  the  only  nitrogenous  group  in  the  lecithins  and  that  there- 
fore the  generally  accepted  formula  for  lecithin  is  incorrect.  Trier3 
has  indeed  obtained  aminoethyl  alcohol  as  a  cleavage  product  from  sev- 
eral phosphatides,  which  he  calls  lecithins,  but  because  of  the  difficulty 
in  preparing  phosphatides  in  a  pure  condition  we  are  not  sure  that  he  was 
"working  with  pure  substances.  Lecithin  is  slowly  decomposed  by 
dilute  acids.  Besides  small  quantities  of  glycerophosphoric  acid  we 
have  large  quantities  of  free  phosphoric  acid  split  off.  The  lecithins 
are  also  decomposed  by  enzymes  (lipase)  with  the  splitting  off  of  fatty 
acids. 

Lecithin  is  optically  active,  and  as  the  glycerophosphoric  acid  which 
can  be  split  off  is  also  active,  Willstatter  and  Ludecke  4  claim  that  the 
phosphoric  acid  is  not  bound  on  the  middle  unsymmetric  CH  group,  but 
rather  at  the  end  CH2  group  of  glycerin. 

Lecithin,  according  to  Hoppe-Seyler,5  is  found  in  nearly  all  animal 
and  vegetable  cells  thus  far  studied,  and  also  in  nearly  all  animal  fluids. 
It  is  especially  abundant  in  the  brain,  nerves,  fish  eggs,  yolk  of  the  egg, 
electrical  organs  of  the  Torpedo  electricus,  semen,  and  pus,  and  also  in 
the  muscles  and  blood-corpuscles,  blood-plasma,  lymph,  milk,  especially 
woman's  milk,  and  bile.  Lecithin  is  also  found  in  different  pathological 
tissues  or  liquids.  As  the  presence  of  lecithin  is  only  indirectly  deter- 
mined by  the  detection  of  phosphorus  in  organic  combinations,  it  must 
be  borne  in  mind  that  the  above  assertions  relate  chiefly  to  the  occur- 
rence of  phosphatides. 

1  Hoppe-Seyler,  Med.  chem.  Unters.,  Heft  2  and  3. 

2  Zeitschr.  f.  physiol.  Chem.,  59  and  Bioch.  Centralbl.,  9. 
3 1.  c,  Zeitschr.  f.  physiol.  Chem.,  73,  76  and  80. 

4  Ber.  d.  d.  chem.  Gesellsch.,  37. 

5  Physiol.  Chem.  Berlin,  1877-81,  p.  57. 


244  ANIMAL  FATS  AND  PHOSPHATIDES. 

The  same  also  applies  to  the  claims  as  to  the  quantity  of  lecithin 
in  various  organs  and  tissues  as  well  as  in  different  ages.  In  these  cases 
the  lecithin  has  not  been  prepared  in  a  pure  state,  and  the  determina- 
tions represent  only  the  approximate  quantity  of  phosphatides.  These 
determinations  of  Siwertzow,  Glikin  and  Nerking,1  show  that  lecithins 
(phosphatides)  occur  abundantly  in  the  bone  marrow,  suprarenal  capsule, 
heart  and  lungs,  besides  in  the  spinal  marrow,  brain,  and  egg,  and  also 
that  the  quantity  varies  strikingly  in  different  varieties  of  animals. 
Nerking  found  41.7  per  cent  lecithin  in  the  bone  marrow  and  21.33 
per  cent  in  the  suprarenal  capsule  of  the  sea-urchin  when  calculated  on 
the  living  organs,  while  the  corresponding  results  in  the  rabbit  were  2.71 
and  2.39  per  cent,  respectively. 

The  statements  as  to  the  properties  of  the  lecithins  apply  chiefly  to 
the  lecithin  of  the  hen's  egg,  which  since  Hoppe-Seyler  and  Diaconow's 
time  has  been  considered  as  distearyl-lecithin  without  any  positive  founda- 
tion. Other  lecithin  preparations  correspond  essentially  with  this,  and 
certain  differences  between  the  various  lecithins  may  be  possibly  due 
to  decomposition  products  or  to  admixture  with  other  phosphatides. 
It  is  still  questioned  whether  the  so-called  distearyl-lecithin  is  a  unit 
body  or  not. 

Lecithin  may  be  obtained  in  grains  or  warty  masses  composed  of  small 
crystalline  plates  by  thoroughly  cooling  its  solution  in  strong  alcohol.  In 
the  dry  state  it  has  a  waxy  appearance,  is  plastic,  but  forms  pulverizable 
masses  when  dried  in  vacuum,  and  is  soluble  in  alcohol,  especially  on 
heating  (to  40-50°  C.) ;  it  is  less  soluble  in  ether.  It  is  dissolved  also  by 
chloroform,  carbon  disulphide,  benzene,  and  fatty  oils.  The  solution 
of  lecithin  from  egg-yolk  is  dextrorotatory  (Ulpiani2).  P.  Mayer3 
claims  to  have  prepared  racemic  lecithin  from  ordinary  lecithin,  and 
Z-lecithin  from  the  r-lecithin  by  cleavage  with  lipase.  As  he  did  not 
make  use  of  pure  lecithin  it  is  difficult  to  judge  his  results.  The  solu- 
tion of  lecithin  in  alcohol-ether  or  chloroform  is  precipitated  by  acetone, 
although  not  completely.  It  swells  in  water  to  a  pasty  mass  which  shows 
under  the  microscope  slimy,  oily  drops  and  threads,  so-called  myelin 
forms  (see  Chapter  XI).  On  warming  this  swollen  mass  or  the  concen- 
trated alcoholic  solution,  decomposition  takes  place  with  the  produc- 
tion of  a  brown  color.  On  allowing  the  solution  or  the  swollen  mass  to 
stand,  decomposition  takes  place  and  the  reaction  becomes  acid.  Accord- 
ing to  the  investigations  of   Long4  the  lecithins  seem  to  be  much  more 

1  Siwertzow,  see  Biochem.  Zeitschr.,  2,  p.  310;  Glikin,  Biochem.  Zeitschr.,  4  and 
7;   Nerking,  ibid.,  10. 

2Chem.  Centralbl.,  1901,  2,  30  and  193. 

3  Biochem.  Zeitschr.,  1. 

4  Journ.  of  Arner.  chem.  Soc,  30,  1908. 


LECITHINS.  245 

resistant  than  was  generally  believed,  and  further  investigations  with 
pure  lecithin  are  desirable. 

With  considerable  water  the  lecithin  gives  an  emulsion  or  colloidal 
solution  which  is  not  only  precipitated  by  salts  with  divalent  cations. 
Ca,  Mg,  and  others  as  claimed  by  W.  KOCH,  but  is  also  precipitated  accord- 
ing to  Long  and  F.  Gephart1  by  salts  with  monovalent  cations,  although 
slowly.  In  putrefaction, lecithins  yield  dvcerophosphoric  acid  and  choline; 
the  latter  further  decomposes  with  the  formation  of  methylamine,  ammonia, 
carbon  dioxide,  and  marsh  gas  (Hasebiioek  2).  If  dry  lecithin  be  heated 
it  decomposes,  takes  fire,  and  burns,  leaving  a  phosphorized  ash.  On 
fusing  with  caustic  alkali  and  saltpetre  it  yields  alkali  phosphates. 

Lecithins  combine  with  acids  and  bases.  The  compound  with  hydro- 
chloric acid  gives  with  platinum  chloride  a  double  salt  which  is  insoluble 
in  alcohol,  soluble  in  ether,  and  which  contains  10.2  per  cent  platinum 
(for  distearyl-lecithin).  The  cadmium-chloride  compound,  whose  com- 
position has  been  found  somewhat  variable  by  different  investigators 
is  soluble  with  difficulty  in  alcohol,  but  dissolves  in  a  mixture  of  carbon 
disulphide  and  ether  or  alcohol.  A  solution  of  lecithin  in  alcohol  is  not 
precipitated  by  lead  acetate  and  ammonia. 

Lecithins  (and  the  same  applies  to  the  phosphatides  in  general)  are 
easily  carried  down  during  the  precipitation  of  other  compounds,  such 
as  the  protein  bodies,  and  may  therefore  very  greatly  change  the  solubil- 
ilities  of  other  bodies.  It  is  not  known  whether  we  are  here  dealing 
with  an  adsorption  or  a  chemical  combination,  and  the  conditions  are 
not  the  same  in  all  cases.  The  combination  with  protein,  the  vitellines 
and  lecithalbumins  have  been  discussed  in  a  previous  chapter,  and 
attention  is  there  called  to  the  necessity  for  more  thorough  investigation 
of  this  subject.  Further  investigations  of  the  so-called  lecithin-sugar 
(Bixg)  is  also  desirable,  as  we  know  nothing  definite  as  to  its  nature. 
According  to  the  investigations  of  Winterstein,  Hiestand  and  E. 
Schulze,  lecithins  (phosphatides)  containing  carbohydrates  occur  in 
the  plant  kingdom,  and  contain  about  20  per  cent  carbohydrate.  We 
are  still  not  decided  whether  we  are  here  dealing  with  combinations 
or  admixtures.3  The  same  is  true  for  the  iron  content  of  the  lecithins  or 
phosphatides  as  observed  by  Glikin.4 


1  W.  Koch.,  Zeitschr.  f.  physiol.  Chem.,  37;    Long  and   Gephart,    Journ.  of  Amer. 
Chem.  Soc,  30;  see  also  Porges  and  Neubauer,  Biochem.  Zeitschr.,  7. 
-  Zeitschr.  f.  physiol.  Chem.,  12. 

3  Winterstein  and  Hiestand,  Zeitschr.  f.  physiol.  Chem.,  47  and  54;    Schulze,  ibid.t 
52  and  55;  V.  Xjegovan,  ibid.,  76. 

4  Ber.  d.  d.  chem.  Gesellsch.,  41. 


246  ANIMAL  FATS  AND  PHOSPHATIDES. 

Various  methods  have  been  suggested  by  Strecker,  Hoppe-Seyler 
and  Diaconow,  Thudichum,  Gilson,  Zuelzer  and  Bergell  x  for  the 
preparation  of  the  lecithins.  As  none  of  these  yield  a  positively  pure 
product  we  will  here  only  mention  them.  According  to  Erlandsen's 
experience  all  methods  which  are  based  upon  the  precipitation  of  the 
lecithin  as  a  metallic  compound  should  be  avoided.  The  best  method 
depends  upon  the  solubility  of  the  lecithin  in  alcohol  and  in  ether  in 
the  cold  and  its  precipitation  by  acetone  (Erlandsen,  H.  E.  Roaf  and 
E.  Edie2).  The  work  of  Erlandsen  is  especially  referred  to  in  the 
preparation  of  lecithins. 

For  the  present  we  have  no  quantitative  method  for  estimating 
lecithin.  The  methods  used  in  the  past,  when  the  amount  of  lecithin 
was  calculated  from  the  amount  of  phosphorus  contained  in  the  alcohol- 
ether  extract  is  useless,  as  in  this  case  the  phosphorous  content  of  all 
the  phosphatides  is  determined  and  not  alone  of  the  lecithins.  Even 
the  detection  of  choline  is  not  evidence,  as  this  base  probably  occurs  also 
in  other  phosphatides.  In  the  detection  of  choline  the  double  platinum 
compound  is  ordinarily  prepared,  and  this  can  be  done  as  described 
below.  In  special  determinations  of  lecithin  and  cephalin  Koch  used 
to  heat  with  hydroiodic  acid,  and  determined  the  methyl  groups  split 
off  below  240°  and  those  at  about  300°.  Instead  of  this  he  recommends 
with  Woods  3  to  separate  the  two  by  precipitation  in  alcoholic  solution, 
while  boiling,  with  alcoholic  lead  acetate  solution  and  a  little  ammonia, 
which  precipitates  only  the  cephalin. 

Of  the  cleavage  products  of  the  lecithins  choline  is  of  especially 
great  interest. 

Choline  (trimethyloxyethyl  ammonium  hydroxide), 

/CH2.CH2.(OH) 
C5H15N02,  =  HO.N< 

X(CH3)3, 

stands   in  close  "relation  to  the  poisonous  base  neurine   (trimethylvinyl 

/(CH3)3 
ammonium  hvdroxide),  HO.N<^  ,  which  according  to  Brieger 

XCH:CH2 

can  be  formed  from  choline  by  the  action  of  bacteria,  and  also  to  mus- 

/(CH3)3 
carine,  HO.N<y  ,  which  is  the  aldehyde  of  choline  and  occurs  in 

XH2CHO 

/   °   \ 
the  fly  agaric,  and  also  to  betaine,  trimethyl  glycocoll,  (CH3)3N<(  /CO, 

XCH-/ 


1  Strecker,  Annal.  d.  Chem.  u.  Pharm.,  148;  Hoppe-Seyler  and  Diaconow,  1.  c; 
Thudichum,  1.  c;  Gilson,  Zeitschr.  f.  physiol.  Chem.,  12;  Zuelzer,  ibid.,  27;  Bergell, 
Per.  d.  d.  chem.  Gesellsch.,  33. 

2  Erlandsen.  1.  c;  Roaf  and  Edie,  Thompson  Yates  Laboratory  Reports,  Vol.  6 
part  I,  1905. 

3  Koch  and  Woods,  Journ.  of  biol.  Chem.,  1. 


CHOLINE.  247 

which  may  be  considered  as  the  anhydride  of  the  arid  corresponding  to 
choline.  Muscarine  and  betaine  can  he  obtained  from  choline  on  oxida- 
tion. Choline  yields  trimethylamine  as  a  decomposition  product,  and  this 
seems  to  be  formed  in  the  transformation  of  choline  in  the  animal  body. 

(  holine  occurs  in  the  plant  kingdom  as  well  as  in  the  animal  kingdom. 
Mott  and  Halliburton  have  repeatedly  found  choline  in  the  blood  in 
degenerative  diseases  of  the  nervous  system.  It  was  first  shown  also  in 
normal  blood  by  Marino  Zuco,  l  and  this  investigator  first  found  it  in 
tbe  suprarenal  capsule,  but  designated  it  neurine.  Lohmann  found  it 
later  in  this  organ,  and  recently  it  has  been  found  in  various  organs  by 
other  investigators,  especially  by  C.  Schwarz  and  v.  Furth.  The  fact 
that  choline  is  a  cleavage  product  of  lecithin  in  the  animal,  and  that  it  is 
antagonistic  to  adrenalin  (of  the  suprarenal  capsule)  by  its  depressing 
action  upon  the  blood  pressure,  and  that  it  has  an  exciting  action  upon 
certain  secretions  (Lohmann,  Theissier  and  Thevenot,  v.  Furth 
and  Schwarz2),  gives  choline  great  physiological  importance.  The 
physiological  action  of  choline  is  still  very  much  disputed. 

Choline  is  a  syrupy  fluid,  readily  miscible  with  absolute  alcohol. 
Hydrochloric  acid  gives  with  it  a  compound  which  is  very  soluble  in  water 
and  alcohol,  but  insoluble  in  ether,  chloroform,  and  benzene.  This  com- 
pound forms  a  double  combination  with  platinum  chloride,  is  soluble  in 
water,  insoluble  in  absolute  alcohol  and  ether,  and  crystallizing  from 
water  in  monoclinic  system  and  this  form  is  strongly  double-refractive. 
From  a  mixture  of  water  and  alcohol  it  crystallizes  in  the  regular  form 
(octahedral).  Both  forms  can  be  changed  from  one  to  the  other 
and  are  used  according  to  Kauffmann  and  Vorlander  3  in  the  detec- 
tion of  choline.  Choline  also  forms  a  crystalline  double  compound 
with  mercuric  chloride  and  with  gold  chloride.  Choline  is  precipitated 
by  potassium  iodide  and  iodine  (Gulewitsch),  and  potassium  triiodide 
can  be  used  for  the  quantitative  estimation  of  this  base  (Stanek4). 
On  heating  the  free  base  it  decomposes  into  trimethylamine,  ethylene 
oxide,  and  water. 

In  preparing  choline  from  lecithins,  and  also  for  the  detection  of 
lecithin  in  an  alcohol-ether  extract,  proceed  as  follows:   The  residue  from 

1  Mott  and  Halliburton,  Philos.  Trans.,  Ser.  B,  191  (1899)  and  194  (1901);  Marino 
Zuco,  see  Maly's  Jahresber.,  24,  pp.  181  and  698. 

2  Lohmann,  Pfluger's  Arch.,  118  and  122;  v.  Furth  and  Schwarz,  ibid.,  124,  which 
also  contains  the  literature. 

3  See  Gulewitsch,  Zeitschr.  f.  physiol.  Chem.,  24;  Kauffmann  and  Vorlander,  Ber. 
d.  d.  chem.  Gesellsch.,  43. 

4  Gulewitsch,  Zeitschr.  f.  physiol.  Chem.,  24;  Stanek,  ibid.,  56.  In  regard  to  the 
quantitative  estimation  see  also  Kiesel,  ibid.,  53;  Stanek,  ibid.,  54;  Moruzzi,  ibid., 
55;  and  MacLean,  ibid.,  55. 


248  ANIMAL  FATS  AND  PHOSPHATIDES. 

the  above,  or  the  solid  lecithin  is  boiled  one  hour  with  baryta-water, 
filtered,  and  the  excess  of  baryta  precipitated  by  CO2;  filter  while  hot, 
concentrate  to  a  syrup,  and  extract  with  absolute  alcohol,  when  the 
insoluble  barium  glycerophosphate  remains;  then  precipitate  the  filtrate 
with  an  alcoholic  platinum  chloride  solution. 

CH2.OH 
Glycerophosphoric  acid,  C3H9P06  =CH.OH       ,  is  a  bibasic  acid  which  prob- 

CH2— 0\ 
OH->PO 
OH/ 
abfy  occurs  in  the  animal  fluids  and  tissues  only  as  a  cleavage  product  of  lecithins. 
According  to  Willstatter  and  Ludecke  1  the  glycerophosphoric  acid  split  off 
from  lecithins  is  optically  active.     Its  barium  and  potassium  salts  are  levorotatory, 
and  behave  in  certain  respects  differently  from  the  corresponding  salts  of  syn- 
thetically prepared  glycerophosphoric  acid.     The  Ba  and  Ca  salts  of  glycero- 
phosphoric acid  are  crystalline  and  are  more  soluble  in  cold  than  in  warm  water. 
The  acid  itself  is  a  syrupy  fluid. 

Cephalin  is  also  a  monoaminophosphatide  whose  formula,  based  upon 
the  investigations  of  Thudichum,  Koch,  Thierfelder  and  Stern,2  is 
probably  C42H82NPO13.  The  views  of  these  investigators  as  to  the  con- 
stitution of  this  body,  which  is  difficult  to  purify,  differ  very  considerably. 
According  to  Thudichum,  on  cleavage  it  yields  neurine,  glycerophosphoric 
acid,  stearic  acid,  and  a  specific  fatty  acid,  cephalic  acid.  According  to 
Koch  it  contains,  on  the  contrary,  only  one  methyl  group  attached  to 
nitrogen,  and  is  therefore  probably  dioxystearylmonomethyl  lecithin. 
Frankel  and  Dimitz  found  no  choline,  while  according  to  Cousin  it 
yields,  like  lecithin,  stearic  acid,  an  unsaturated  fatty  acid,  glycero- 
phosphoric acid  and  choline  as  decomposition  products.  The  glycero- 
phosphoric acid  from  brain  cephalin  gives,  according  to  Frankel  and 
Dimitz,  a  dextrorotatory  Ba  salt  and  is  therefore  not  identical  with  the 
glycerophosphoric  acid  from  lecithin.  According  to  these  investigators 
the  cephalin  of  the  human  brain  is  a  mixture  of  palmityl  and  stearyl- 
cephalin.  Besides  these  two  fatty  acids  cephalin  also  contains  an  unsat- 
urated fatty  acid,  cephalinic  acid,  which  according  to  Parnas3  is  related 
to  leinoleic  acid  or  perhaps  identical  therewith. 

From  the  investigations  carried  on  thus  far  we  can  conclude  that 
cephalin  differs  from  lecithin  in  that  it  contains  cephalinic  acid,  another 
glycerophosphoric  acid  and  probably  no  choline  but  a  monomethyl 
base.      Cephalin    has   probably    never    been   obtained    in    a  pure  form. 


1  Ber  d.  d.  chem.  Gesellsch.,  37. 

*  Thudichum,  1.  c;  Koch,  Zeitschr.  f.  physiol.  Chem.,  36;  Thierfelder  and  Stern, 
ibid.  53. 

3  Frankel  and  Dimitz,  Bioch.  Zeitschr.,  21;  Parnas,  ibid.,  22;  Cousin,  Compt. 
Rend.  soc.  biol.,  62. 


CEPHALIN  AND   CUOBIN.  249 

The  cephalin  from  the  brain  has,  according  to  Falk,1  a  different  composi- 
tion than  that  of  the  nerves  and  certain  observations  indicate  that  there 
are  several  cephalins. 

Cephalin  occurs  quite  abundantly  in  the  brain  and  also  in  nerves 
and  in  the  egg-yolk.  The  statements  as  to  its  further  occurrence  in  the 
animal  kingdom  require  substantiation. 

Cephalin  is  amorphous,  not  very  plastic,  and  more  easily  triturated 
than  lecithin.  It  is  readily  soluble  in  cold  ether,  in  chloroform  and 
benzene  but  differs  from  lecithin  by  being  insoluble  or  soluble  with  difficulty 
in  alcohol.  As  unsaturated  phosphatide  it  gives,  like  lecithin,  a  positive 
reaction  with  Pettenkofer's  bile-acid  test.  The  cadmium-  and  plat- 
inum chloride  combinations  are  soluble  in  ether.  Cephalin  is  obtained 
from  the  brain,  after  dehydration  with  acetone,  by  extracting  with  ether 
and  precipitating  the  concentrated  ethereal  extract  with  alcohol.  In 
regard  to  the  preparation  and  detection  of  cephalin  we  must  refer  to  more 
extensive  hand-books. 

The  purest  phosphatide  prepared  thus  far  seems  to  be  cuorin,  dis- 
covered by  Erlandsen. 

Cuorin,  C71H125NP2O21,  is  a  monaminodiphosphatide  prepared  by 
Erlandsen  2  from  the  heart  muscle  of  the  ox,  and  which  has  an  iodine 
equivalent  of  101.  It  yields  as  cleavage  products  3  molecules  fatty  acids 
of  unknown  nature,  partly  or  entirely  belonging  to  the  series  CnH2n-402 
and  CnH2n-602;  also  glycerin,  phosphoric  acid  and  a  base  which  is 
not  well  known,  but  it  is  not  choline.  Cuorin  is  autooxidizable,  and  gives 
Pettenkofer's  bile-acid  test. 

Cuorin  is  amorphous,  yellowish-brown  and  similar  to  rosin.  It 
gives  a  neutral  solution  with  water  which  is  like  an  emulsion.  Cuorin 
does  not  reduce  Fehling's  solution,  even  after  boiling  with  acids.  It  is 
soluble  in  ether,  chloroform,  petroleum  ether  and  carbon  disulphide. 
It  dissolves  with  difficulty  in  benzene ;  it  is  insoluble  in  ethyl  and  methyl 
alcohol  and  in  acetone.  Cuorin  is  precipitated  from  its  alcohol-ether 
solution  by  cadmium  or  platinum  chloride. 

1  Bioch.  Zeitschr.,  13  and  16. 

2  Zeitschr.  f.  physiol.  Chem.,  51,  where  the  method  of  preparation  is  described. 


CHAPTER  V. 

THE    BLOOD 

The  blood  is  to  be  considered  from  a  certain  standpoint  as  a  fluid 
tissue;  it  consists  of  a  transparent  liquid,  the  blood-plasma,  in  which 
a  vast  number  of  solid  particles,  the  red  and  white  blood-corpuscles  (and 
the  blood-plates),  are  suspended. 

Outside  of  the  organism  the  blood,  as  is  well  known,  coagulates  more 
or  less  quickly;  but  this  coagulation  is  accomplished  generally  in  a 
few  minutes  after  leaving  the  body.  All  varieties  of  blood  do  not  coagulate 
with  the  same  degree  of  rapidity.  Some  coagulate  more  quickly,  others 
more  slowly.  In  vertebrates  with  nucleated  blood-corpuscles  (birds, 
reptiles,  batrachia,  and  fishes)  Delezenne  has  shown  that  the  blood 
coagulates  very  slowly  if  it  is  collected  under  such  precautions  that  it 
does  not  come  in  contact  with  the  tissues.  On  contact  with  the  tissues  or 
with  their  extracts  it  coagulates  in  a  few  minutes.  The  blood  with 
non-nucleated  blood-corpuscles  (mammals),  on  the  contrary,  coagulates 
very  rapidly.  The  coagulation  of  the  blood  in  these  cases  may  also  be 
somewhat  retarded  by  preventing  the  blood  from  coming  in  contact 
with  the  tissues  (Spangaro,  Arthus1).  Among  the  varieties  of  blood 
of  mammals  thus  far  investigated  the  blood  of  the  horse  coagulates  most 
slowly.  The  coagulation  may  be  more  or  less  retarded  by  quickly  cool- 
ing; and  if  we  allow  equine  blood  to  flow  directly  from  the  vein  into  a 
glass  cylinder  which  is  not  too  wide  and  which  has  been  cooled,  and  let  it 
stand  at  0°  C,  the  blood  may  be  kept  fluid  for  several  days.  An  upper 
amber-yellow  layer  of  plasma  gradually  separates  from  a  lower  red  layer 
composed  of  blood-corpuscles  with  only  a  little  plasma.  Between  these 
is  observed  a  whitish-gray  layer  which  consists  of  white  blood-corpuscles. 

The  plasma  thus  obtained  and  filtered  is  a  clear  amber-yellow  alkaline 
(toward  litmus)  liquid  which  remains  fluid  for  some  time  when  kept 
at  0°  C,  but  soon  coagulates  at  the  ordinary  temperature. 

The  coagulation  of  the  blood  may  be  prevented  in  other  ways.  After 
the    injection    of  peptone,   or,   more   correctly,   proteose   solutions   into 

1  Drlezenne,  Corr.pt.  rend.  soc.  de  biol.,  49;  Spangaro,  Arch.  ital.  de  Biol.,  32; 
Arthus,  Journ.  de  Physiol,  et  Pathol.,  4. 

250 


PREVENTION  OF  COAGULATION.  251 

the  blood  (in  the  living  dog),  it  does  not  coagulate  on  leaving  the  veins 
(Fano,  Schmidt-Mulheim  1).  The  plasma  obtained  from  such  blood 
by  means  of  centrifugal  force  is  called  peptone-plasma.  According  to 
Arthus  and  Htjber  2  the  caseoses  and  gelatoses  act  like  fibrin  proteose 
in  dogs.  Eel  serum  and  certain  lymph-forming  extracts  of  organs 
(see  Chapter  VI)  have  an  analogous  action.  The  coagulation  of  the 
blood  of  warm-blooded  animals  is  prevented  by  the  injection  of  an 
effusion  of  the  mouth  of  the  officinal  leech  or  a  solution  of  the  active 
substance  of  such  an  infusion,  hirudin  (Franz),  into  the  blood  current 
(Haycraft3).  If  the  blood  is  allowed  to  flow  directly,  while  stirring 
it,  into  a  neutral  salt  solution — best  a  saturated  magnesium-sulphate 
solution  (1  vol.  salt  solution  and  3  vols,  blood) — we  obtain  a  mixture 
of  blood  and  salt  which  remains  uncoagulated  for  several  days.  The 
blood-corpuscles,  which,  because  of  their  adhesiveness  and  elasticity, 
would  otherwise  easily  pass  through  the  pores  of  the  filter-paper,  are 
made  solid  and  stiff  by  the  salt,  so  that  they  may  be  easily  filtered 
off.  The  plasma  thus  obtained,  which  does  not  coagulate  spontaneously, 
is  called  salt-plasma. 

An  especially  good  method  of  preventing  coagulation  of  blood  con- 
sists in  drawing  the  blood  into  a  dilute  solution  of  potassium  oxalate, 
so  that  the  mixture  contains  0.1  per  cent  oxalate  (Arthus  and  Paces4). 
The  soluble  calcium  salts  of  the  blood  are  precipitated  by  the  oxalate, 
and  hence  the  blood  loses  its  coagulability.  On  the  other  hand,  Horne  5 
found  that  chlorides  of  calcium,  barium,  and  strontium,  when  present 
in  large  amounts  (2-3  per  cent),  may  prevent  coagulation  for  several 
days.  According  to  Arthus  6  a  non-coagulable  blood-plasma  may  be 
obtained  by  drawing  the  blood  into  a  sodium-fluoride  solution  until  it 
contains  0.3  per  cent  NaFl. 

On  coagulation  there  separates  in  the  previously  fluid  blood  an  insoluble 
or  a  very  difficultly  soluble  protein  substance,  fibrin.  When  this  separa- 
tion takes  place  without  stirring,  the  blood  coagulates  in  a  solid  mass, 
which,  when  carefully  severed  from  the  sides  of  the  vessel,  contracts, 
and  a  clear,  generally  yellow-colored  liquid,  the  blood-serum,  exudes. 
The  solid  coagulum  which  encloses  the  blood-corpuscles  is  called  the 
blood-clot  (placenta  sanguinis).  If  the  blood  is  beaten  during  coagula- 
tion, the  fibrin  separates  in  elastic  threads  or  fibrous  masses,  and  the 

^ano,  Arch  f..  (anat.  u.)  Physiol.,  1881;  Schmidt-Mulheim,  ibid.,  1880. 

2  Arch,  de  Physiol.  (5),  8. 

3  Haycraft,  Proc.  Physiol.  Soc,  1SS4,  13,  and  Arch.  f.  exp.  Path.  u.  Pharm.,  18; 
Franz,  Arch.  f.  exp.  Path.  u.  Pharm.,  49. 

4  Archives  de  Physiol.  (5),  2,  and  Compt.  Rend.,  112. 

5  Journ.  of  Physiol.,  19. 

6  Journ.  de  Physiol,  et  Path.,  3  and  4. 


252  THE  BLOOD. 

djibrinated  blood  which  separates  is  sometimes  called  cruor  l  and  con- 
sists of  blood-corpuscles  and  blood-serum,  while  uncoagulated  blood 
consists  of  blood-corpuscles  and  blood-plasma.  The  essential  chemical 
difference  between  blood-serum  and  blood-plasma  is  that  the  blood- 
serum  does  not  contain  even  traces  of  the  mother-substance  of  fibrin, 
the  fibrinogen,  which  exists  in  the  blood-plasma,  while  the  serum  is  pro- 
portionally richer  in  another  body,  the  fibrin  ferment  (see  below). 

I.    BLOOD-PLASMA  AND   BLOOD-SERUM. 
The  Blood-plasma. 

In  the  coagulation  of  the  blood  a  chemical  transformation  takes 
place  in  the  plasma.  A  part  of  the  proteins  separate  as  insoluble  fibrin. 
The  albuminous  bodies  of  the  plasma  must  therefore  be  first  described. 
They  are,  as  far  as  we  know  at  present,  fibrinogen,  nucleoprotein,  ser- 
globulins,  and  ser  albumins. 

Fibrinogen  occurs  in  blood-plasma,  chyle,  lymph,  certain  transudates 
and  exudates,  in  bone-marrow  (P.  Muller),  and  perhaps  also  in  other 
lymphoid  organs.  The  seats  of  formation  of  fibrinogen  are,  according 
to  Mathews,  the  leucocytes,  especially  of  the  intestine,  according  to 
Muller,  the  bone-marrow  and  probably  other  lymphoid  organs  such 
as  the  spleen  and  lymph  glands,  and  according  to  Doyon  and  Nolf, 
the  liver.  The  statement  that  the  intestinal  wall  is  a  seat  of  formation 
of  fibrinogen,  a  view  that  had  been  held  by  Dastre,  is  substan- 
tiated not  only  by  the  direct  researches  of  Mathews,  but  also  by  the 
older  and  substantiated  opinion  that  the  blood  from  the  mesentery  vein 
is  richer  in  fibrinogen  than  the  arterial  blood.  This  origin  of  fibrinogen 
has  been  shown  to  be  improbable  by  the  recent  researches  of  Doyon,  Cl. 
Gautier  and  Morel.  The  occurrence  of  fibrinogen  in  the  bone-marrow 
and  other  lymphoid  organs  as  shown  by  Muller,  and  an  increase  of 
fibrinogen  in  the  blood  as  well  as  in  the  bone-marrow  of  animals  immunized 
with  certain  bacteria,  especially  pus-staphylococci,  indicates  the  forma- 
tion of  fibrinogen  in  this  tissue.  The  relation  between  the  quantity  of 
fibrin  and  leucocytosis  as  shown  by  many  investigators  such  as  Lang- 
stein  and  Mayer,  Morawitz  and  Rehn,  also  indicate  such  a  formation 
of  fibrinogen.  The  observations  of  Doyon,  Gautier  and  Mawas  that 
a  rapid  re-formation  of  fibrinogen  takes  place  in  splenectomized  animals 

1  The  name  cruor  is  used  in  different  senses.  We  sometimes  mean  thereby  only 
the  blood  when  coagulated  in  a  red  solid  mass,  in  other  cases  the  blood-clot  after  the 
separation  of  the  serum,  and  again  the  sediment  consisting  of  red  blood-corpuscles 
which  is  obtained  from  defibrinated  blood  by  means  of  centrifugal  force  or  by  letting 
it  stand. 


FIBRINOGEN.  253 

without  any  changes  in  the  bone-marrow  speak  against  the  especially 
great  importance  of  the  spleen  and  bone-marrow  for  the  formation  of 
fibrinogen.  That  the  liver  takes  part  in  the  formation  of  fibrinogen 
is  implied  by  the  fact  that  the  quantity  of  fibrinogen  in  the  blood 
strongly  diminishes  after  the  extirpation  of  the  liver  (Nolf),  and  that 
fibrinogen  may  indeed  be  entirely  absent  in  the  blood  in  phosphorus 
poisoning  (Corin  and  Ansiaux,  Jacoby,  Doyon,  Morel,  and  Kareff  l), 
and  that  the  blood  of  the  hepatic  vein,  according  to  Doyon,  Morel 
and  Kareff,  is  richer  in  fibrinogen  than  the  blood  from  other  vessels, 
and  finally  according  to  Whipple  and  Hurwitz2  in  chloroform  poison- 
ing the  fibrinogen  content  of  the  blood  diminishes  with  the  injury  to 
the  liver  and  rises  again  with  restitution  of  the  organ. 

Fibrinogen  has  the  general  properties  of  the  globulins,  but  differs 
from  other  globulins  as  follows:  In  a  moist  condition  it  forms  white 
flakes  which  are  soluble  in  dilute  common  salt  solutions,  and  which 
easily  conglomerate  into  tough,  elastic  masses  or  lumps.  The  solution 
in  5-10  per  cent  NaCl  coagulates  on  heating  at  52-55°  C,  and  the  faintly 
alkaline  or  nearly  neutral  weak  salt  solution  coagulates  at  56°  C,  or  at 
exactly  the  same  temperature  at  which  the  blood-plasma  coagulates. 
Fibrinogen  solutions  are  precipitated  by  an  equal  volume  of  a  saturated 
common  salt  solution,  and  are  completely  precipitated  by  adding  an  excess 
of  NaCl  in  substance  (thus  differing  from  serglobulin) .  A  salt-free 
solution  of  fibrinogen  in  as  little  alkali  as  possible  gives  with  CaCb 
a  precipitate  which  contains  calcium  and  soon  becomes  insoluble.  In 
the  presence  of  NaCl  or  by  the  addition  of  an  excess  of  CaCk  the  precipitate 
does  not  appear.3  A  neutral  solution  of  fibrinogen  is  precipitated  by 
a  concentrated  solution  of  sodium  fluoride  when  added  in  a  sufficient  quan- 
tity. Fibrinogens  from  different  kinds  of  blood  behave  somewhat  dif- 
ferently in  this  regard.  According  to  Huiskamp  4  fibrinogen  from  horse- 
blood  hardly  dissolves  in  NaCl  of  3-5  per  cent  at  ordinary  temperatures, 
while  it  does  dissolve  at  40-45°.     It  also  dissolves  in  ammonia  of  0.05 

1  P.  Miiller,  Hofmeister's  Beitrage,  6;  Mathews,  Amer.  Journ.  of  Physiol.,  3;  Nolf, 
Bull.  Acad.,  Roy.  Belg.,  1905,  and  Arch,  intern,  de  Physiol.,  3,  1905;  Langstein,  and 
Mayer,  Hofmeister's  Beitrage,  5;  Morawitz  and  Rehn,  Arch.  f.  exp.  Path.  u.  Pharm., 
58;  Corin  and  Ansiaux,  Mary's  Jahresber.,  24;  Jacoby,  Zeitschr.  f.  physiol.  Chem., 
30;  Doyon,  Morel  and  Kareff,  Compt.  Rend.,  140;  Do3'on,  Morel,  and  P6ju,  Comp. 
rend.  soc.  biolog.,  58;  Doyon,  CI.  Gautier,  and  Morel  ibid.,  62;  Doyon,  Gautier 
and  Mawas,  ibid.,  64. 

2  Doyon,  Morel  and  Kareff,  Journ.  de  Physiol.,  8  (1906);  Whipple  and  Hurwitz, 
Journ.  of  exp.  Med.  13.     See  also  Meek,  Amer.  Journ.  of  Physiol.,  30. 

3  See  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  22;  Cramer,  ibid.,  23. 

4  Huiskamp,  ibid.,  44  and  46.  In  regard  to  fibrinogen  the  reader  is  referred  to 
the  author's  investigations.  Pfluger's  Archiv.,  19  and  22,  and  Zeitschr.  f.  physiol. 
Chem.,  28. 


254  THE   BLOOD. 

per  cent,  and  on  the  addition  of  3-5  per  cent  NaCl  this  solution  can  be 
neutralized.  The  fibrinogen  prepared  by  Huiskamp  in  this  way  retained 
its  typical  properties.  Fibrinogen  differs  from  the  myosin  of  the  muscles, 
which  coagulates  at  about  the  same  temperature,  and  from  other  pro- 
tein bodies,  in  the  property  of  being  converted  into  fibrin  under  certain 
conditions.  Fibrinogen  has  a  strong  decomposing  action  on  hydrogen 
peroxide.  It  is  quickly  made  insoluble  by  precipitation  with  water  or 
with   dilute  acids.     Its  specific  rotation  is   (a)D=— 52.5°   according  to 

MlTTELBACH.1 

Fibrinogen  may  be  easily  separated  from  the  salt-plasma  or  oxalate- 
plasma  by  precipitation  with  an  equal  volume  of  a  saturated  NaCl  solu- 
tion. It  must  be  observed  that  the  oxalate-plasma  can  only  be  employed 
after  the  precipitate,  containing  proenzymes,  and  produced  by  exposure 
to  cold,  has  settled  and  been  filtered  off.  If  this  is  not  done  then  the 
fibrinogen  is  always  impure.  For  further  purification  the  precipitate 
is  pressed,  redissolved  in  an  8-per  cent  salt  solution,  the  filtrate  pre- 
cipitated by  a  saturated  salt  solution  as  above,  and  after  being  treated 
in  this  way  three  times,  the  precipitate  at  last  obtained  is  pressed  between 
filter-paper  and  finely  divided  in  water.  The  fibrinogen  dissolves  with 
the  aid  of  the  small  amount  of  NaCl  contained  in  itself,  and  the  solution 
may  be  made  salt-free  by  dialysing  with  very  faintly  alkaline  water.  The 
fibrinogen  can  be  almost  freed  from  fibrin-globulin,  -which  will  be  spoken 
of  later,  by  precipitating  with  double  the  volume  of  saturated  sodium- 
fluoride  solution,  redissolving  in  water  with  0.05-per  cent  ammonia, 
and  then  neutralizing  this  solution,  treated  with  NaCl,  and  repeating 
this  several  times.  Fibrinogen  may  also,  according  to  Reye,2  be  prepared 
by  fractionally  precipitating  the  plasma  with  a  saturated  solution  of 
ammonium  sulphate.  We  have  no  knowledge  as  to  the  purity  of  the 
fibrinogen  so  prepared.  The  methods  for  the  detection  and  quantitative 
estimation  of  fibrinogen  in  a  liquid  were  formerly  based  on  its  property 
of  yielding  fibrin  on  the  addition  of  a  little  blood,  of  serum,  or  of  fibrin 
ferment.  Reye  has  suggested  the  fractional  precipitation  with  ammonium 
sulphate  as  a  quantitative  method.  The  value  of  this  method  has  not 
been  sufficiently  tested. 

Fibrinogen  stands  in  close  relation  to  its  transformation  product, 
fibrin. 

Fibrin  is  the  name  of  that  protein  body  which  separates  on  the  so- 
called  spontaneous  coagulation  of  blood,  lymph,  and  transudates  as  well 
as  in  the  coagulation  of  a  fibrinogen  solution  after  the  addition  of  serum 
or  fibrin  ferment  (see  below). 

If  the  blood  is  beaten  during  coagulation,  the  fibrin  separates  in 
elastic,  fibrous  masses.     The  fibrin  of  the  blood-clot  may  be  beaten  to 

1  Zcitschr.  f.  physiol.  Chem.,  19. 

2  W.  Reye,  Ueber  Nachweifl  und  Bestimmung  des  Fibrinogens,  Inaug-Diss.  Strass- 
burg,  1898. 


FIBRIN.  255 

small,  less  elastic,  and  not  particularly  fibrous,  lumps.  The  typical 
fibrous  and  elastic  white  fibrin,  after  washing,  stands,  in  regard  to  its 
solubility,  close  to  the  coagulated  proteins.  It  is  insoluble  in  water, 
alcohol,  or  ether.  It  expands  in  hydrochloric  acid  of  1  p.  m.,  as  also  in 
caustic  potash  or  soda  of  1  p.  m.,  to  a  gelatinous  mass,  which  dissolves  at 
the  ordinary  temperature  only  after  several  days;  but  at  the  temperature 
of  the  body  it  dissolves  more  readily,  although  still  slowly.  Fibrin  may 
be  dissolved  by  dilute  salt  solutions,  after  a  long  time,  at  the  ordinary 
temperature,  or  much  more  readily  at  40°  C,  and  this  solution  takes  place, 
according  to  Arthus  and  Hubert  and  also  Dastre,1  without  the  aid 
of  micro-organisms.  This  action  is  due  to  proteolytic  enzymes  car- 
ried down  by  the  fibrin  or  enclosed  within  the  leucocytes  (Rulot2). 
According  to  Green  and  Dastre  3  two  globulins  are  formed  in  the  solu- 
tion of  fibrin  in  neutral  salt  solution,  and  according  to  Rulot  also  pro- 
teoses (and  peptones)  on  the  solution  of  fibrin  containing  leucocytes. 
Fibrin,  like  fibrinogen,  decomposes  hydrogen  peroxide,  due  to  a  con- 
tamination with  catalases,  but  this  property  is  destroyed  by  heating  or 
by  the  action  of  alcohol. 

What  has  been  said  of  the  solubility  of  fibrin  relates  only  to  the  typical 
fibrin  obtained  from  the  arterial  blood  of  oxen  or  man  by  whipping 
and  washing  first  with  water  and  with  common  salt  solution,  and  then 
with  water  again.  The  blood  of  various  kinds  of  animals  yields  fibrin 
with  somewhat  different  properties,  and  according  to  Fermi4  pig-fibrin 
dissolves  much  more  readily  than  ox-fibrin  in  hydrochloric  acid  of  5  p.  m. 
Fibrins  of  varying  purity  or  originating  from  blood  from  different  parts 
of  the  body  have  unlike  solubilities. 

The  fibrin  obtained  by  beating  the  blood,  and  purified  as  above 
described,  is  alwaj'S  contaminated  by  secluded  blood-corpuscles  or 
remains  thereof,  and  also  by  lymphoid  cells.  It  can  be  obtained  pure 
only  from  filtered  plasma  or  filtered  transudates.  For  the  preparation 
of  pure  fibrin,  as  well  as  for  the  quantitative  estimation  of  it,  the  spon- 
taneously coagulating  liquid  is  at  once,  or  the  non-spontaneously  coagu- 
lating liquid  only  after  the  addition  of  blood-serum  or  fibrin  ferment, 
thoroughly  beaten  with  a  whale-bone,  and  the  separated  coagulum  is 
washed  first  in  water  and  then  with  a  5-per  cent  common  salt  solution, 
and  again  with  water,  and  finally  extracted  with  alcohol  and  ether.  If 
the  fibrin  is  allowed  to  stand  for  some  time  in  contact  with  the  blood 
from  which  it  was  formed,  it  partly  dissolves  (fibrinolysis —  Dastre.5). 
This  fibrinolysis  must  be  prevented  in  the  exact  quantitative  estimation 

Arthus  and  Hubert,  Arch,  de  Physiol.  (5)  5;  Dastre,  ibid.,  (5)  7. 

2  Arch,  intern,  de  Physiol.,  1. 

3  Green,  Journ.  of  Physiol.,  8;  Dastre,  1.  c. 

4  ZeiHcl.r.  f.  Biologie,  28. 

5  Archives,  de  Physiol.  (5),  5  and  6. 


256  THE  BLOOD. 

of  fibrin  (Dastre).  The  blood  constituents  that  are  active  in  fibrinolysis 
are  still  unknown,  but  they  are  without  doubt  of  enzymotic  nature. 
It  must  be  mentioned  that  a  strong  fibrinolysis  takes  place  in  blood 
after  acute  phosphorus  poisoning  (Jacoby  and  others),  after  extirpation 
of  the  liver  (Nolf),  and  also  when  the  coagulability  of  the  blood  has  been 
reduced  by  the  injection  of  proteoses  (Nolf,  Rulot  1). 

A  pure  fibrinogen  solution  may  be  kept  at  the  ordinary  temperature 
until  putrefaction  begins  without  showing  a  trace  of  fibrin  coagula- 
tion. But  if  to  this  solution  is  added  a  water-washed  fibrin-clot  or  a 
little  blood-serum,  it  immediately  coagulates,  and  may  yield  a  perfect 
typical  fibrin.  The  transformation  of  the  fibrinogen  into  fibrin  requires 
the  presence  of  another  body  contained  in  the  blood-clot  and  in  the  serum. 
This  body,  whose  importance  in  the  coagulation  of  fibrin  was  first  observed 
by  Buchanan  2,  was  later  rediscovered  by  Alexander  Schmidt,3  and 
designated  as  fibrin  ferment  or  thrombin.  The  nature  of  this  enzymotic 
body  has  not  been  ascertained  with  certainty.  Even  after  careful 
purification  it  gives  very  faint  protein  reactions  and  it  is  a  much  disputed 
question  whether  it  is  a  globulin  or  a  nucleoprotein.  It  is  a  fact  that 
powerfully  active  solutions  of  thrombin  can  be  obtained  that  do 
not  give  either  the  reactions  for  globulins  or  nucleoproteins.  Fibrin  fer- 
ment is  produced,  according  to  Pekelharing,4  by  the  influence  of  soluble 
calcium  salts  on  a  preformed  zymogen  existing  in  the  non-coagulated 
plasma.  Schmidt  admits  the  presence  of  such  a  mother-substance 
of  the  fibrin  ferment  in  the  blood,  and  calls  it  prothrombin.  The  con- 
version of  this  mother-substance  into  thrombin  is  a  very  complicated 
process,  which  will  be  discussed  under  the  coagulation  of  the  blood. 
Thrombin  behaves  like  other  enzymes  in  that  the  very  smallest  amount  of 
it  produces  an  action,  and  its  solution  becomes  inactive  on  heating.  The 
velocity  of  coagulation  is  dependent  upon  the  quantity  of  thrombin, 
and  indeed  a  time  law  has  been  proposed  for  the  action  of  thrombin. 
According  to  Fuld  the  action  of  thrombin,  at  least  within  certain  limits, 
follows  Schutz's  law,  and  according  to  Stromberg  the  thrombin  follows 
in  its  action  a  time  law,  which  at  least  in  the  beginning,  corresponds  to 

1  Jacoby,  Zeitschr.  f.  physiol.  Chem.,  30;  Nolf,  Arch,  intern,  de  Physiol.,  3,  1905; 
Rulot,  1.  c. 

2  London  Med.  Gazette,  1845,  617.     Cit.  by  Gamgee,  Journal  of  Physiol,  1879. 

3  Pfliiger's  Arch.,  6;  see  also  Zur  Blutlehre,  1892,  and  Weitere  Beitriige  zur  Blut- 
lehre,  1895. 

*  Pekelharing,  Verhandl.  d.  Kon.  Akad.  d.  Wetensch.  te  Amsterdam,  1892,  Deel  1; 
ibid.,  1895,  and  Centralbl.  f.  Physiol.,  9;  Wright,  Proc.  Roy.  Irish  Acad.  (3),  2;  The 
Lancet,  1892,  and  On  Wooldridge's  Method,  etc.,  British  Med.  Journal,  1891;  Lilien- 
feld,  Hamatol.  Untersuch.  Arch.  f.  (Anat.  u.)  Physiol.,  1892;  Ueber  Leukocyten  und 
Blutgerinnung,  ibid.;  Halliburton  and  Brodie,  Journal  of  Physiol.,  17  and  18;  Huis- 
kamp,  Zeitschr.  f.  physiol.  Chem.,  32;  Pekelharing  and  Huiskamp,  ibid.,  39. 


FIBRIN  FORMATION.  257 

ScHtJTz's  law  while  on  increasing  dilution  deviates  more  and  more 
and  finally  shows  a  proportionally  slow,  and  more  irregular  procedure. 
Martin  l  has  found  another  law  from  experiments  with  plasma  and  snake- 
poisons  containing  thrombin.  According  to  him  the  behavior  is  as 
follows:  As  in  the  casein  coagulation  with  rennin,  the  celerity  of  coagula- 
tion is  inversely  proportional  to  the  quantity  of  ferment;  and  Loeb 
has  observed  a  similar  conduct  with  invertebrates.  The  optimum  of  the 
thrombin  action  lies  at  about  40°  C;  at  70-75°  C,  in  neutral  solution, 
the  enzyme  is  destroyed.  According  to  Howell  and  Rettger  2  throm- 
bin, under  proper  conditions,  can  withstand  boiling  for  a  short  time.  The 
question  as  to  whether  the  thrombin  found  in  different  animals  is  the 
same  substance  or  whether  we  have  several  thrombins,  has  not  been 
decided.  The  latter  is  not  improbable;  nevertheless  a  definite  specificity 
of  different  thrombins  has  not  been  observed  with  certainty. 

The  isolation  of  thrombin  has  been  tried  in  several  ways.  Ordinarily, 
it  may  be  prepared  by  the  following  method,  proposed  by  Alex.  Schmidt: 
Precipitate  the  serum  or  defibrinated  blood  with  15-20  vols,  of  alcohol 
and  allow  it  to  stand  a  few-  months.  The  precipitate  is  then  filtered 
off  and  dried  over  sulphuric  acid.  The  ferment  may  be  extracted  from 
the  dried  powder  by  means  of  water.  Other  methods  have  been  suggested 
by  Hammarsten,  Pekelharing,  and  Howell.3  According  to  a  method 
suggested  by  Hammarsten  a  solution  of  thrombin  so  poor  in  lime  salts 
that  it  contains  only  0.3-0.4  p.  m.  solids  and  about  0.0007  p.  m.  CaO 
can  be   prepared. 

If  a  fibrinogen  solution  containing  salt,  as  above  prepared,  is  treated 
with  a  solution  of  thrombin,  it  coagulates  at  the  ordinary  temperature 
more  or  less  quickly  and  yields  a  typical  fibrin.  Besides  the  thrombin, 
the  presence  of  neutral  salts  is  necessary,  for  Alex.  Schmidt  has  shown 
that  fibrin  coagulation  does  not  take  place  without  them.  The  presence 
of  soluble  calcium  salts  is  not,  as  is  generally  assumed,  a  positive  con- 
dition for  the  formation  of  fibrin,  because,  thrombin  can  transform 
fibrinogen  into  typical  fibrin  in  the  absence  of  lime  salts  precipitable  by 
oxalate.4  The  fibrin  is  not  richer  in  lime  than  the  fibrinogen  used  in  its 
preparation  if  the  fibrinogen  and  thrombin  solutions  are  employed  as  lime- 
free  as  possible,  and  the  view  that  the  fibrin  formation  is  connected  with 
a  taking  up  of  lime  has  been  shown  to  be  untenable  (Hammarsten). 
The  quantity  of  fibrin  obtained  on  coagulation  is  always  smaller  than 

1  Martin,  Journ.  of  Physiol.,  32;  Fuld,  Hofmeister's  Beitrage,  2;  Loeb,  ibid.,  9; 
Stromberg,  Biochem.  Zeitschr.,  37. 

2  Howell,  Amer.  Journ.  of  Physiol.,  26;  Rettger,  ibid.,  24. 

3  Hammarsten,  ibid.,  18;  Pekelharing,  1.  c;  Howell,  1.  c. 

*  See  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  22,  which  also  cites  the  works  of 
Schmidt  and  Pekelharing,  and  ibid.,  28. 


258  THE  BLOOD. 

the  amount  of  fibrinogen  from  which  the  fibrin  is  derived,  and  we  always 
find  a  small  amount  of  protein  substance  in  the  solution.  It  is  therefore 
not  improbable  that  the  fibrin  coagulation,  in  accordance  with  the  views 
first  proposed  by  Denis,  is  a  cleavage  process  in  which  the  soluble  fibrinogen 
is  split  into  an  insoluble  protein,  the  fibrin,  which  forms  the  chief  mass, 
and  a  soluble  protein  substance  which  is  produced  only  in  small  amounts. 
We  find  a  globulin-like  substance  which  coagulates  at  about  64°  C.  in 
blood-serum  as  well  as  in  the  serum  from  coagulated  fibrinogen  solutions. 
This  substance  is  called  fibrin-globulin  by  Hammarsten.  The  investiga- 
tions of  Huiskamp  have  shown  that  this  substance  is  not  formed  as  a 
cleavage  product  from  pure  fibrinogen,  but  occurs  in  plasma  or  in  fibrinogen 
solutions  not  purified  from  sodium  fluoride  or  perhaps  in  loose  com- 
bination with  fibrinogen.  The  view  that  a  cleavage  takes  place  in  the 
coagulation  of  the  fibrinogen  has  not  been  supported  by  these  investi- 
gations.1 

Opinions  are  not  unanimous  in  regard  to  the  enzyme  nature  of  throm- 
bin and  the  enzymotic  formation  of  fibrin,  and  there  are,  indeed,  investiga- 
tors who  consider  the  coagulation  as  another  process.  A  more  thorough 
discussion  of  this  subject  can  take  place  only  in  connection  with  the 
coagulation  of  the  blood. 

Nucleoprotein.  This  substance,  which,  as  above-mentioned,  is  considered 
by  Pekelharing  and  Huiskamp  as  identical  with  the  prothrombin  or  thrombin, 
occurs  in  the  blood-plasma  as  well  as  in  the  serum,  and  is  precipitated  from  the 
latter  with  the  globulin.  It  is  similar  to  the  globulin  in  that  it  is  readily  soluble 
in  neutral  salt  solution,  and  can  be  completely  salted  out  on  saturation  with 
magnesium  sulphate,  and  separates  only  incompletely  on  dialysis.  It  is  much 
less  soluble  than  serglobulin  in  an  excess  of  dilute  acetic  acid,  and  coagulates 
at  65-69°  C.  C.  G.  Liebermeister  2  found  only  0.08-0.09  per  cent  phosphorus 
in  the  nucleoprotein,  which  indicates  that  the  nucleoprotein  was  contaminated 
with  other  proteins.  He  also  found  that  the  substance  was  soluble  in  acetic  acid 
with  difficulty,  a  property  which  is  used  by  Pekelharing  as  an  important  means 
of  separating  the  compound  proteins  from  the  globulins. 

Serglobulins,  also  called  paraglobulin  (Kuhne),  fibrinoplastic  substance 
(Alex.  Schmidt),  serum-casein  (Panum3),  occur  in  the  plasma,  serum, 
lymph,  transudates  and  exudates,  in  the  white  and  red  corpuscles,  and 
probably  in  many  animal  tissues  and  form-elements,  though  in  small 
quantities.     They  are  also  found  in  the  urine  in  many  diseases. 

The  so-called  serglobulin  is  without  doubt  not  an  individual  sub- 
stance, but  consists  of  a  mixture  of  two  or  more  protein  bodies  which 

1  See  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  28;  Heubner,  Arch.  f.  exp.  Path, 
u.  Pharm.,  4'J,  ami  Zeitschr.  f.  physiol.  Chem.,  45;  Huiskamp,  ibid.,  44  and  40. 

-  Hofineister'.s  Beitnijie,  8;  Pekelharing  and  Huiskamp,  1.  c.  footnote  1,  page  256. 

:t  Kii!, no,  Lehrbucb  d.  physiol.  Chem.,  Leipzig.,  1866-68;  Alex.  Schmidt,  Arch.  f. 
(Anat.  n. )  Physiol.,  1861-62;  Panum,  Virchow's  Arch.,  3  and  4. 


SEKGLOBULINS.  259 

cannot  be  completely  and  positively  separated  from  each  other.  The 
mixture  of  globulins  obtained   from   blood-plasma  or  blood-serum   hy 

saturation  with  magnesium  sulphate  or  half-saturation  with  ammonium 
sulphate  consists  of  nucleoprotein,  fibrin-globulin,  and  the  true  serglobulin 
or  mixture  of  globulins. 

The  nucleoprotein  has  been  previously  discussed.  The  fibrin-globulin, 
which  occurs  in  the  serum  only  in  small  amounts,  can  be  completely  pre- 
cipitated by  NaCl.  It  has  the  general  properties  of  the  globulins,  but 
differs  from  the  serglobulins  by  a  lower  coagulation  temperature,  64- 
66°  C,  and  also  in  that  it  is  precipitated  by  (NH^SCU  even  in  28  per 
cent  solution. 

Serglobulins.  If  the  globulin  obtained  by  saturation  with  magnesium 
sulphate  is  dialyzed,  then,  as  has  been  known  for  a  long  time  and  further 
substantiated  by  Marcus,  only  a  part  of  the  globulin  separates  out, 
while  a  portion  remains  in  solution  and  cannot  be  precipitated  by  the 
addition  of  acid.  For  this  reason  Marcus  1  also  differentiates  between 
a  water-soluble  globulin  and  one  insoluble  in  water.  According  to  the 
later  investigations  of  Hofmeister  and  Pick  2  the  part  insoluble  in 
water  corresponds  chiefly  to  a  globulin  fraction  readily  precipitated  by 
(NH4)2S04  (by  28-36  vols,  per  cent  saturated  solution),  and  the  part 
soluble  in  water  corresponds  to  a  fraction  difficult  to  precipitate  (by 
36-44  vols,  per  cent  saturated  solution).  The  first  fraction  is  called 
euglobulin  and  the  second  pseudoglobulin.  According  to  Porges  and 
Spiro  3  the  serglobulins  can  be  separated  by  (NHU^SO-t  into  three 
fractions  whose  precipitation  limits  are  28-36,  33-42,  and  40-46  vols, 
per  cent  saturated  solution.  All  three  fractions  contain  globulin  insoluble 
in  water.  Freund  and  Joachim4  have  found  that  the  euglobulin  as 
well  as  the  pseudoglobulin  fraction  is  a  mixture  of  globulin  soluble  in 
water  and  globulin  insoluble  in  water,  and  consequently  the  number 
of  different  globulins  in  the  serum  may  be  still  greater. 

It  follows  from  all  these  investigations  that  either  the  difference  between 
the  globulin  soluble  in  water  and  that  insoluble  is  not  sufficient  or  that  the  frac- 
tional precipitation  with  ammonium  sulphate  is  not  suited  for  the  separation 
of  the  various  globulins.  This  latter  seems  to  be  the  case,  as  shown  by  Mellanby 
Haslam  and  recently  by  Wiener.5  It  must  not  be  forgotten  that  the  globulin 
fractions  are  always  contaminated  with  other  serum  constituents,  and  that  these 
may  influence  the  solubility  and  precipitability.  As  Hammarsten  has  shown, 
a  water-soluble  globulin,  can  be  transformed  into  a  globulin  insoluble  in  water 

1  Zeitschr.  f.  physiol.  Chem.,  28. 

2  Hofmeister's  Beit  rage,  1. 
■  Hofmeister's  Beit  rage,  3. 

4  Zeitschr.  f.  physiol.  Chem.,  36. 

5  Mellanby,  Journ.  of  Physiol.,  36;  Haslam,  ibid.,  32;  Wiener,  Zeitschr.  f.  physiol 
Chem.,  74. 


260  THE  BLOOD. 

by  careful  purification,  and  also  the  reverse,  namely,  a  globulin  insoluble  in 
water  can  sometimes  be  converted  into  one  soluble  in  water  by  allowing  it  to  lie 
in  the  air.  An  insoluble  protein  like  casein  can  also,  according  to  Hammarsten,1 
have  the  solubilities  of  a  globulin  due  to  contamination  with  constituents  of  the 
serum,  and  K.  Morner  -  has  also  shown  that  a  contamination  of  the  serum- 
globulins  with  soap  can  essentially  modify  the  precipitation  of  these  globulins. 
Under  these  circumstances  the  above  assumptions  in  regard  to  the  different 
globulin  fractions  must  be  accepted  with  great  caution. 

The  investigations  made  thus  far  upon  the  so-called  serglobulin, 
have  not  led  to  any  positive  results.  That  this  globulin,  with  the 
exception  of  the  enzymes,  antienzymes,  immune  bodies,  and  other  . 
unknown  substances  which  are  carried  down  by  the  various  fractions,  is 
a  mixture  of  globulins  there  seems  to  be  no  doubt.  The  serglobulin  or 
the  globulin  mixture  which  is  obtained  from  the  serum  by  the  methods 
to  be  described  has  the  following  properties: 

In  a  moist  condition  it  forms  snow-white  flaky  masses,  neither  tough 
nor  elastic,  which  always  contain  thrombin  and  hence  can  bring  about 
coagulation  in  a  fibrinogen  solution.  The  neutral  solution  is  only  incom- 
pletely precipitated  by  NaCl  added  to  saturation,  and  is  not  precipitated 
by  an  equal  volume  of  a  saturated  salt  solution.  It  is  only  partly 
precipitated  by  dialysis  or  by  the  addition  of  acid.  On  saturation  with 
magnesium  sulphate,  or  one-half  saturation  with  ammonium  sulphate 
a  complete  precipitation  is  obtained.  The  coagulation  temperature 
is,  writh  5-10  per  cent  NaCl  in  solution,  69-76°,  but  more  often  75°  C. 
The  specific  rotation  of  the  solution  containing  salt  is  (a)D=— 47.8° 
for  the  serglobulin  from  ox-blood  (Fredericq3).  The  various  globulin 
fractions  do  not  differ  essentially  from  each  other  in  their  coagulation 
temperatures,  specific  rotation,  refraction  coefficient  (Reiss4),  and  their 
elementary  composition.  The  average  composition  is,  according  to 
Hammarsten,  C.  52.71,  H  7.01,  N  15.85,  S  1.11  per  cent.  K.  Morner5 
found  1.02  per  cent  sulphur  and  0.67  per  cent  lead-blackening  sulphur. 
All  the  sulphur  seems  to  exist  as  cystine. 

Serglobulin  contains,  as  K.  Morner  first  showed,  a  carbohydrate 
group  wdiich  can  be  split  off.  Langstein  6  has  obtained  several  car- 
bohydrates   from    the    blood-globulin,    namely,    glucose,    glucosamine, 


1  See  Hammarsten,  Ergebnisse,  d.  Physiol.,  1,  Abt.  1. 

2  Zeitschr.  f.  physiol.  Chem.,  34. 

3  Bull.  Acad.  Roy.  de  Belg.  (2),  50.  In  regard  to  paraglobulin,  see  Hammarsten, 
Pfliiger's  Arch.,  17  and  18,  and  Ergebnisse  d.  Physiol,  1,  Abt.  1. 

4  Hofmeister's  Beitrage,  4. 

6  Zeitsrhr.  f.  physiol.  Chem.,  34. 

8  Morner,  Centralbl.  f.  Physiol.,  7;  Langstein,  Munch,  med.  Wochenschr.,  1902, 
1876,  and  Wien.  Sitzungsber.,  112,  Abt.  lib,  1903;  Monatsheft  f.  Chem.,  25;  Hof- 
meister's  Beitrage,  6;  see  also  footnote  5,  p.  84. 


SERALBUMIN*.  261 

and  carbohydrate  acids  of  unknown  kinds.  It  has  not  boon  shown 
whether  these  small  amounts  of  carbohydrate  are  derived  from  the  globulin 
or  from  other  contaminating  bodies.  According  to  Zanetti  and  also 
Bywaters,  the  blood-serum  contains  a  glucoproteid,  seromucmd,  and 
the  investigations  of  Eichholz  *  seem  to  show  that  the  globulins  are 
contaminated  by  a  glucoproteid.  According  to  Langstein  the  sugar 
is  not  only  mixed  with  the  globulin,  but  it  exists  in  a  combined  form, 
probably  in  loose  combination. 

Serglobulin  (the  euglobulin)  may  be  easily  separated  as  a  fine  floc- 
eulent  precipitate  from  blood-serum  by  neutralizing  or  making  faintly 
acid  with  acetic  acid  and  then  diluting  with  10-20  vols  of  water.  For 
further  purification  this  precipitate  is  dissolved  in  dilute  common  salt 
solution,  or  in  water  with  the  aid  of  the  smallest  possible  amount  of 
alkali,  and  then  reprecipitated  by  diluting  with  water  or  by  the  addition 
of  a  little  acetic  acid.  All  the  serglobulin  may  also  be  separated  from 
the  serum  by  means  of  magnesium  or  ammonium  sulphate;  in  these 
cases  it  is  difficult  to  completely  remove  the  salt  by  dialysis.  As  long 
as  we  are  not  agreed  as  to  the  number  of  globulins  in  the  serum,  it  is 
not  necessary  to  give  a  method  of  separating  the  various  globulins  in  this 
mixture.  Thus  far  the  fractional  precipitation  with  (NH^SCU  has 
chiefly  been  used.  The  serglobulin  from  blood-serum  is  always  contam- 
inated with  lecithin  and  thrombin.  A  serglobulin  free  from  thrombin 
may  be  prepared  from  ferment-free  transudates,  as  sometimes  from 
hydrocele  fluids,  and  this  shows  that  serglobulin  and  thrombin  are  dif- 
ferent bodies.  For  the  detection  and  the  quantitative  estimation  of 
serglobulin  we  may  use  the  precipitation  by  magnesium  sulphate  added 
to  saturation  (Hammarsten),  or  by  an  equal  volume  of  a  saturated 
neutral  ammonium-sulphate  solution  (Hofmeister  and  Kauder  and 
Pohl2).  In  the  quantitative  estimation  the  precipitate  is  collected  on  a 
weighed  filter,  washed  with  the  salt  solution  employed,  dried  with  the 
filter  at  about  115°  C,  then  washed  with  boiling-hot  water,  so  as  to 
completely  remove  the  salt,  extracted  with  alcohol  and  ether,  dried, 
weighed,  and  incinerated  to  determine  the  ash.  This  method,  according 
to  the  investigations  of  Wiener,3  can  only  be  used  when  the  serum  is 
sufficiently  diluted  with  water. 

Seralbumins  are  found  in  large  quantities  in  blood-serum,  blood- 
plasma,  lymph,  transudates,  and  exudates.  Probably  they  also  occur 
in  other  animal  fluids  and  tissues.  The  proteins  which  pass  into  the 
urine  under  pathological  conditions  consist  largely  of  seralbumin. 

The  seralbumin,  like  the  serglobulin,  seems  also  to  be  a  mixture  of 
at  least  two  protein  bodies.     The  preparation  of  crystalline  seralbumin 


1  Zanetti,  Chem.  Centralbl.,  189S,  I,  p.  624;    Bywaters,  Journ.  of  Physiol.,  35,  and 
Biochem.  Zeitschr.,  15;  Eichholz,  Journ.  of  Physiol.,  23. 

2  Hammarsten,  1.  c;  Hofmeister,  Kauder  and  Pohl,  Arch.  f.  exp.  Path.  u.  Pharm.,  20. 

3  Zeitschr.  f.  physiol.  Chem.,  74. 


262  THE  BLOOD. 

(from  horse-serum)  was  first  performed  by  Gurber.  It  crystallizes 
with  difficulty  from  other  blood-sera  (Gruzewska).  Even  from  horse- 
serum  only  a  portion,  according  to  Robertson  1  not  more  than  40  per 
cent,  of  the  albumin  can  be  obtained  as  crystals,  and  it  is  also  pos- 
sible that  the  amorphous  albumin,  which  is  precipitated  by  ammo- 
nium sulphate  with  difficulty,  represents  two  seralbumins  (Maximo- 
witsch).  According  to  Gurber  and  Michel  it  would  seem  that  the 
crystalline  seralbumin  is  also  a  mixture,  but  this  is  disproved  by  the  obser- 
tions  of  Schulz,  Wichmann,  and  Krieger2.  We  know  nothing  as 
to  the  behavior  of  the  amorphous  fraction  of  the  seralbumin  in  this  respect. 
Because  of  the  different  coagulation  temperatures,  Halliburton  claims 
the  existence  of  three  different  albumins  in  the  blood-serum,  a  view 
which  has  been  disputed  by  several  experimenters,  and  recently  by 
Hougardy.  Oiv.  the  other  hand,  the  earlier  investigations  of  Kauder, 
as  well  as  the  more  recent  work  of  Oppenheimer,3  seem  to  indicate  a 
non-unit  nature  of  the  seralbumins,  but  this  question  is  still  an  open 
one. 

The  crystalline  seralbumin  may  perhaps  be  a  combination  with 
sulphuric  acid  (K.  Morner,  Inagaki).  The  coagulated  albumin  obtained 
from  the  aqueous  solution  of  the  crystals  with  the  aid  of  alcohol  has 
almost  the  same  elementary  composition  (Michel)  as  the  amorphous 
mixture  of  albumin  prepared  from  horse-serum  (Hammarsten  and 
K.  Starke4).  The  average  composition  was  C  53.06,  H  6.98.  N  15.99, 
S  1.84  per  cent.  K.  Morner,  after  the  removal  of  the  sulphuric  acid 
from  crystalline  albumin,  found  1.73  per  cent  total  sulphur,  which  prob- 
ably exists  only  as  cystine.  Langstein  5  has  been  able  to  split  off  a  nitrog- 
enous carbohydrate  (glucosamine)  from  crystalline  seralbumin.  The 
quantity  was  so  small  that  the  question  is  still  undecided  whether  or 
not  the  carbohydrate  was  a  contamination.  The  fact  that  Abder- 
halden,  Bergell,  and  Dorpinghaus  6  were  able  to  prepare  a  seral- 
bumin entirely  free  from  carbohydrate  and  which  did  not  respond  to 
Molisch's  very  delicate  reaction,  seems  to  be  decisive  on  this  point. 
The  specific  rotation  of  crystalline  seralbumins  from  horse-serum  was 
found  by  Michel  to  be  (a)D=— 61  to  61.2°,  and  by  Maximowitsch  on 
the  contrary  (o:)D= —47.47°. 

1  Journ.  of  biol.  Chem.,  13. 

2  In  regard  to  the  literature  on  the  crystalline  seralbumins,  see  Schulz,  Die  Kristal- 
lisation  von  Eiweissstoffen,  Jena,  1901;  Maximowitsch,  Maly's  Jahresber.,  31,  35. 

3  Halliburton,  Journ.  of  Physiol.,  5  and  7;  Hougardy,  Centralbl.  f.  Physiol.,  15, 
665;  Oppenheimer,  Verhandl.  d.  physiol.  Gesellsch.,  Berlin,  1902. 

4  Michel,  Verhandl.  d.  phys-med.  Gesellsch.  zu  Wiirzburg,  29,  No.  3;  K.  Starke,. 
Maly's  Jahresber.,  11;  K.  Morner,  1.  c;  Inagaki,  Biochem,  Centralbl.,  4,  p.  515. 

6  K.  Morner,  1.  c;  Langstein,  Hofmeister's  Beitrage,  1. 
•  Zeitschr.  f.  physiol.  Chem.,  41. 


SERUM    PROTEINS.  2G3 

The  crystalline  and  amorphous  seralbumin  in  aqueous  solution  give 
the  ordinary  albumin  reactions.  The  coagulation  temperature  of  a 
1-per  cent  solution,  poor  in  salts  is  about  50°  C,  but  rises  with  the  quan- 
tity of  salt.  The  coagulation  of  the  mixture  of  albumins  from  serum 
generally  takes  place  at  70-85°  C,  but  is  essentially  dependent  upon 
the  reaction  and  the  amount  of  salt  present.  Up  to  the  present  time  no 
seralbumin  solution  has  been  prepared  free  from  mineral  bodies.  A 
solution  as  free  from  salts  as  possible  does  not  coagulate  either  on  boil- 
ing or  on  the  addition  of  alcohol.  On  the  addition  of  a  little  common 
salt  it  coagulates  in  both  cases.1 

Seralbumin  differs  from  the  albumin  of  the  white  of  the  hen's  egg  in 
the  following  particulars:  It  is  more  levogyrate;  the  precipitate  formed 
by  hydrochloric  acid  easily  dissolves  in  an  excess  of  the  acid;  it  is  rendered 
less  insoluble  by  alcohol. 

In  preparing  the  seralbumin  mixture,  first  remove  the  globulins, 
according  to  Johansson,  by  saturating  with  magnesium  sulphate  at 
about  30°  C.  and  filtering  at  the  same  temperature.  The  cooled  filtrate 
is  separated  from  the  crystallized  salt  and  is  treated  with  acetic  acid  so 
that  it  containes  about  1  per  cent.  The  precipitate  formed  is  filtered 
off,  pressed,  dissolved  in  water  with  the  addition  of  alkali  to  neutral 
reaction,  and  the  solution  freed  from  salt  by  dialysis.  The  mixture  of 
albumins  may  be  obtained  in  a  solid  form  from  the  dialyzed  solution, 
either  by  evaporating  the  solution  at  a  gentle  temperature  or  by  pre- 
cipitating with  alcohol,  which  must  be  quickly  removed.  Starke  2 
has  suggested  another  method,  which  is  also  to  be  recommended.  The 
crystalline  seralbumin  may  be  prepared  from  serum  freed  from  globulin 
by  half  saturating  with  ammonium  sulphate,  by  the  addition  of  more 
salt  until  a  cloudiness  appears,  and  then  proceeding  according  to  the 
suggestion  of  Gurber  and  Michel.  On  acidifying  with  acetic  acid 
or  sulphuric  acid  the  crystallization  may  be  considerably  accelerated.3 
The  quantity  of  seralbumin  is  best  calculated  as  the  difference  between 
the  total  proteins  and  the  globulins.  A  method  for  the  quantitative 
estimation  of  globulins  and  albumins  in  blood  serum  by  refractometric 
means  has  been  suggested  by  Robertson.4 

Summary  of  the  elementary  composition  of  the  above-mentioned  and  described 
proteins  (from  horse-blood): 

c            h  n  s  0 

Fibrinogen 52 .  93  6 .  90  16 .  66  1 .  25        22 .  26      (Hammarsten) 

Fibrin 52.68  6.83  16.91  1.10        22.48 

Fibrin-globulin 52.70  6.98  16.06         

Serglobulin 52.71  7.01  15.85  1.11         23.32 

Seralbumin 53 .  CS  7.10  15 .  93  1 .  90        21 .86      (Michel) 

'In  regard  to  the  relationship  of  neutral  salts  to  heat  coagulation,,  see  J.  Starke, 
Sitzungsber.  d.  Gesellsch.  f.  Mori>h.  u.  Physiol,  in  Munehen,  1897. 

2  Johansson,  Zeitschr.  f.  fhvsiol.  Chem.,  9;  K.  Staike,  Maly's  Jahresber.,  11. 

3  See  Hopkins  and  Pinkus,  Journ.  of  Physiol.,  23;  Krieger,  I'eber  die  Darstellung. 
krystallinscher  tierischer  Eiweissstoffe,  Inaug. -Dissert.  Strassburg,  1899. 

*  Journ.  of  biol.  Chem.,  11. 


204  THE  BLOOD. 

Proteose-like  substances  have  been  found  in  blood-serum  by  several 
investigators,  and  Nolf  !  has  shown  that  after  the  abundant  introduc- 
tion of  proteoses  into  the  intestine,  they  pass  into  the  blood.  Bor- 
chardt  2  has  also  been  able  to  show  that  not  only  after  the  introduction 
of  elastin-proteose  per  os,  but  also  after  feeding  dogs  with  not  over- 
abundant quantities  of  elastin,  a  proteose,  hemielastin,  passes  into  the 
blood  and  can  indeed  be  eliminated  in  the  urine.  The  question  whether 
the  proteoses  are  normal  constituents  of  the  blood  under  ordinary  con- 
ditions is  still  much  disputed.  The  difficulty  in  deciding  this  ques- 
tion lies  in  the  fact  that  in  the  removal  of  the  proteins  a  small  amount 
of  proteose-like  substance  is  formed  from  other  proteins  (namely  from  the 
globin  of  the  blood  pigment),  and  on  the  other  hand  the  proteoses  can  be 
precipitated  with  the  other  bodies.  The  question  as  to  the  physiological 
occurrence  of  proteoses  in  the  blood  or  plasma  must  be  considered  as 
still  undecided.3 

In  close  relation  to  the  proteoses  stands  perhaps  the  above-men- 
tioned seromucoid,  which  was  discovered  by  Zanetti  and  especially 
studied  by  Bywaters.  It  is  a  glycoprotein  which  is  soluble  in  water, 
and  precipitated  by  alcohol.  Seromucoid  contains,  according  to  By- 
waters,4  11.6  per  cent  N,  1.8  per  cent  S,  and  yields  approximately  25 
per  cent  glucosamine.     The  quantity  in  the  blood  is  0.2-0.9  p.  m. 

The  Blood-serum. 

As  above  stated,  the  blood-serum  is  the  clear  liquid  which  is  pressed 
out  by  the  contraction  of  the  blood-clot.  It  differs  chiefly  from  the 
plasma  in  the  absence  of  fibrinogen  and  in  containing  an  abundance 
of  fibrin  ferment.  Otherwise  considered  qualitatively,  the  blood-serum 
contains  the  same  chief  constituents  as  the  blood-plasma. 

Blood-serum  is  a  sticky  liquid  which  is  more  alkaline  toward  litmus 
than  the  plasma.  The  specific  gravity  in  man  is  1.027  to  1.032,  average 
1.028.  The  color  is  more  or  less  yellow;  in  human  blood-serum  it  is 
pale  yellow  with  a  shade  toward  green,  and  in  horses  it  is  often  amber- 
yellow.  The  serum  is  ordinarily  clear;  after  a  meal  it  may  be  opales- 
cent, cloudy,  or  milky  white,  according  to  the  amount  of  fat  contained 
in  the  food. 

Besides  the  above-mentioned  bodies,  the  following  constituents  are 
found  in  the  blood-plasma  or  blood-serum: 

1  Bull.  Acad.  Roy.  Belg.,  1903  and  1904. 

2  Zeitschr.  f.  physiol.  Chem.,  51  and  57. 

3  See  especially  Abderhalden,  Zeitschr.  f.  physiol.  Chem.,  51,  and  Biochem.  Zeitschr., 
8  and  10,  and  E.  Freund,  ibid.,  7  and  9,  which  also  contains  the  literature. 

4  Biochem.  Zeitschr.,  15. 


BLOOD  SERUM.  265 

Fat  occurs  from  1-7  p.  m.  in  fasting  animals.  After  partaking  of 
food  the  amount  is  increased  to  a  great  extent.  Fatty  acids,  or  soaps, 
glycerin  (Nicloux,  Fr.  Tangl,  and  St.  Weiser  l)  phosphatides  and 
cholesterin  are  also  found.  Cholesterin  occurs,  according  to  Hurthle  2, 
at  least  in  part,  as  fatty-acid  esters  (serolin  according  to  Boudet).  Accord- 
ing to  Letsche3  free  cholesterin  probably  also  occurs  in  the  serum. 

Sugar  seems  to  be  a  physiological  constituent  of  the  plasma  and 
serum.  According  to  the  investigations  of  many  workers4  the  sugar 
found  is  glucose.  Strauss5  has  also  detected  fructose  in  blood-serum 
and  in  transudates  and  exudates.  The  question  as  to  the  occurrence 
of  other  varieties  of  sugar,  such  as  isomaltose  (Pavy  and  Siau)  and  pen- 
tose (Lepine  and  Boulud6),  in  blood  serum  is  still  undecided.  Asher 
and  Rosenfeld  and  Michaelis  and  Rona  in  a  more  conclusive  manner, 
have  shown  that  at  least  a  considerable  part  of  the  sugar  can  1  le  removed 
from  the  blood  by  dialysis,  hence  it  must  exist  in  solution  in  the  free 
state.  These  observations  do  not  exclude  the  possibility  of  the  existence 
of  another  part  of  the  sugar  which  is  in  combination  with  protein. 
Lepine  and  Boulud  7  could  only  obtain  a  diffusion  of  the  sugar  by  a 
short  dialysis  from  serum  12  hours  old,  but  not  from  perfectly  fresh 
serum,  an  observation  which  somewhat  diminishes  the  conclusiveness 
of  Michaelis  and  Rona's  experiment  with  24-hour  dialysis.  A  fur- 
ther testing  of  this  question  is  therefore  very  desirable. 

The  quantity  of  sugar  in  the  serum  or  plasma  is  for  man  0.6-1  p.  m. 
calculating  the  total  reduction  as  glucose,  and  in  animals  about  the  same 
but  in  rabbits  considerably  higher  or  2.2  p.  m.8  Besides  the  sugar,  the 
blood  contains,  as  first  shown  by  J.  Otto,  also  another  or  perhaps  several 
reducing  substances,  a  part  existing  in  the  serum  and  another  part  in  the 
blood-corpuscles.  We  will  discuss  the  nature  of  these  bodies  as  well 
as  the  so-called  virtual  sugar  and  glycolysis  in  speaking  of  the  division 
of   the  sugar  in  the  blood-corpuscles    and  plasma    in    connection   with 


1  Nicloux,  Compt.  Rend.  soc.  biol.,  55;  Tangl  and  St.  Weiser,  Pfluger's  Arch.,  115. 

2  Hurthle,  Zeitschr.  f.  physiol.  Chem.,  21,  where  Boudet  is  also  cited.     In  regard 
to  the  quantity  of  these  esters  in  bird-serum,  see  Brown,  Amer.  Journ.  of  Physiol.,  2. 

3  Zeitschr.  f.  physiol.  Chem.,  53. 

4  See  v.  Mering,  Arch.  f.  (Anat.  u.)  Physiol.,  1877  (this  article  contains  numerous 
references);  Seegen,  Pfluger's  Arch.,  40;  Miura,  Zeitschr.  f.  Biologie,  32. 

8  Fortschritte  d.  Mediz.,  1902. 

6  Pavy  and  Siau,  Journ.  of  Physiol.,  26;   Lepine  and  Boulud,  Compt.  Rend.,  133, 
135,  and  136. 

7  Rosenfeld,  Centralbl.  f.  Physiol.,  19,  p.  449;    Lupine  and  Boulud,  Compt.  Rend., 
143;  Asher,  Biochem.  Zeitschr.,  3;  Michaelis  and  Rona,  ibid.,  14. 

8  See  E.  Frank,  Zeitschr.  f.  physiol.  Chem.,  70;    Lyttkens  and  Sandgren,  Bioch. 
Zeitschr.,  21  and  26. 


266  THE  BLOOD. 

the  total  blood.     The  same  applies   to  the  conjugated  glucuronic  acids, 
which  it  seems,  originate  from  the  form-elements. 

The  blood-plasma  and  the  serum,  as  well  as  the  lymph  also  contain 
enzymes  of  various  kinds.  According  to  Rohmann,  Bial,  Hamburger,1 
and  others,  diastases,  which  convert  starch  and  glycogen  into  maltose  or 
isomaltose,  as  well  as  a  maltase,  are  found  in  the  blood.  The  diastase, 
whose  quantity  is  very  variable  in  the  blood  of  different  animals,  seems 
at  least  in  great  part,  to  originate  in  the  pancreas  but  can  also  come 
from  other  organs  and  according  to  Haberlandt  also  from  the  leucocytes.2 
Hanriot  and  others  have  detected,  in  the  serum,  lipases  or  esterases  which 
decompose  butyrin  and  neutral  fats  and  other  esters.  The  occurrence 
of  butyrinases  which  split  mono-  as  well  as  tributyrin  has  been  recently 
substantiated  by  Rona  and  Michaelis,  while  the  property  of  this  lipase 
of  splitting  olein  and  other  neutral  fats  is  not  generally  acknowledged 
(Arthus,  Doyon  and  Morel3). 

This  lipolytic  property,  if  it  exists  to  the  extent  that  Hanriot  ascribes  to 
it,  must  not  be  confounded  with  the  transformation  of  fat  into  unknown  sub- 
stances soluble  in  water,  a  phenomenon  first  observed  by  Connstein  and  Michae- 
lis and  further  studied  by  Weigert.  The  occurrence  of  such  a  body  is  positively 
denied  by  G.  Mansfeld.4 

Besides  the  above-mentioned  enzymes  and  thrombin  and  the  gly- 
colytic enzymes  that  will  be  discussed  later,  several  other  enzymes  have 
been  found  in  the  blood-serum,  namely,  oxidases,  catalases,  proteolytic 
enzymes,  among  which  we  must  mention  the  polypepiide-splitting 
enzymes  studied  by  Abderhalden  and  collaborators,5  also  rennin  and 
several  antienzymes.  We  cannot  enter  into  the  discussion  of  these,  nor 
of  the  many  not  chemically  characterized  bodies  which  have  been  called 
toxines  and  antitoxines,  immune  bodies,  alexines,  Iwmoly sines,  cytotoxines, 
etc.,  and  which  have  been  discussed  in  Chapter  I.  The  various  enzymes 
and  antienzymes,  and  the  above  mentioned  bodies  are  as  a  rule  pre- 
cipitated with  the  globulin,  but  differ  among  each  other  in  that  some  are 

'Rohmann:  Rohmann  and  Hamburger,  Ber.  d.  deutsch.  chem.  Gesellsch.,  25 
and  27;  Pfliiger's  Arch.,  52  and  60;  Bial.  Ueber  das  diast.  Ferm.,  etc.,  Inaug.-Diss. 
Breslau,  1892  (older  literature).  See  also  Pfliiger's  Arch.,  52,  54,  and  55;  Wohlgemuth, 
Bioch.  Zeitschr.,  21. 

2  Wohlgemuth,  1.  c;  Moeckel  and  Rost,  Zeitschr.  f.  physiol.  Chem.,  67;  Clerk 
and  Loeper,  Compt.  Rend.  soc.  biol..  66:   Haberlandt,  Pfliiger's  Arch..  132. 

3  Hanriot,  Compt.  Rend.  soc.  biol..  48  and  54.  Compt.  Rend.  123  and  132;  Rona, 
and  Michaelis,  Bioch.  Zeitschr.,  31;  Rona,  ibid..  33;  Arthus,  Journ.  de  Physiol,  et 
de  Pathol.,  4;  Doyon  and  Morel,  Compt.  Rend.  soc.  biol.,  54;  Achard  and  Clerc 
(Lipase  in  Disease),  Compt.  Rend.,  129,  and  Arch.  d.  med.  expe>.,  14. 

*  Connstein  and  Michaelis,  Pfliiger's  Arch.,  65  and  69:  Weigert,  ibid.,  82;  Mansfeld, 
Centralbl.  f.  Physiol,  21. 

s  Zeitschr.  f.  physiol.  Chem.,  51,  53,  55. 


BLOOD  SERUM.  207 

carried  down  by  the  euglobulin,  while  the  others  are  carried  down  by 
the  pseudoglobulin  fraction. 

The  non-protein  organic  constituents  of  the  serum  have  been  given 
especial  and  careful  study  by  E.  Letsche  l  and  he  has  found,  besides  the 
previously  known  bodies,  that  the  serum  contains  several  acids,  among 
which  there  are  twro  nitrogeneous  acids  whose  nature  has  not  been  studied. 
These,  including  other  nitrogenous  substances  found  by  him,  represent 
a  part  of  the  so-called  rest  nitrogen,  i.e.,  that  nitrogen  which  remains  in 
the  serum  after  the  complete  removal  of  the  coagulable  proteins.  As 
representatives  of  the  bodies  occurring  as  rest  nitrogen  in  the  serum  we 
must  in  the  first  place  mention  area,  also  creatine,  carbarriic  acid,  ammonia, 
hippuric  acid,  phosphocarnic  acid  (Panella),  traces  of  indol  (Hervieux), 
perhaps  also  uric  acid  found  by  Abeles  2  in  human  blood,  while  Letsche 
could  not  find  any  in  horse-blood. 

According  to  Browtnski  proteic  acids  (see  Chapter  XIV)  occur  in 
the  serum  and  Czernecki3  has  investigated  the  quantity  of  proteic 
acid  nitrogen  in  serum  and  transudates  under  different  conditions.  The 
occurrence  of  proteoses  is,  as  above  mentioned,  somewhat  disputed. 
We  have  several  investigations  on  the  occurrence  of  amino-acids  (v. 
Bergmann,  Howell,  Letsche,  Abderhalden  and  others)  which  make 
the  occurrence  of  these  very  probable,  and  recently  Bingel  has  been  able 
to  show  the  presence  of  glycocoll  in  normal  ox-blood.  Otherwise  the 
amino-acids  have  often  been  sought  for  in  normal  blood  but  in  vain; 
still  recently  certain  investigators  like  van  Slyke  and  Meyer4  have 
showrn  the  presence  of  amino-acids  in  the  blood  under  normal  con- 
ditions. In  dog  blood  after  24  hours'  starvation  they  found  3-5  milli- 
grams of  amino-acid  nitrogen  in  100  parts  blood.  Under  pathological 
conditions  lysine  (Neuberg  and  Richter5),  leucine  and  tyrosine  have 
been  found.  Also  purine  bases  and  bile  acids  have  been  found  in  the 
serum  under  pathological  conditions.  That  the  quantity  of  rest  nitro- 
gen is  larger  during  digestion  than  in  starvation  requires  further  con- 
firmation.6 


1  Zeitschr.  f.  physiol.  Chem.,  53. 

2  Panella,  cited  in  Virehow's  Jahresb.,  1902,  150,  Hervieux  Compt.  Rend.  soc. 
biol.,  56;  Abeles,  Wien.  med.  Jahrb.,  1887. 

3  Browinski,  Zeitschr.  f.  physiol.  Chem.,  54  and  58;  Czernecki,  Mary's  Jahresb., 
39  and  40. 

4  v.  Bergmann,  Hofmeister's  Beitrage,  6;  Howell,  Amer.  Journ.  of  Physiol.,  17; 
Letsche,  1.  c;  Abderhalden,  Zeitschr.  f.  physiol.  Chem.,  72;  Bingel,  ibid.,  57;  D. 
v.  Slyke  and  Meyer,  Journ.  of  biol.  Chem.,  12. 

5  Deutsch.  med.  Wochenschr.,  1904. 

6  v.  Bergmann  and  Langstein,  Hofmeister's  Beitrage,  6;  Hohlweg  and  Meyer, 
ibid.,  11. 


268  THE  BLOOD. 

As  rest-carbon.  Mancini  x  designates  that  carbon  which  is  not  precipitated 
by  phosphotungstic  acid.  It  originates  in  great  part  from  the  urea  and  sugar 
and  amounts  to  0.076-0.089  gram  in  100  cc. 

The  pigments  of  the  blood-serum  are  very  little  known.  Besides 
other  pigments  horse-serum  contains,  as  first  shown  by  Hammarsten, 
bilirubin,  which,  according  to  Ranc,  is  the  only  pigment  of  the  serum  of 
this  animal.  This  pigment  occurs,  although  in  small  amounts,  sometimes 
in  the  serum  of  other  animals  and,  according  to  Biffi  and  Galli,2  is 
especially  abundant  in  the  blood  of  new-born. 

Urobilin  is  not,  according  to  Auche,  Roth  and.  Herzfeld,  a  physi- 
ological serum-pigment.  Urobilinogen  may  occur  in  extraordinary 
cases  according  to  Hildebrandt,3  and  on  allowing  the  blood  in  such 
cases  to  stand  urobilin  may  be  formed  therefrom.  The  yellow  coloring- 
matter  of  the  serum  seems  to  belong  to  the  group  of  luteins,  which 
are  often  called  lipochromes  or  fat-coloring  matters.  From  ox-serum 
Krukenberg  4  was  able  to  isolate  with  amyl  alcohol  a  so-called  lipo- 
chrome  whose  solution  shows  two  absorption-bands,  of  which  one  encloses 
the  line  F  and  the  other  lies  between  F  and  G. 

The  mineral  bodies  in  serum  and  plasma  are  qualitatively,  but  not 
quantitatively,  the  same.  A  part  of  the  calcium,  magnesium,  and 
phosphoric  acid  is  removed  on  the  coagulation  of  the  fibrin.  By  means 
of  dialysis,  the  presence  of  sodium  chloride,  which  forms  the  chief  mass 
or  60-70  per  cent  of  the  total  mineral  bodies,  lime-salts,  sodium  car- 
bonate, and  traces  of  sulphuric  and  phosphoric  acids  and  of  potassium, 
may  be  directly  shown  in  the  serum.5  Traces  of  silicic  acid,  fluorine, 
copper,  iron,  and  manganese,  are  claimed  to  have  been  found  in  the  serum. 
As  in  most  animal  fluids,  the  chlorine  and  sodium  are  in  the  blood- 
serum  in  excess  of  the  phosphoric  acid  and  potassium  (the  occurrence 
of  which  in  the  serum  is  even  doubted).  The  acids  present  in  the  ash 
are  not  sufficient  to  saturate  the  bases  found,  a  condition  which  shows 
that  a  part  of  the  bases  is  combined  with  organic  substances,  perhaps 
proteins.  This  also  coincides  with  the  fact  that  the  great  part  of 
the  alkalies  does  not  exist  in  the  serum  as  diffusible  alkali  compounds, 
carbonate  and  phosphate,  but  as  non-diffusible  compounds,  protein 
combinations.  According  to  Hamburger  37  per  cent  of  the  alkali  of 
the  serum  from  horse-blood  was  diffusible  and  63  per  cent  non-diffusible. 

1  Bioch.  Zeitschr.,  26  and  32. 

2  Hammarsten,  see  Maly's  Jahresb.,  8  (1878);  Ranc,  Compt.  Rend.  soc.  biol.,  62; 
Biffi  and  Galli,  Journ.  de  Physiol,  et  Path.,  9  (1907). 

'  Auch6,  Compt.  Rend.  soc.  biol.,  67;  Roth  and  Herzfeld,  Deutsch.  med.  Wochenschr., 
37;  Hildebrandt,  Munch.  Med.  Wochenschr.,  57. 
*  Sitz.-Ber.  d.  Jen.  Gesellsch.  f.  Med.,  1885. 
6  See  Giirber,  Verhandl.  d.  phys.-med.  Gesellsch.  zu  Wurzburg,  23. 


BLOOD  SERUM.  269 

According  to  Rona  and  Takahashi  1  25-30  per  cent  of  the  calcium  is 
non-diffusible,  probably  combined  with  proteins. 

Iodine,  which  seems  to  be  habitually  found,  is  also  considered  as 
a  mineral  constituent  of  the  plasma  or  serum  (Gley  and  Bourcet), 
while  arsenic,  although  not  found  in  all  blood,  occurs  in  human  blood 
(Gautier,  Bourcet2).  Iodine  occurs  to  a  greater  extent  in  menstrual 
blood  than  in  other  blood  and  does  not  exist  as  a  salt,  but  as  an  organic 
compound  (Bourcet). 

The  gases  of  the  blood-serum,  which  consists  chiefly  of  carbon  dioxide 
with  only  a  little  nitrogen  and  oxygen,  will  be  described  when  treating 
of  the  gases  of  the  blood. 

We  have  only  a  few  analyses  of  blood-plasma.  As  an  example  the 
results  of  the  analyses  of  the  blood-plasma  of  the  horse  will  be  given 
below.  The  analysis  No.  1  was  made  by  Hoppe-Seyler.3  No.  2  is  the 
average  of  the  results  of  three  analyses  made  by  Hammarsten.  The 
figures  are  given  for  1000  parts  of  the  plasma. 

No.  1.  No.  2. 

Water 908.4  917.0 

Solids 91.0  82.4 

Total  proteins 77 . 0  09 . 5 

Fibrin 10.1  0.5 

Globulin 38 . 4 

Seralbumin 24 . 0 

Fat 1.2] 

Extractive  substances 4.01  ,on 

Soluble  salts 0.4  12-9 

Insoluble  salts 1 . 7  J 

Lewinsky4  has  determined  the  total  proteins  and  the  individual 
proteins  in  the  blood-plasma  of  man  and  animals  with  the  following 
results : 

Total  Protein.  Albumin.  Globulin.  Fibrinogen. 

Man 72.0  40.1  28.3  4.2 

Dog 00.3  31.7  22.0  0.0 

Sheep 72.9  38.3  30.0  4.0 

Horse 80.4  28.0  47.9  4.5 

Pig 80.5  44.2  29.8  0.5 

Abderhalden  has  made  complete  analyses  of  the  blood-serum  of 
several  domestic  animals.  From  these  analyses,  as  well  as  from  those 
made  by  Hammarsten  of  the  serum  from  human,  horse,  and  ox-blood, 
it  follows  that  the  amount  of  solids  ordinarily  varies  between  70-97 
p.  m.  The  chief  mass  of  the  solids  consists  of  proteins,  about  55-84 
p.    m.     In   hens   Hammarsten   found  much   lower   values,   namely,    54 

1  Hamburger,  Arch.  f.  (Anat.  u.)  Physiol.,  1898;  Rona  and  Takahashi,  Bioch. 
Zeitschr.,  31. 

2  Gley  and  Bourcet,  Compt.  Rend.,  130;  Bourcet,  ibid.,  131;  Gautier,  ibid.,  131. 

3  Cit.  from  v.  Gorup-Besanez's  Lehrbuch  der  physiol.  Chem.,  4.  Aufl.,  340. 
*  Pfluger's  Arch.,  100. 


270  THE  BLOOD. 

p.  m.  solids,  with  only  39.5  p.  m.  protein,  and  Halliburton  found  only 
25.4  p.  m.  protein  in  frog's  blood.  The  relation  between  globulin  and 
seralbumin  is,  as  shown  by  the  analyses  of  Hammarsten,  Hallibur- 
ton, and  Rubbrecht,1  very  different  for  various  animals,  but  may  also 
vary  considerably  in  the  same  species  of  animal.  In  human  blood- 
serum  Hammarsten  found  more  seralbumin  than  globulin,  and  the 
relation  of  serglobulin  to  seralbumin  was  as  1:1.5.  Lewinsky  found  the 
relationship  in  man  greater  than  1,  indeed  1:1.39-2.13.  In  regard  to  the 
quantity  of  the  remaining  organic  constituents  of  the  serum  we  refer 
the  reader  to  Abderhalden's  complete  analyses. 

In  starvation  it  seems,  as  first  found  by  Burckhardt  and  then  sub- 
stantiated by  other  investigators,  that  the  quantity  of  globulins  relative 
to  that  of  albumin  in  dogs  and  also  in  rats  (Robertson2),  is  increased. 
According  to  Robertson,  in  the  horse,  ox  and  rabbit  the  reverse  exists, 
namely,  the  amount  of  albumin  relative  to  the  globulin  increases  in 
starvation.  A  change  in  the  relation  with  a  decrease  in  the  albumin 
and  an  increase  in  the  globulin  may  also  occur  in  animals  which  have 
been  made  sick  or  in  part  immune  by  inoculation  with  pathogenic 
micro-organisms  (Langstein  and  Mayer  3) .  The  total  protein  content 
is  raised  in  nearly  all  cases.  The  amount  of  fibrinogen  in  the  plasma 
is  especially  increased  by  pneumococci,  streptococci,  and  pus-staphy- 
lococci  (P.  Muller4). 

The  quantity  of  mineral  bodies  in  the  serum  has  been  determined  by 
many  investigators.  The  conclusion  drawn  from  the  analyses  is  that 
there  exists  a  rather  close  correspondence  between  human  and  animal 
blood-serum,  and  it  is  therefore  sufficient  to  here  give  the  analysis  of  C. 
Schmidt5  of  (1)  human  blood,  and  Bunge  and  Abderhalden's  analyses 
(2)  of  serum  of  ox,  bull,  sheep,  goat,  pig,  rabbit,  dog,  and  cat.  The  results 
correspond  to  1000  parts  by  weight  of  the  serum. 

1  2 

K20 0.387-0.401  0.226-0.270 

Na20 4.290-4.290  4.251-4.442 

CI 3.565-3.659  3.627-4.170 

CaO 0.155-0.155  0.119-0.131 

MgO 0.101    0.040-0046 

P,05  (inorg.) 0.052-0.085 


1  Abderhalden',  Zeitschr.  f.  physiol.  Chem.,  25;  Hammarsten,  Pfliiger's  Arch.,  17; 
Halliburton,  Journ.  of  Physiol.,  7;  Rubbrecht,  Travaux  du  laboratoire  de  l'institut 
de  physiologie  de  Liege,  5,  1896. 

2  Burckhardt,  Arch.  f.  exp.  Path.  u.  Pharm.,  16;  Githens,  Hofmeister's  Beitrage, 
5;  see  also  Morawitz,  ibid.,  7,  and  Inagaki,  Zeitschr.  f.  Biol.,  49;  Robertson,  Journ. 
of  biol.  Chem.,  13. 

3  Hofmeister's  Beitrage,  5. 
'  Ibid.,  8. 

*  Cit.  from  Hoppe-Seyler,  Physiol.  Chem.,  1881,  p.  439. 


BLOOD  SERUM.  271 

A  Macallum  l  has  determined  the  quantity  of  mineral  bodies  in 
the  serum  of  certain ,  cold-blooded  animals  (fishes,  shark,  lobster  and 
others).  The  amount  of  sodium  and  chlorine  in  the  serum  of  these  animals 
living  in  sea-water  was  much  greater  than  in  warm-blooded  animals. 

Even  if  we  bear  in  mind  that  certain  bodies,  such  as  carbon  dioxide, 
arc  driven  oft"  during  incineration,  and  that  other  bodies,  such  as  sul- 
phuric acid  and  phosphoric  acid,  are  formed  from  sulphurized  and 
phosphorized  organic  substances,  still  quantitative  analyses  like  the 
above  are  not  sufficient  for  the  scientific  demands  of  to-day.  They 
do  not  show  the  true  composition,  and  especially  do  not  give  an  explana- 
tion of  the  number  of  different  ions  present  in  the  serum  or  in  other  fluids, 
a  question  which  is  of  the  greatest  physiological  importance.  An 
answer  to  these  questions  is  obtainable  only  by  physico-chemical  investiga- 
tions, which  have  thus  far  been  used  chiefly  in  determining  the  molecular 
concentration,  the  amount  of  electrolytes  and  non-electrolytes,  and  the 
degree  of  dissociation. 

The  average  depression  of  the  freezing-point  of  mammalian  blood 
corresponds,  as  given  in  Chapter  I,  closely  to  a  9  p.  m.  (A  =  about 
—0.56°)  solution  of  common  salt,  and  at  the  present  time  such  a  solution 
is  considered  as  a  physiological  salt  solution  for  man  and  other  mammalia. 
In  lower  animals  and  fish  the  conditions  are  otherwise,  as  shown  in 
the  above-mentioned  chapter. 

There  are  recorded  a  great  number  of  investigations  on  the  changes 
in  the  osmotic  pressure  or  the  molecular  concentration  of  the  blood- 
serum  under  various  physiological  conditions  as  wrell  as  in  disease,  but 
still  it  is  no  doubt  too  early  to  draw  any  definite  conclusions  from  these 
observations. 

The  degree  of  dissociation  (see  Chapter  I)  of  sera  has  been  determined 
by  several  investigators,  and  according  to  Hamburger2  it  lies  between 
0.65  and  0.82.  The  molecular  concentration,  which  represents  the 
total  number  of  molecules  and  ions  per  liter,  is  according  to  Bvrgarsky 
and  Tangl,  on  an  average  about  0.320  mol.  per  liter.  They  also 
found  that  about  three-fourths  of  the  total  number  of  dissolved  mole- 
cules in  blood-serum  were  electrolytes,  although  the  serum  contained 
about  70-80  p.  m.  protein  and  10  p.  m.  inorganic  bodies,  and  also  that 
three-fourths  of  the  quantity  of  electrolytes  consisted  of  NaCl. 

In  the  determination  of  the  alkalinity  of  blood  and  blood-serum, 
up  to  the  present  time,  we  have  estimated  the  amount  of  alkali  by  titra- 
tion with  an  acid.     We  cannot  dispense  v  ith  such  d<  terminations,  although 


iProc.  Roy.  Soc,  ser.  B.,  82. 

2  Osmotisher  Druck  und  Ionenlehre,  Wiesbaden,    1902-1904,  where  the  literature 
on  the  physical  chemistry  of  the  blood  can  be  found. 


272  THE  BLOOD. 

they  do  not  yield  any  information  as  to  the  true  alkalinity,  apart  from 
the  fact  that  the  results  are  dependent  upon  the  indicator  used,  because 
we  understand  as  true  alkalinity  the  concentration  of  the  hydroxyl 
ions.  The  Na2C03  is  in  aqueous  solution  more  or  less  dissociated  into 
2Na+  and  C03=,  depending  upon  the  dilution.  The  C03°  ions  com- 
bine partly  with  the  H+  ions  of  the  dissociated  water,  forming  HCO3-, 
and  the  corresponding  HO~  ions  produce  the  alkaline  reaction.  If  now 
by  the  addition  of  a  little  acid,  a  few  of  the  HO"  ions  are  removed, 
the  equilibrium  is  then  disturbed,  a  new  quantity  of  Na2C03  is  dissociated, 
and  this  process  is  repeated  every  time  a  new  quantity  of  acid  is  added 
until  all  the  carbonate  is  dissociated.  The  dissociation  of  the  carbonate 
existing  in  the  original  concentration,  upon  which  the  number  of  HO~ 
ions  is  dependent,  cannot  therefore  be  determined  by  titration. 

For  these  reasons  we  generally  determine  the  quantity  of  HO 
and  H  ions  in  the  serum  and  blood  by  methods  based  upon  Nernst's 
theory  for  the  electromotive  force  of  gas-chains.  According  to  these 
investigations  it  has  been  found  that  the  concentration  of  the  hydroxyl 
ions  in  blood-serum  and  blood  is  only  a  little  higher  than  in  distilled 
water  (see  Chapter  I  page  76,  and  the  reaction  of  the  blood  below). 

H.   THE  FORM-ELEMENTS  OF  THE  BLOOD. 
The  Red  Blood-corpuscles. 

The  blood-corpuscles  are  round,  biconcave  disks  without  membrane 
and  nucleus,  in  man  and  mammalia  (with  the  exception  of  the  llama, 
the  camel,  and  their  congeners).  In  the  latter  animals,  as  also  in  birds, 
amphibia,  and  fish  (with  the  exception  of  the  Cyclostoma)  the  cor- 
puscles have  in  general  a  nucleus,  are  biconvex  and  more  or  less  ellip- 
tical. The  size  varies  in  different  animals.  In  man  they  have  an  average 
diameter  of  7  to  8  ju  (/x  =  0.001  mm.)  and  a  maximum  thickness  of  1.9  /x. 
They  are  heavier  than  the  blood-plasma  or  serum,  and  therefore  sink 
in  these  liquids.  In  the  discharged  blood  they  may  sometimes  lie  with 
their  fiat  surfaces  together,  forming  a  cylinder  like  a  roll  of  coin  (rouleaux). 
The  reason  for  this  phenomenon,  which  is  considered  as  an  agglutination, 
has  not  been  sufficiently  studied,  but  as  it  may  be  observed  in  defibrinated 
blood  it  seems  probable  that  the  formation  of  fibrin  has  nothing  to 
do  with  it. 

The  number  of  red  blood-corpuscles  is  different  in  the  blood  of  various 
animals.  In  the  blood  of  man  there  are  generally  5  million  red  cor- 
puscles in  1  c.mm.,  and  in  woman  4  to  4.5  million. 

The  blood-corpuscles  consist  principally  of  two  chief  constituents, 
the  stroma,  which  forms  the  real  protoplasm,  and  the  intraglobular 
contents,    whose    chief    constituent    is    haemoglobin.     We    cannot    state 


RED  BLOOD-CORPUSCLES.  273 

anything  positive  for  the  present  in  regard  to  a  more  detailed  arrange- 
ment, and  the  views  on  this  subject  are  somewhat  divergent.  The  two 
following  views  are  more  or  less  related  to  each  other.  According  to 
one  view  the  blood-corpuscles  consist  of  a  membrane  which  encloses  a 
haemoglobin  solution,  while  the  other  view  considers  the  stroma  as  a  proto- 
plasmic structure  soaked  with  haemoglobin.  This  latter  view  is  in  accord 
with  the  assumption  as  to  an  outside  boundary-layer.  Thus  accord- 
ing to  Hamburger  the  stroma  forms  a  protoplasmic  net  in  whose  meshes 
there  exists  a  red  fluid  or  semi-fluid  mass  which  consists  in  great  meas- 
ure of  haemoglobin.  This  mass  represents  the  water-attracting  force 
of  the  blood-corpuscles,  and  besides  this  it  is  also  considered  that  the 
outer  protoplasmic  boundary  is  semi-permeable,  i.  e.,  permeable  to  water 
but  not  permeable  to  certain  crystalloids.  The  researches  of  Koppe, 
Albrecht,  Pascucci,  Rywosch,1  and  others  indicate  the  presence  of  a 
special  envelope  or  boundary-layer,  and  there  is  no  doubt  that  the  outer 
layer  contains  so-called  lipoids,  such  as  cholesterin,  lecithin,  and  similar 
bodies. 

The  red  blood-corpuscles  retain  their  volume  in  a  salt  solution  which 
has  the  same  osmotic  pressure  as  the  serum  of  the  same  blood,  although 
they  may  change  their  form  in  such  solutions,  becoming  more  spherical, 
and  may  also  undergo  a  chemical  change.  Such  a  salt  solution  is  iso- 
tonic with  the  blood-serum,  and  its  concentration  for  a  NaCl  solution  is 
approximately  9  p.  m.  for  human  and  mammalian  blood.  A  solution 
of  greater  concentration,  a  hyperisotonic  solution,  abstracts  water  from 
the  blood-corpuscles  until  osmotic  equilibrium  is  established,  hence  the 
corpuscles  shrink  and  their  volumes  become  smaller.  In  solutions  of 
less  concentration,  hypisotonic  solutions,  the  corpuscles  swell,  due  to 
the  taking  up  of  water,  and  this  swelling  may  be  so  great,  on  diluting 
the  blood  with  water,  that  the  haemoglobin  is  separated  from  the  stroma 
and  passes  into  the  watery  solution.  This  process  is  called  haemolysis 
(see  Chapter  I). 

A  haemolysis  may  also  be  brought  about  by  alternately  freezing  and 
thawing  the  blood,  as  well  as  by  the  action  of  various  chemical  substances, 
which  act  as  protoplasmic  poisons.  These  bodies  are  ether,  chloroform 
alkalies,  bile-acids,  solanin,  saponin,  and  also  the  saponin  substances, 
which  have  a  very  strong  haemoloytic  action,  also  metabolic  products  of 
bacteria,  higher  plants  and  animals  (snakes,  toads,  bees,  spiders  and 
others)  and  also  bodies  occurring  in  blood  serum  of  normal  or  immunized 
animals. 


xSee  Hamburger,  Osmotischer  Druck  und  Ionenlehre,  1902;  Koppe,  Pfluger's 
Arch.,  99  and  107;  Albrecht,  Centralbl.  f.  Physiol.,  19;  Pascucci,  Hofmeister's 
Beitrage,  6;  Rywosch,  Centralbl.  f.  Physiol.,  19. 


274  THE  BLOOD. 

When  the  haemoglobin  is  separated  from  the  so-called  stroma  by  a 
sufficiently  strong  dilution  with  water  the  stroma  is  found  in  the  solution 
in  a  swollen  condition.  By  the  action  of  carbon  dioxide,  by  the  careful 
addition  of  acids,  acid  salts,  tincture  of  iodine,  or  certain  other  bodies, 
this  residue,  rich  in  proteins,  condenses,  and  in  many  cases  the  form  of 
the  blood-corpuscles  may  be  again  obtained.  This  residue,  the  so- 
called  ghosts  or  stromata  of  the  blood-corpuscles,  can  also  be  directly 
colored  in  dilute  blood  by  methyl  violet  and  in  this  way  detected,  and 
attempts  have  been  made  to  isolate  it  for  chemical  investigation.  In 
the  following  pages  we  mean  by  the  name  stroma  only  that  residue  which 
remains  after  the  removal  of  haemoglobin  and  other  bodies  soluble  in 
water. 

To  isolate  the  stromata  from  the  blood-corpuscles,  they  are  washed 
first  by  diluting  the  blood  with  10-20  vols,  of  a  1-2  per  cent  common 
salt  solution  and  then  separating  the  mixture  by  centrifugal  force  or 
by  allowing  it  to  stand  at  a  low  temperature.  This  is  repeated  a  few 
times  until  the  blood-corpuscles  are  freed  from  serum.  These  purified 
blood-corpuscles  are,  according  to  Wooldridge,  mixed  with  5-6  vols, 
of  water  and  then  a  little  ether  is  added  until  complete  solution  is  obtained. 
The  leucocytes  gradually  settle  to  the  bottom,  a  movement  which  may 
be  accelerated  by  centrifugal  force,  and  the  liquid  which  separates  there- 
from is  very  carefully  treated  with  a  1-per  cent  solution  of  KHSO4  until 
it  is  about  as  dense  as  the  original  blood.  The  separated  stromata  are 
collected  on  a  filter  and  quickly  washed.  Pasctjcci,1  on  the  contrary, 
treats  the  mass  of  corpuscles  with  15-20  vols,  of  a  one-fifth  saturated 
ammonium-sulphate  solution,  allows  the  corpuscles  to  settle,  siphons 
off  the  fluid,  repeatedly  centrifuges,  allows  the  residue  to  dry  quickly 
(on  porcelain  plates)  at  the  ordinary  temperature,  and  then  washes 
with  water  until  the  blood-pigments  and  the  other  soluble  bodies  are 
dissolved  out. 

Wooldridge  found  as  constituents  of  the  stromata  lecithin,  choles- 
terin,  nucleoalbumin,  and  a  globulin  which,  according  to  Halliburton, 
is  probably  a  nucleoproteid  which  he  calls  cell-globulin.  No  nuclein 
substances  or  seralbumin  or  proteoses  could  be  detected  by  Hallibur- 
ton and  Friend.  According  to  Pascucci,  the  stromata  (from  horse- 
blood)  consists  of  one-third  cholesterin  and  lecithin  (besides  a  little 
cerebroside),  and  two-thirds  protein  substances  and  mineral  bodies. 
The  nucleated  red  blood-corpuscles  of  the  bird  contain,  according  to 
Plosz  and  Hoppe-Seyler,2  a  protein  (nucleoprotein)  which  swells  to  a 
slimy  mass  in  a  10-per  cent  common  salt  solution,  and  which  seems  to 

1  Hofmeister'fi  Beitrage,  0. 

*  Wooldridge,  Arch.  f.  (Anat.  u.)  Physiol.,  1881,387;  Halliburton  and  Friend, 
Journal  of  Physiol.,  10;  Halliburton,  ibid.,  18;  P16sz,  Hoppe-Seyler's  Med.  chem. 
Untersuch.,  510. 


RED  BLOOD-CORPUSCLES.  275 

be  closely  related  to  the  hyaline  substance  (hyaline  substance  of  Rovida), 
occurring  in  the  lymph-cells.  In  the  mass  extracted  by  alcohol  from 
the  blood-corpuscles  of  the  hen,  Ackermann  found  3.93  per  cent  phos- 
phorus and  17.2  per  cent  nitrogen,  which  on  calculation  gave  42.10  per 
cent  nucleic  acid  and  57.82  per  cent  histone.  Piettre  and  Vila  *  found, 
in  the  stromata,  0.3  per  cent  phosphorus  in  the  horse  and  2.3-2.6  per 
cent  in  birds  (ducks  and  hens),  calculated  on  the  ash-free  substance.  They 
found  the  quantity  of  nitrogen  to  be  11.7  and  13.21  per  cent  for  the  horse 
and  dog  respectively.  The  non-nucleated  red  blood-corpuscles  are, 
as  a  rule,  very  poor  in  protein,  but  rich  in  haemoglobin;  the  nucleated 
corpuscles  are  richer  in  protein  and  poorer  in  haemoglobin  than  the 
non-nucleated.  The  reducing  substances,  and  in  certain  animal  sugars, 
probably  also  conjugated  glucuronic  acids  and  several  enzymes,  among 
which  occurs  the  proteolytic  enzyme  studied  by  Abderhalden  and  col- 
laborators,2 belong  to  the  stromata.  It  is  difficult  to  decide  in  many 
cases  whether  the  enzymes  found  in  the  blood  belong  to  the  fluid  or  to 
the  various  kinds  of  form-elements. 

A  gelatinous,  fibrin-like  protein  body  may  be  obtained  from  the  red 
blood-corpuscles  under  certain  circumstances.  This  fibrin-like  mass 
has  been  observed  on  freezing  and  then  thawing  the  sediment  of  the 
blood-corpuscles,  or  on  discharging  the  spark  from  a  large  Ley  den  jar 
through  the  blood,  or  on  dissolving  the  blood-corpuscles  of  one  kind  of 
animal  in  the  serum  of  another  (Landois,  stroma-fibrin) ;  i.e.,  in  the  so- 
called  hemagglutination,  a  clumping  of  the  red  blood-corpuscles  into 
clusters  takes  place.  This  agglutination  can  be  brought  about  by  bodies 
similar  to  the  haemolysines  and  also  by  serum  constituents  produced 
normally  or  by  immunization.  It  has  not  been  shown  that  a  fibrin  for- 
mation from  the  stroma  takes  place,  nor  is  it  probable.  Fibrinogen 
has  been  detected  only  in  the  red  corpuscles  of  frog's  blood  (Alex.  Schmidt 
and  Semmer3).  *•« 

Closely  related  to  the  anatomical  and  chemical  structure  of  the  erythro- 
cytes is  the  question,  which  is  important  for  the  metabolism  in  the  blood, 
as  to  the  permeability  of  the  erythrocytes,  that  is,  their  power  of  tak- 
ing up  substances  of  different  kinds.  This  question  as  well  as  the  per- 
meability of  the  blood-corpuscles  for  anions  under  the  influence  of  carbon 
dioxide  has  been  discussed  in  Chapter  I,  pages  7  and  8. 

The  mineral  bodies  of  the  red  corpuscles  will  be  treated  in  connection 
with  their  quantitative  constitution. 


1  Ackermann,  Zeitschr.  f.  physiol.  Chem.,  43;  Piettre  and  Vila,  Compt.  Rend.,  143. 
-  Zeitschr.  f.  physiol.  Chem.,  51,  53  and  55. 

3  Landois,  Centralbl.  f.  d.  med.  Wissensch.,  1874,  421;    Schmidt,    Pfliiger's  Arch., 
11,  550-559. 


276  THE  BLOOD. 

The  constituent  of  the  blood-corpuscles  existing  in  greatest  quantity 
is  the  red  pigment  haemoglobin. 

Blood-pigments. 

According  to  Hoppe-Seyler  the  coloring-matter  of  the  red  blood- 
corpuscles  is  not  in  a  free  state,  but  combined  with  some  other  sub- 
stance. The  crystalline  coloring-matter,  the  haemoglobin  or  oxyhaemo- 
globin,  which  may  be  isolated  from  the  blood,  is  considered,  according 
to  Hoppe-Seyler,  as  a  cleavage  product  of  this  compound,  but  it  acts 
in  many  ways  unlike  the  questionable  compound  itself.  This  compound 
is  insoluble  in  water  and  uncrystallizable.  It  strongly  decomposes 
hydrogen  peroxide  without  being  oxidized  itself;  it  shows  a  greater  resist- 
ance to  certain  chemical  reagents  (as  potassium  ferricyanide)  than  the 
free  coloring-matter;  and,  lastly,  it  gives  off  its  loosely  combined  oxygen 
much  more  easily  in  vacuum  than  the  free  pigment.  To  distinguish 
between  the  cleavage  products,  the  haemoglobin,  and  the  oxyhaemoglobin, 
Hoppe-Seyler  calls  the  compound  of  the  blood-coloring  matter  of  the 
venous  blood-corpuscles  phlebin,  and  that  of  the  arterial  arterin.  Other 
investigators,  such  as  H.  U.  Kobert  and  Bohr,2  the  latter  calling  the 
pigment  of  the  blood-corpuscles  hcemochrom,  are  of  a  similar  opinion. 
Since  the  above-mentioned  combinations  of  the  blood-coloring  matters 
with  other  bodies,  for  example  (if  they  really  do  exist)  with  lecithin,  have 
not  been  closely  studied,  the  following  statements  will  apply  only  to  the 
free  pigment,  the  haemoglobin. 

The  color  of  the  blood  depends  in  part  on  haemoglobin  and  in  part 
on  a  molecular  combination  of  this  substance  with  oxygen,  the  oxy- 
hemoglobin. We  find  in  blood  after  asphyxiation  almost  exclusively 
haemoglobin,  in  arterial  blood  disproportionately  large  amounts  of 
oxyhemoglobin,  and  in  venous  blood  a  mixture  of  both.  Blood-color- 
ing matters  are  also  found  in  striated  as  well  as  in  certain  smooth  muscles, 
and  lastly  in  solution  in  different  invertebrates,  although  this  pigment 
is  not  quite  identical  with  that  from  higher  animals.  The  quantity  of 
haemoglobin  in  human  blood  may  indeed  be  somewhat  variable  under 
different  circumstances,  but  amounts  to  about  14  per  cent  on  an  average, 
or  8.5  grams  for  each  kilo  of  the  weight  of  the  body. 

Haemoglobin  belongs  to  the  group  of  compound  proteins,  and  yields 
as  cleavage  products,  besides  very  small  amounts  of  volatile  fatty  acids 
and  other  bodies,  chiefly  a  protein  globin,  and  a  coloring-matter,  hcemo- 


2  Hoppe-Seyler,  Zeitschr.  f.  physiol.  Chem.,  13,  479;  H.  U.  Kobert,  Das  Wirbeltier- 
blut  in  mikro-kristallogr.  Hinsicht,  Stuttgart,  1901;  Bohr,  Centralbl.  f.  Physiol.,  17, 
p.  688. 


BLOOD-PIGMENTS. 


277 


chromogen  (about  4  per  cent),  containing  iron,  which  in  the  presence  cf 
oxygen  is  easily  oxidized  into  hcematin. 

As  first  shown  by  Schunck  and  Marchlewski,  and  especially  by  the 
work  of  the  latter,  a  close  relation  exists  between  chlorophyll  and  the 
blood-pigment,  because  a  derivative  of  the  first,  phylloporphyrin, 
stands  very  close  in  certain  respects  to  a  derivative  of  the  blood-pigment 
haematoporphyrin.  By  the  investigations  of  Nencki  in  conjunction 
with  Marchlewski  and  Zaleski,1  it  was  shown  that  haemopyrrol  could 
be  prepared  from  the  derivatives  of  both  the  leaf-pigment  and  the  blood- 
pigments  by  reduction,  and  also  the  investigations  of  Piloty  and  Will- 
statter  on  chlorophyll  and  blood  pigments  have  further  developed 
the  interesting  biological  fact  that  the  chlorophyll  and  blood  pigments 
are  closely  related  bodies. 

The  haemoglobin  prepared  from  different  kinds  of  blood  has  not 
exactly  the  same  composition,  which  seems  to  indicate  the  presence 
of  different  haemoglobins.  The  analyses  by  different  investigators  of 
the  haemoglobin  from  the  same  kind  of  blood  do  not  always  agree  with 
one  another,  which  probably  depends  upon  the  somewhat  varying  methods 
of  preparation.  The  following  analyses  are  given  as  examples  of  the 
constitution  of  different  haemoglobins: 

Hemoglobin  from  the        C  H  N  S  Fe  0 

Dog 53.85  7.32  16.17  0.390  0.430  21.84  (Hoppe-Seyler) 

"    54.57  7.22  16.38  0.568  0.336  20.93  (Jaquet) 

Horse 54.87  6.79  17.31  0.650  0.470  19.73  (Kossel) 

"      51.15  6.76  17.94  0.390  0.335  23.43  (Zinoffsky) 

Ox 54.66  7.25  17.70  0.447  0.400  19.543  (Hufner) 

Pig 54.17  7.38  16.23  0.660  0.430  21.360  (Otto) 

" 54.71  7.38  17.43  0.479  0.399  19.602  (Hufner) 

Guinea-pig 54.12  7.36  16.78  0.580  0.480  20.680  (Hoppe-Seyler) 

Squirrel 54.09  7.39  16.09  0.400  0.590  21.440 

Goose 54.26  7.10  16.21  0.540  0.430  20.690 

Hen 52.47  7.19  16.45  0.857  0.335  22.500  (Jaquet) 

That  the  repeatedly  observed  quantity  of  phosphorus  in  the  haemo- 
globin of  birds  (Inoko  and  others)  is  due  to  a  contamination  has  been 
proved  by  Abderhalden  and  Medigreceanu.  In  the  haemoglobin 
from  the  horse  (Zinoffsky),  the  pig,  and  the  ox  (Hufner),  we  have 
1  atom  of  iron  to  2  atoms  of  sulphur,  while  in  the  haemoglobin  from  the 
dog  (Jaquet)  the  relation  is  1  to  3.  From  the  data  of  the  elementary 
analysis,  as  also  from  the  amount  of  loosely  combined  oxygen,  Hufner  ' 
has  calculated  the  molecular  weight  of  dog-haemoglobin  as  14.129,  and 


Schunck  and  Marchlewski,  Annal.  d.  Chem.  u.  Pharm.,  278,  284,  288,  290; 
Nencki,  Ber.  d.  deutsch.  chem.  Gesellsch.,  29;  Marchlewski,  and  Nencki,  Ber.  d.  d. 
chem.,  Gesellsch.,  34;  Nencki  and  Zaleski,  ibid.,  Marchlewski,  Chem.  Centralbl., 
1902,  I,  1016;  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  37.  The  literature  and  the  works 
of  Willstatter  and  Piloty  will  be  given  under  haemopyrrol,  page  297. 


278  THE  BLOOD. 

the  formula  Ce36Hi625Ni64FeS3Cisi.  According  to  the  more  recent 
determinations  of  Hufner  and  Jaquet,  ox-haemoglobin  contains  an 
average  of  0.336  per  cent  iron,  and  the  human  haemoglobin,  according 
to  Butterfield  1  contains  0.334  per  cent  iron.  From  the  iron  a  molec- 
ular weight  of  16,669  may  be  calculated.  Barcroft  and  Hill  have 
arrived  at  exactly  the  same  value  by  using  an  entirely  different  method 
and  Hufner  and  Gansser  2  have  attempted  to  learn  the  size  of  the  molec- 
ular weight  of  haemoglobin  by  means  of  osmotic  pressure  determinations, 
and  they  found  the  following  approximate  results:  for  horse-haemoglobin 
15,115  and  for  ox-hsemoglobin  16,321.  The  haemoglobin  from  various 
kinds  of  blood  not  only  shows  a  diverse  constitution,  but  also  a  different 
solubility  and  crystalline  form,  and  a  varying  quantity  of  water  of  crys- 
tallization; hence  we  infer  that  there  are  several  kinds  of  haemoglobin. 
Bohr  is  a  very  zealous  advocate  of  this  supposition.  He  has  been  able 
to  obtain  haemoglobins  from  clog-  and  horse-blood,  by  fractional  crystalliza- 
tion, which  had  different  powers  of  combining  with  oxygen  and  contained 
different  quantities  of  iron.  Hoppe-Seyler  had  already  prepared  two 
different  forms  of  haemoglobin  crystals  from  horse-blood,  and  Bohr 
concludes  from  all  these  observations  that  the  ordinary  haemoglobin 
consists  of  a  mixture  of  different  haemoglobins.  In  opposition  to  this 
statement,  Hufner  3  has  shown  that  only  one  haemoglobin  exists  in  ox- 
blood,  and  that  this  is  probably  true  for  the  blood  of  many  other  animals. 
Oxyhemoglobin,  which  has  also  been  called  h^ematoglobulin  or 
HjEMatocrystallin,  is  a  molecular  combination  of  haemoglobin  and 
oxygen.  For  each  molecule  of  haemoglobin  1  molecule  of  oxygen  is 
present,  as  shown  by  the  investigations  of  Hufner  as  well  as  Hufner 
and  Gansser,  and  the  amount  of  loosely  combined  oxygen  which  is  united 
to  1  gram  of  haemoglobin  (of  the  ox)  has  been  determined  by  Hufner4 
as  1.34  cc.  (calculated  at  0°  C.  and  760  mm.  mercury). 

According  to  Bohr,  the  facts  are  different.  He  differentiates  between  four 
oxyhemoglobins,  according  to  the  quantity  of  oxygen  which  they  absorb,  namely 

1  Hoppe-Seyler,  Med.  chem.  Untersuch.,  370;  Jaquet,  Zeitschr.  f.  physiol.  Chem., 
14,  296;  Kossel,  ibid,.  2,  150;  ZinofTsky,  ibid.,  10;  Hufner,  Beitr.  z.  Physiol.,  Festschr. 
f.  C.  Ludwig,  1887,  74-81,  Journ.  f.  prakt.  Chem.  (N.  F.),  22;  Otto,  Zeitschr.  f.  physiol. 
Chem.,  7;  Inoko,  ibid.,  18;  Abderhalden  and  Medigreceanu,  ibid.,  59;  Hufner  and 
Jaquet,  Arch.  f.  (Anat.  u.)  Physiol.,  1894;  E.  Butterfield,  Zeitschr.  f.  physiol.  Chem., 
02. 

•-'  Barcroft  and  Hill,  Journ.  of  Physiol.  30;  Hufner  and  Gansser,  Arch.  f.  (Anat.  u.) 
Physiol.,  1907. 

:'  Bohr,  "Sur  les  combinaisons  de  l'h6moglobine  avec  I'oxygene,"  Extrait  du 
Bulletin  de  I'Academie  Royale  Danoise  des  sciences,  1890;  also  Centralbl.  f.  Physiol. 
1890,  '-'4'.).  Boppe-Seyler,  Zeitschr.  f.  physiol.  Chem.,  2;  Hufner,  Arch.  f.  (Anat.  u.) 
Physiol.,  L894 

1  Arch.  f.  (Anat.  u.)  physiol.,  1901,  Suppl. 


OXYHEMOGLOBIN.  279 

a.t  fi.)  y.  and  6-oxyhsemoglobin,  all  having  the  same  absorption-spectrum,  and  1 
pram  combining  with  respectively  0.4,  0.8,  1.7,  and  2.7  cc.  oxygen  at  the  tem- 
perature of  the  room  and  with  an  oxygen  pressure  of  150  mm.  mercury.  The 
7-oxyha>moglobin  is  the  ordinary  one  obtained  by  the  customary  method  of 
preparation.  Bohk  designates  as  a-oxyhaunoglobin  the  crystallin  powder 
obtained  by  drying  Y-oxyhsemoglobin  in  the  air.  On  dissolving  a-oxyhaemo- 
globin  in  water  it  is  converted  into  j3-oxylurmoglobin  without  decomposition, 
and  the  quantity  of  iron  is  increased.  On  keeping  a  solution  of  7-oxyhaimoglobin 
in  a  sealed  tube  it  is  transformed  into  5-oxhyaemoglobin,  although  the  exact 
conditions  under  which  this  change  takes  place  are  not  known.  According  to 
Hufner  '  these  are  nothing  but  mixtures  of  genuine  and  partly  decomposed 
haemoglobins. 

The  ability  of  haemoglobin  to  take  up  oxygen  seems  to  be  a  function 
of  the  iron  it  contains,  and  when  this  is  calculated  as  about  0.33-0.40 
per  cent,  then  1  atom  of  iron  in  the  haemoglobin  corresponds  to  about 
2  atoms  or  1  molecule  of  oxygen.  By  increasing  the  partial  pressure  as 
well  as  by  increasing  the  quantities  of  oxygen,  the  haemoglobin  in  solu- 
tion takes  up  more  oxygen,  until  it  is  completely  saturated,  when  1  mole- 
cule of  haemoglobin  is  combined  with  1  molecule  of  oxygen.  With  reduced 
oxygen  pressure  a  dissociation  must  naturally  take  place  and  oxygen 
is  given  off,  and  a  re-formation  of  haemoglobin  takes  place,  and  this  makes 
it  possible  to  expel  completely  the  oxygen  from  an  oxyhaemoglobin  solu- 
tion or  blood  by  means  of  vacuum,  or  by  passing  an  indifferent  gas 
through  the  solution.  The  equilibrium  between  oxyhaemoglobin,  haemo- 
globin, and  oxygen  depends,  therefore,  according  to  Hufner,  upon 
a  mass  action,  corresponding  to  the  formula  Hb-f-02<=^Hb02.  Bohr2 
has  arrived  at  the  conclusion  that  not  only  a  double  dissociation  takes 
place,  in  which  a  dissociation  of  the  oxygen-iron  combination  in  the 
oxyhaemoglobin  occurs,  but  also  a  dissociation  of  the  haemoglobin 
into  a  ferruginous  as  well  as  into  a  non-ferruginous  part.  Correspond- 
ingly he  has  suggested  another  formula  and  hence  the  dissociation  curves 
for  oxyhaemoglobin  given  by  Hufner  and  Bohr  are  different. 

Important  investigations  have  recently  been  carried  out  on  this 
question  by  Barcroft  and  his  co-workers  Camis  and  Roberts  from 
which  it  follows  that  a  generally  valid  dissociation  curve  cannot  be  given, 
as  the  curve  direction  is  dependent  upon  the  nature  and  concentration 
of  the  salts  present  in  the  solution.  A  haemoglobin  solution  with  the  salts 
of  the  blood-corpuscles  of  the  dog  gives  a  dissociation  curve  of  dog-blood 
while  with  the  salts  from  human  blood-corpuscles  it  gives  a  curve  like 
human  blood.  In  the  presence  of  salts  the  dissociation  follows  Bohr's 
formula,  and  on  the  contrary  while  a  salt-free  haemoglobin  solution  follows 
the  oxygen  combination  according  to  the  mass-action  law  of  Hufner. 


1  Arch.  f.  (Anat.  u.)  physiol.,  1894. 

2  Bohr,  Centralbl.  f.  physiol.,  17,  pp.  682  and  688. 


280  THE  BLOOD. 

Thus  far  there  does  not  seem  to  be  any  necessity  for  considering  the 
gas  combination  in  the  blood  and  in  haemoglobin  solutions  to  be  adsorp- 
tion processes  as  suggested  by  W.  Ostwald.1 

That  the  gas  combining  ability  of  an  isolated  pure  haemoglobin  cannot 
be  compared  with  the  gas  combining  ability  of  the  so-called  native  haemo- 
globin of  the  blood  has  been  suggested  by  many  experimenters.  In  this 
connection  we  must  mention  the  observations  of  Manchot  2  who  found 
that  the  combining  ability  of  the  blood  for  gases  such  as  O2,  CO,NO,C2H4 
could  be  increased  at  least  to  a  certain  limit  by  increasing  the  dilution 
so  that  at  8-10  times  the  dilution  the  combining  power  was  close  to  the 
limit  value  of  2  mol.  gas  for  each  atom  of  iron. 

The  elucidation  of  these  mentioned  conditions  is  of  the  greatest 
importance,  as  the  knowledge  of  the  various  conditions  which  influence 
the  taking  up  and  the  giving  up  of  oxygen  by  the  haemoglobin  is  of  the 
greatest  importance  for  our  knowledge  of  the  taking  up  of  oxygen  in  the 
lungs  and  the  giving  up  of  the  same  to  the  tissues. 

Oxyhaemoglobin  which  is  generally  considered  as  a  weak  acid,  is 
according  to  Gamgee,3  dextrorotatory.  The  specific  rotation  for  light 
of  medium  wave-length  of  C  is  (a) C  =  about  +10°,  which  corresponds 
also  for  carbon-monoxide  haemoglobin.  The  haemoglobin  is  also,  like 
carbon-monoxide  haemoglobin  (COHb)  and  methaemoglobin  (MHb), 
diamagnetic,  while  the  haematin,  which  is  richer  in  iron,  is  strongly  mag- 
netic (Gamgee4).  On  passing  an  electric  current  through  an  oxyhaemo- 
globin solution,  the  pigment  first  separates  unchanged  at  the  anode  in  a 
colloidal  but  still  soluble  form,  and  is  then  gradually  transferred  to  the 
cathode  in  the  colloidal  state  (Gamgee  5) .  According  to  Gamgee,  the 
haemoglobin  probably  exists  in  such  a  colloidal  condition  in  the  blood- 
corpuscles. 

Oxyhaemoglobin  has  been  obtained  in  crystals  from  several  varieties 
of  blood.  These  crystals  are  blood-red,  transparent,  silky,  and  may 
be  2-3  mm.  long.  The  oxyhaemoglobin  from  squirrel's  blood  crystallizes 
in  six-sided  plates  of  the  hexagonal  system;  the  other  varieties  of  blood 
yield  needles,  prisms,  tetrahedra,  or  plates  which  belong  to  the  rhombic 
system.6    The    quantity    of    water    of    crystallization    varies    between 


1  Barcroft  with  Camis,  Journ.  of  Physiol.,  39;  with  Roberts,  ibid.;  W.  Ostwald, 
Kolloid-Zeitschr.,  2,  cited  in  Maly's  Jahresb.,  38,  187. 

2  Annal.  d.  Chem.  u.  Pharm.,  370  and  Zeitschr.  f.  physiol.  Chem.,  70. 

3  Hofmeister's  Beitrage,  4. 

4  Proceedings  of  Roy.  Society,  68. 
« Itnd.,  70. 

6  The  observation  of  Uhlik  (Pfliiger's  Arch.,  104)  that  the  haemoglobin  from  horse- 
blood  can  also  crystallize  in  hexagonal  plates  seems  to  be  due  to  the  fact  that  he  had 
haemoglobin  and  not  oxyhaemoglobin. 


OXYHEMOGLOBIN.  281 

3-10  per  cent  for  the  different  oxyhemoglobins.  When  completely 
dried  at  a  low  temperature  over  sulphuric  acid  the  crystals  may  be 
heated  to  110-115°  C.  without  decomposition.  At  higher  temperatures, 
somewhat  above  160°  C,  they  decompose,  giving  an  odor  of  burned  horn, 
and  leave,  after  complete  combustion,  an  ash  consisting  of  oxide  of 
iron.  The  oxyhemoglobin  crystals  from  difficultly  crystallizable  blood, 
for  example  from  such  as  ox's,  human,  and  pig's  blood,  are  easily 
soluble  in  water.  The  oxyhemoglobins  from  easily  crystallizable 
blood,  as  from  that  of  the  horse,  dog,  squirrel,  and  guinea-pig,  are  soluble 
with  difficulty  in  the  order  above  given.  The  oxyhemoglobin  dissolves 
more  easily  in  a  very  dilute  solution  of  alkali  carbonate  than  in  pure  water, 
and  this  solution  may  be  kept.  The  presence  of  a  little  too  much  alkali 
causes  the  oxyhemoglobin  to  decompose  quickly.  The  crystals  are 
insoluble  in  absolute  alcohol  without  decolorization.  According  to 
Nencki  l  it  is  converted  into  an  isomeric  or  polymeric  modification, 
called  by  him  parahoemoglobin.  Oxyhemoglobin  is  insoluble  in  ether, 
chloroform,  benzene,  and  carbon  disulphide. 

A  solution  of  oxyhemoglobin  in  water  is  precipitated  by  many  metallic 
salts,  but  is  not  precipitated  by  sugar  of  lead  or  basic  lead  acetate.  On 
heating  the  watery  solution  it  decomposes  at  about  70°  C,  and  splits 
off  protein  and  hematin  when  sufficiently  heated.  It  is  also  readily 
decomposed  by  acids,  alkalies,  and  many  metallic  salts.  It  gives  the 
ordinary  reactions  for  proteins  with  those  protein  reagents  which  first 
decompose  the  oxyhemoglobin  with  the  splitting  off  of  protein.  Oxy- 
hemoglobin, like  the  other  blood-pigments,  has  a  direct  oxidizing  action 
upon  tincture  of  guaiacum.  It  has,  on  the  other  hand,  like  all  blood- 
pigments  containing  iron,  the  property  of  an  "  ozone  transmitter  "  in 
that  it  turns  tincture  of  guaiacum  blue  in  the  presence  of  reagents  con- 
taining peroxide,  such  as  old  turpentine. 

A  sufficiently  dilute  solution  of  oxyhemoglobin  or  arterial  blood 
shows  a  spectrum  with  two  absorption-bands  between  the  Fraun- 
hofer  lines  D  and  E  (spectrum  Plate  1).  The  one  band,  a,  which  is  nar- 
rower but  darker  and  sharper,  lies  on  the  line  D;  the  other,  broader, 
less  defined  and  less  dark  band,  /3,  lies  at  E.  The  middle  of  the  first 
band  corresponds  to  a  wave-length  X  =  579  and  the  second  X  =  542.  On 
dilution  the  band  /3  first  disappears.  By  increased  concentration  of  the 
solution  the  two  bands  become  broader,  the  space  between  them  smaller 
or  entirely  obliterated,  and  at  the  same  time  the  blue  and  violet  part 
of  the  spectrum  is  darkened.     Besides  these  two  bands  we  can  also  observe 


1  Nencki  and  Sieber,  Ber.  d.  d.  chem.  Gesellsch.,  18.  According  to  Kriiger  (see 
Biochem.  Centralbl.,  I,  40,  463)  haemoglobin  is  somewhat  changed  by  alcohol  as  well 
as  by  chloroform. 


282  THE  BLOOD. 

by  the  aid  of  special  appliances  (L.  Lewin,  Miethe,  and  Stenger)  the 
band  first  described  by  Soret  and  then  by  Gamgee  in  the  ultra-violet 
portion.  This  violet  band,  X  =  415,  is  of  importance  in  the  detection 
of  very  small  quantities  of  blood.  While  the  two  oxyhemoglobin  bands 
are  still  detectable  in  a  dilution  of  1 :  14, 700  the  violet  band  may  be  seen, 
according  to  Lewin,  Miethe  and  Stenger  l  in  a  dilution  of  1 :40,000. 

The  observation  of  Piettre  and  Vila  that  so-called  laky  blood  and  oxyhsemo- 
globin  solutions  in  thick  layers  also  show  a  third  band  in  the  red  (X  =  634)  depends 
in  all  probability,  as  also  claimed  fey  Ville  and  Derrien,  upon  a  partial  forma- 
tion of  niethsemoglobin  winch  according  to  Aron  2  exists  preformed  in  all  blood. 

A  great  many  methods  have  been  proposed  for  the  preparation  of 
oxyhemoglobin  crystals,  but  in  their  chief  features  they  all  agree  with 
the  following  one  suggested  by  Hoppe-Seyler:  The  washed  blood- 
corpuscles  (best  those  from  the  dog  or  the  horse)  are  stirred  with  2 
vols,  of  water  and  then  shaken  with  ether.  After  decanting  the  ether  and 
allowing  the  ether  which  is  retained  by  the  blood  solution  to  evaporate 
in  an  open  dish  in  the  air,  cool  the  filtered  blood  solution  to  0°  C,  add 
while  stirring  one-fourth  vol.  of  alcohol  also  cooled,  and  allow  to  stand 
a  few  days  at  —5°  to  —10°  C.  The  crystals  which  separate  may  be 
repeatedly  recrystallized  by  dissolving  in  water  of  about  35°  C,  cooling, 
and  adding  cooled  alcohol  as  above.  Lastly,  they  are  washed  with 
cooled  water  containing  alcohol  (one-quarter  vol.  alcohol)  and  dried 
in  vacuum  at  0°  C.  or  a  lower  temperature.3 

For  the  preparation  of  oxyhemoglobin  crystals  in  small  quantities 
from  easily  crystallizable  blood,  it  is  often  sufficient  to  stir  a  drop  of 
blood  with  a  little  water  on  a  microscope  slide  and  allow  the  mixture 
to  evaporate  so  that  the  drop  is  surrounded  by  a  dried  ring.  After 
covering  with  a  cover-glass,  the  crystals  gradually  appear  radiating  from 
the  ring.  These  crystals  are  formed  more  surely  if  the  blood  is  first 
mixed  with  some  water  in  a  test-tube  and  shaken  with  ether,  and  a  drop 
of  the  lower  deep-colored  liquid  treated  as  above  on  the  slide. 

Haemoglobin,  also  called  reduced  haemoglobin  or  purple  cruorin 
(Stokes4),  occurs  only  in  very  small  quantities  in  arterial  blood,  in 
larger  quantities  in  venous  blood,  and  is  almost  the  only  blood-coloring 
matter  after  asphyxiation. 

Hemoglobin  is  much  more  soluble  than  the  oxyhemoglobin,  and 
it   can   therefore   be   obtained   as   crystals   only  with   difficulty.     These 

1  Soret,  cited  in  Maly's  Jaresb.,  8;  Gamgee,  Zeitschr.  f.  Biol.,  34;  Lewin,  Miethe 
and  Stenger,  Pfliiger's  Arch.,  118;  Lewin  and  Miethe,  ibid.,  121. 

2  Piettre  and  Vila,  Compt.  Rend.,  140;  Ville  and  Derrien,  ibid.,  140;  Aron,  Biochem. 
Zeitschr.,  3. 

3  In  regard  to  the  preparation  of  oxyhemoglobin,  see  also  Hoppe-Seyler-Thier- 
felder's  Handbuch,  8.  Aufl.;  also  the  works  cited  in  footnote  1,  p.  278;  also  Schuur- 
manns-Stekhoven,  Zeitschr.  f.  physiol.  Chem.,  33,  296;  see  also  Bohr,  Skand.  Arch, 
f.  Physiol.,  3;  J.  Offringa,  Bioch.  Zeitschr.,  28. 

*  Philosophical  Magazine,  28,  No.  190,  Nov.,  1864. 


METHEMOGLOBIN.  283 

crystals  are  as  a  rule  isomorphous  with  the  corresponding  oxyhemo- 
globin crystals,  but  are  darker,  having  a  shade  toward  blue  or  purple, 
and  are  decidedly  more  pleochromatic.  The  haemoglobin  from  horse- 
blood  has  also  been  obtained  by  Uhlik  x  in  hexagonal  plates.  Its 
solutions  in  water  are  darker  and  more  violet  or  purplish  than  solu- 
tions of  oxyhemoglobin  of  the  same  concentration.  They  absorb  the 
blue  and  the  violet  rays  of  the  spectrum  in  a  less  marked  degree,  but 
strongly  absorb  the  rays  lying  between  C  and  D.  In  proper  dilution 
the  solution  shows  a  spectrum  with  one  broad,  not  sharply  defined  band 
between  D  and  E,  whose  darkest  part  corresponds  to  the  wave-length 
X  =  559  (spectrum  Plate,  2).  This  band  does  not  lie  in  the  middle 
between  D  and  E,  but  is  toward  the  red  end  of  the  spectrum,  a  little 
over  the  line  D.  This  pigment  also  gives  a  band  in  the  ultra-violet, 
X  =  429.  A  haemoglobin  solution  actively  absorbs  oxygen  from  the  air 
and  is  converted  into  an  oxyhemoglobin  solution. 

A  solution  of  oxyhemoglobin  may  be  easily  converted  into  a  solution 
having  the  spectrum  of  hemoglobin  by  means  of  a  vacuum,  by  passing 
an  indifferent  gas  through  it,  or  by  the  addition  of  a  reducing  substance, 
as,  for  example,  an  ammoniacal  ferrous-tartrate  solution  (Stokes'  reduc- 
tion liquid).  If  an  oxyhemoglobin  solution  or  arterial  blood  is  kept  in 
a  sealed  tube,  we  observe  a  gradual  consumption  of  oxygen  and  a  reduc- 
tion of  the  oxyhemoglobin  into  hemoglobin.  If  the  solution  has  a 
proper  concentration,  a  crystallization  of  hemoglobin  may  occur  in  the 
tube  at  lower  temperatures  (Hufner  2). 

Methaemoglobin.  This  name  has  been  given  to  a  coloring-matter 
which  is  easily  obtained  from  oxyhemoglobin  as  a  transformation  prod- 
uct and  which  has  been  correspondingly  found  in  transudates  and  cystic 
fluids  containing  blood,  in  urine  in  hematuria  or  hemoglobinuria,  and 
also  in  urine  and  blood  on  poisoning  with  potassium  chlorate,  amyl 
nitrite  or  alkali  nitrite,  and  many  other  bodies. 

Methemoglobin  does  not  contain  any  oxygen  in  molecular  or  dis- 
sociable combination,  but  still  the  oxygen  seems  to  be  of  importance  in 
the  formation  of  methemoglobin,  because  it  is  formed  from  oxyhemo- 
globin and  not  from  hemoglobin  in  the  absence  of  oxygen  or  oxidizing 
agents.  If  arterial  blood  be  sealed  up  in  a  tube,  it  gradually  consumes 
its  oxygen  and  becomes  venous,  and  by  this  absorption  of  oxygen  a  little 
methemoglobin  is  formed.  The  same  occurs  on  the  addition  of  a  small 
quantity  of  acid  to  the  blood.  By  the  spontaneous  decomposition 
of  blood  some  methemoglobin  is  formed,  and  by  the  action  of  ozone, 
potassium    permanganate,    potassium     ferricyanide,    chlorates,    nitrites, 

1  Pfluger's  Arch.,  104. 

2  Zeitschr.  f.  physiol.  Chem.,  4;  see  also  Uhlik,  1.  c. 


284  THE  BLOOD. 

nitrobenzene,  pyrogallol,  pyrocatechin,  acetanilide,  and  certain  other 
bodies  on  the  blood  an  abundant  formation  of  methsemoglobin  takes  place. 
By  the  action  of  light,  Hasselbach,1  especially  by  the  use  of  rays  hav- 
ing a  wave-light  below  310  n  n,  obtained  methsemoglobin  from  oxyhemo- 
globin, but  not  from  haemoglobin  in  the  absence  of  oxygen,  and  by  this 
behavior  pure  methsemoglobin  can  be  prepared. 

According  to  the  investigations  of  Hufner,  Kulz,  and  Otto  2 
methsemoglobin  contains  just  as  much  oxygen  as  oxyhsemoglobin,  but 
it  is  more  strongly  combined,  a  view  which  is  accepted  by  most  investiga- 
tors. According  to  Hufner  and  v.  Zeynek  we  can  admit  in  the  methsemo- 
globin formation  of  an  expulsion  of  oxygen  and  a  combination  of  two 

,OH 
hydroxyl  groups;     methsemoglobin    would    then    be  Hb<^       .    Accord- 

NOH 
ing  to  others,  Hoppe-Seyler,  Kuster,  Letsch  the  methsemoglobin 
contains  less  oxygen  than  the  oxyhsemoglobin  and  is  HbO  or  HbOH. 
A  methsemoglobin  solution  is  converted  into  a  haemoglobin  solution 
by  reducing  substances.  The  reaction  taking  place  in  the  forma- 
tion of  methsemoglobin  from  oxyhsemoglobin  by  the  action  of  potassium 
ferricyanide  has  been  quantitatively  followed  by  v.  Reinbold.3  He 
found  that  one  molecule  of  K3Fe(CN)6  was  necessary  to  transform  1 
molecule  oxyhsemoglobin  or  to  drive  off  1  molecule  of  oxygen  from  the 
oxyhsemoglobin.     The  reaction  takes  place  according  to  the  equation: 

/° 
Hb<  |  +K3Fe(CN)6-|-H20  =  Hb.OH+K3HFe(CN)6+02 

xO 

and  from  his  investigations  he  gives  the  formula  Hb.OH  to  methsemo- 
globin, in  correspondence  to  the  views  of  Kuster. 

According  to  Hufner  and  Reinbold4  1  gram  methsemoglobin  can 
take  up  2.685  cc.  nitric  oxide. 

Methsemoglobin  crystallizes,  as  first  shown  by  Hufner  and  Otto, 
in  brownish-red  needles,  prisms,  or  six-sided  plates.  It  dissolves  easily 
in  water;  the  solution  has  a  brown  color  and  becomes  a  beautiful  red 
on  the  addition  of  alkali.  The  solution  of  the  pure  substance  is  not 
precipitated  by  basic  lead  acetate  alone,  but  by  basic  lead  acetate  and 


1  Bioch.  Zeitschr.,  19. 

*See  Otto,  Zeitschr.  f.  physiol.  Chem.,  7;   v.  Zeynek,  Arch.  f.  (Anat.  u.)  Physiol., 
1899;  Hufner,  ibid. 

3  Kuster,  Zeitschr.  f.  physiol.  Chem.,  66;  Letsche,  ihid.,  80;   v.  Reinbold,  Zeitschr. 
f.  physiol.  Chem.,  85. 

4  Arch.  f.  (Anat.  u.)  Physiol.,  1904,  Suppl. 


METH.EMOGOBLV     OYANMETHiEMOGLOBIN.  285 

ammonia.  The  a l»<>rpt ion-spectrum  of  a  watery  or  acidified  solution 
of  met  haemoglobin  is,  according  to  Jaderholm  and  Bertin-Sans,  very 
similar  to  that  of  haematin  in  acid  solution,  but  is  easily  distinguished 
from  the  latter  since,  on  the  addition  of  a  little  alkali  and  a  reducing 
substance,  the  former  passes  over  to  the  spectrum  of  reduced  haemoglobin, 
while  a  haematin  solution  under  the  same  conditions  gives  the  spectrum 
of  an  alkaline  haemochromogen  solution  (see  below).  According  to 
Araki  and  Dittrich,  a  neutral  or  faintly  acid  methaemoglobin  solution 
shows  only  one  characteristic  band,  a,  between  C  and  D,  whose  middle 
corresponds  to  about  X  =  634.  The  two  bands  between  D  and  E  are 
only  due  to  contamination  with  oxyhsemoglobin  (Menzies,  Lewin, 
Miethe  and  Stenger.  According  to  Hasselbach's  l  experience  a 
pure  neutral  solution  of  methaemoglobin  gives  four  absorption  bands  cor- 
responding to  a  maxima  X  =  630,  580,  540  and  500.  Methaemoglobin 
in  alkaline  solution  shows  two  absorption-bands  which  are  like  the 
two  oxy haemoglobin  bands,  but  they  differ  from  these  in  that  the  band 
/S  is  stronger  than  a.  By  the  side  of  the  band  a  and  united  with  it  by  a 
shadow  lies  a  third  fainter  band  between  C  and  D,  near  to  D.  (Spec- 
trum Plate,  4.) 

The  claims  as  to  the  action  of  sodium  fluoride  upon  haemoglobin  and  methaemo- 
globin are  somewhat  contradictory.2 

Crystallized  methaemoglobin  may  be  easily  obtained  by  treating  a 
concentrated  solution  of  oxyhemoglobin  with  a  sufficient  quantity  of 
concentrated  potassium-ferricyanide  solution  to  give  the  mixture  a  porter- 
brown  color.  After  cooling  to  0°  C.  add  cne-fourth  vol.  cooled  alcohol 
and  allow  the  mixture  to  stand  a  few  days  in  the  cold.  The  crystals 
may  be  easily  purified  by  recrystallizing  from  water  by  the  addition 
of  alcohol.  According  to  Hasselbach  this  method  ordinarily  gives 
imnure  products  while  a  pure  preparation  can  be  obtained  by  the  action 
of  i^ht  (see  above). 

Cyanmethaemoglobin  (cyanhaemoglobin)  is,  according  to  Haldaxe.  identical 
with  photomethaemoglobin  (Bock),  which  is  produced  by  the  influence  of  sun- 
light upon  a  methaemoglobin  solution  containing  potassium  ferricyanide.  It 
wu-  first  carefully  described  by  R.  Robert  and  obtained  in  a  crystalline  form 
by  v.  Zeyxek.3  It  is  immediately  formed  in  the  cold  by  the  action  of  a  hydro- 
cyanic-acid solution  upon  methaemoglobin,  but  is  formed  by  its  action  upon  oxy- 

1  Jaderholm,  Zeitschr.  f.  Biol.  16;  Bertin-Sans.  Comp.  Rend.,  106;  Araki,  Zeitschr.  f. 
physiol.  Chem.,  14;  Dittrich.  Arch.  f.  exp.  Path.  u.  Pharm..  29:  Menzies,  Journ.  of 
Physiol.,  17;  Lewin  and  collaborators,  footnote  1,  page  282:  Hasselbach,  Bioch. 
Zeitschr.,  19,  and  Proceedings  of  the  7th  Internat.  Congr.  of  Appl.  Chem.,  London, 
1909.      Important  references  on  methaemoglobin  are  given  by  Otto,  Pfluger's  Arch.,  31. 

2  Piettre  and  Vila,  Compt.  Rend.,  140;  Ville  and  Derrien,  ibid.,  140. 

3  Haldane,  Journ.  of  Physiol.,  25;  Bock,  Skand.  Arch.  f.  Physiol.,  6:  Robert, 
Pfluger's  Arch..  82;  v.  Zeynek,  Zeitschr.  f.  physiol.  Chem.,  33.  See  also  Leers, 
Biochem.  Zeitschr.,  12. 


286  THE  BLOOD. 

hsemoglobin  only  at  the  body  temperature.  The  neutral  or  faintly  alkaline 
solutions  show  a  spectrum  which  is  very  similar  to  the  hsemoglobin  spectrum. 
The  question  as  to  a  special  cyanmethsemoglobin  is  still  disputed. 

Acid  haemoglobin  is  a  coloring-matter  produced  by  the  action  of  very  weak 
acids  upon  oxyhemoglobin,  which  according  to  Harnack  *  is  not,  as  used  to  be 
admitted,  identical  with  methsemoglobin. 

Carbon-monoxide  Haemoglobin  2  is  the  molecular  combination  between 

1  molecule  of  hsemoglobin  and  1  molecule  of  CO,  according  to  Hufner,3 
which  contains  1.34  cc.  of  carbon  monoxide  (at  0°  and  760  mm.  Hg) 
for  1  gram  haemoglobin.  This  combination  is  stronger  than  the  oxygen 
combination  of  hsemoglobin.  The  oxygen  is  for  this  reason  easily  driven 
out  of  oxyhsemoglobin  by  carbon  monoxide,  and  this  explains  the  poison- 
ous action  of  this  gas,  which  kills  by  the  expulsion  of  the  oxygen  of  the 
blood.  In  regard  to  the  division  of  the  blood-pigments  between  the  car- 
bon monoxide  and  oxygen  under  different  partial  pressures  of  both  gases 
in  the  air,  we  must  refer  to  the  investigations  of  Hufner,  Douglas  and 
Haldane.4 

The  carbon  monoxide  can  be  driven  out  by  a  vacuum  as  well  as  by 
passing  an  indifferent  gas,  or  oxygen,  or  nitric  oxide,  through  the  solu- 
tion for  a  long  time,  and  in  these  cases  hsemoglobin,  oxyhsemoglobin, 
or  nitric-oxide  hsemoglobin  are  formed.  The  carbon-monoxide  is  also 
expelled  by  potassium  ferricyanide  and  methaemoglobin  is  formed 
(Haldane  5) .  The  above-mentioned  behavior  found  by  Manchot  for 
the  absorption  of  oxygen,  namely,  that  the  amount  of  gas  taken  up 
increases  writh  the  dilution  of  the   blood  so  that  for  every  atom  of  iron 

2  mol.  of  gas  are  absorbed  applies  also  for  the  carbon-monoxide  hsemo- 
globin as  well  as  for  the  nitric-oxide  hsemoglobin,  which  will  be  discussed 
further  on. 

Carbon-monoxide  hsemoglobin  is  formed  by  saturating  blood  or 
a  hsemoglobin  solution  with  carbon  monoxide,  and  may  be  obtained 
as  crystals  by  the  same  means  as  oxyhsemoglobin.  These  crystals  are 
isomorphous  with  the  oxyhsemoglobin  crystals,  but  are  less  soluble  and 
more  stable,  and  their  bluish-red  color  is  more  marked.     For  the  detec- 


1  Zeitschr.  f.  physiol.  Chem.,  26. 

2  In  reference  to  carbon-monoxide  haemoglobin,  see  especially  Hoppe-Seyler,  Med.- 
chem.  Untersuch.,  201;  Centralbl.  f.  d.  med.  Wissensch.,  1S64  and  1865;  Zeitschr. 
f.  physiol.  Chem.,  1  and  13. 

3  Arch.  f.  (Anat.  u.)  Physiol.,  1894.  On  the  dissociation  constant  of  carbon- 
monoxide  hsemoglobin,  see  ibid.,  1895.  In  regard  to  the  contradictory  statements 
of  Saint-Martin  and  others  and  their  disapproval,  see  Hufner,  Arch.  f.  (Anat.  u.) 
Physiol.,  1903. 

4  Hufner,  Arch.  f.  exp.  Path.  u.  Pharm.,  48;  Douglas  and  Haldane,  Journ.  of 
Physiol.,  44. 

6  Journ.  of  Physiol.,  22. 


CARBON-MONOXIDE  HAEMOGLOBIN.  287 

tion  of  carbon-monoxide  haemoglobin,  its  absorption-spectrum  is  of  the 

greatest  importance.  This  spectrum  shows  two  bands  which  are  very 
similar  to  those  of  oxyhemoglobin,  hut  they  occur  more  toward  the  violet 
part  of  the  spectrum.  The  middle  of  the  first  hand  corresponds  to 
X  =  570,  and  the  second  to  X  =  542  (Lewix,  Miethe  and  Stenger). 
These  bands  do  not  change  noticeably  on  the  addition  of  reducing 
substances;  this  constitutes  an  important  difference  between  carbon- 
monoxide  haemoglobin  and  oxyhemoglobin.  If  the  blood  contains  oxy- 
hemoglobin and  carbon-monoxide  haemoglobin  at  the  same  time,  we 
obtain  on  the  addition  of  a  reducing  substance  (ammoniacal  ferro-tar- 
trate  solution)  a  mixed  spectrum  originating  from  the  haemoglobin  and 
carbon-monoxide  haemoglobin.  Carbon-monoxide  haemoglobin  also  gives 
a  band  in  the  violet  X  =  dd6. 

A  great  many  reactions  have  been  suggested  for  the  detection  of 
carbon-monoxide  haemoglobin  in  medico-legal  cases.  A  simple  and  at 
the  same  time  a  good  one  is  Hoppe-Seyler's  alkali  test.  The  blood  is 
treated  with  double  its  volume  of  caustic-soda  solution  of  1.3  sp.  gr., 
by  which  ordinary  blood  is  converted  into  a  ding}'  brownish  mass,  which 
when  spread  out  on  porcelain  is  brown  with  a  shade  of  green.  Carbon- 
monoxide  blood  gives  under  the  same  conditions  a  red  mass,  which  if 
spread  out  on  porcelain  shows  a  beautiful  red  color.  Several  modifica- 
tions of  this  test  have  been  proposed.  Another  very  good  reagent  is  tan- 
nic acid,  which  gives  with  dilute  normal  blood  a  brownish-green  precip- 
itate and  with  carbon-monoxide  blood  a  pale  crimson-red  precipitate.1 

As  according  to  Bohr  there  are  several  oxyhemoglobins,  so  also  accord- 
ing to  Bohr  and  Bock,'-  there  are  several  carbon-monoxide  hemoglobins,  with 
different  amounts  of  carbon  monoxide.  As  haemoglobin  can  unite  with  oxygen 
and  carbon  dioxide  simultaneously,  as  shown  by  Bohr  and  Troup,  so  also  can  it 
unite  with  carbon  monoxide  and  carbon  dioxide  simultaneously  and  independently 
of  each  other. 

Carbon-monoxide  methaemoglobin  has  been  prepared  by  Weil  and  v.  Axrep 
by  the  action  of  potassium  permanganate  on  carbon-monoxide  haemoglobin, 
but  this  is  contradicted  by  Bertix-Saxs  and  Moitessier.3  Sulphur  methaemo- 
globin is  the  name  given  by  Hoppe-Seyler  to  that  coloring-matter  which  is 
formed  by  the  action  of  sulphureted  hydrogen  upon  oxyhemoglobin  and  which 
is  generally  designated  sidphcemoglobin.  The  solution  has  a  greenish-red,  dirty 
color,  and  shows  two  absorption-bands  between  C  and  D.  This  coloring-matter 
is  claimed  to  be  the  greenish  color  seen  on  the  surface  of  putrefying  flesh.  Accord- 
ing to  Harxack  the  conditions  are  different  when  H>S  is  passed  through  an 
oxygen-free   solution   of   hemoglobin    (or   carbon-monoxide   haemoglobin).     The 

1  In  regard  to  this  test  (as  suggested  by  Kunkel)  and  others  we  refer  to  Kostin, 
Pfliiger's  Arch.,  S4,  which  contains  a  very  excellent  summary  of  the  literature  on  the 
subject.     See  also  de  Domenicis,  Chem.  Centralbl.,  1908,  2,  p.  06. 

2  Centralbl.  f.  Physiol.,  8,  and  Mary's  Jahresber.,  25. 

3  v.  Anrep,  Arch.  f.  (Anat.  u.)  Physiol.,  1880;  Sans  and  Moitessier,  Compt.  Rend., 
113. 


288  THE  BLOOD. 

sulphsemoglobin  thus  formed  shows  one  band  in  the  red  between  C  and  D. 
According  to  Clarke  and  Hurtley  '  the  formation  of  sulphsemoglobin  takes 
place  after  the  reduction  to  haemoglobin. 

Carbon-dioxide  Haemoglobin,  Carbohcemoglobin.  Haemoglobin,  accord- 
ing to  Bohr  and  Torup,2  also  forms  a  molecular  combination  with 
carbon  dioxide  whose  spectrum  is  similar  to  that  of  haemoglobin.  Accord- 
ing to  Bohr  there  are  three  different  carbohsemoglobins,  namely,  a-, 
/3-,  and  7-carbohsemoglobin,  in  which  1  gram  combines  with  respectively 
1.5,  3,  and  6  cc.  CO2  (measured  at  0°  C.  and  760  mm.)  at  18°  C.  and  a 
pressure  cf  60  mm.  mercury.  If  a  hsemoglobin  solution  is  shaken  with  a 
mixture  of  oxygen  and  carbon  dioxide,  the  hsemoglobin  combines  loosely 
with  the  oxygen  as  well  as  with  the  carbon  dioxide,  independently  of 
each  other,  just  as  if  each  gas  existed  alone  (Bohr).  He  considers 
that  the  two  gases  are  combined  with  different  parts  of  the  hsemoglobin, 
that  is,  the  oxygen  with  the  pigment  nucleus  and  the  carbon  dioxide 
with  the  protein  component.  Attention  must  be  called  to  the  fact  that, 
as  observed  by  Torup,  hsemoglobin  is  in  part  readily  decomposed  by 
the  carbon  dioxide  with  the  splitting  off  of  some  protein. 

Nitric-oxide  Haemoglobin  is  also  a  crystalline  molecular  combina- 
tion which  is  even  stronger  than  the  carbon-monoxide  hsemoglobin. 
Its  solution  shows  two  absorption-bands,  which  are  paler  and  less  sharp 
than  the  carbon-monoxide  hsemoglobin  bands,  and  they  do  not  dis- 
appear on  the  addition  of  reducing  bodies.  Hsemoglobin  also  forms  a 
molecular  combination  with  acetylene  and  ethylene. 

Haemorrhodin  is  the  name  given  by  Lehmann  to  a  beautiful  red  pigment 
soluble  in  alcohol  and  ether,  which  is  extracted  from  meat  and  meat  products 
by  boiling  alcohol  and  which  seems  to  be  produced  by  the  action  of  small  amounts 
of  nitrites.  Another  pigment  isolated  by  Lewin  3  from  the  blood  of  animals 
poisoned  by  phenylhydrazine,  has  been  called  hcenioverdin.  By  heating  a  solu- 
tion of  blood-pigment  treated  with  caustic  potash  and  mixed  with  alcohol  to 
60°  C.  we  obtain,  according  to  v.  Klaveren,  a  pigment  which  he  calls  kathoemo- 
globin,  but  called  by  Arnold,4  who  first  obtained  it,  neutral  hopmatin,  which  is 
produced  by  the  splitting  off  of  a  ferruginous  complex.  This  pigment  still  con- 
tains protein,  but  is  poorer  in  iron  than  the  hsemoglobin  or  methsemoglobin  and 
probably  forms  an  intermediary  product  in  the  conversion  of  the  above  into 
hsematin. 

Decomposition  products  of  the  blood-pigments.  By  its  decomposi- 
tion, hsemoglobin  yields,  as  previously  stated,  a  protein   which  has  Leon 

1  Hoppe-Seyler,  Med. -chem.  Untersuch.,  151.  See  Araki,  Zeitschr.  f.  physiol. 
Chem.,  14;  Harnack,  1.  c;  Clarke  and  Hurtley,  Journ.  of  Physiol.,  36. 

2  Bohr,  Extrait,  du  Bull,  de  l'Acad.  Danoise,  1890;  Centralbl.  f.  Physiol.,  4  and 
17;  Torup,  Maly'e  Jahresber.,  17. 

3  K.  B.  Lehmann,  Sitzungsber.  d.  phys.-med.  Gesellsch.  Wiirzburg,  1899;  Lewin, 
Compt.  Rend.,  133. 

*  v.  Klaveren,  Zeitschr.  f.  physiol.  Chem.,  33;  Arnold,  ibid,  29. 


HSEMOCHROMOGEN.  289 

called  globin  (Preyer,  Schulz),  and  a  ferruginous  pigment  as  chief  prod- 
ucts. According  to  Lawrow  94.09  per  cent  protein,  4.47  per  cent 
hsematin,  and  1.44  per  cent  other  bodies  are  produced  in  this  decom- 
position. The  globin,  which  was  isolated  and  studied  by  Schulz,1 
differs  from  most  other  proteins  by  containing  a  high  amount  of  carbon, 
54.97  per  cent.,  with  16.98  per  cent  of  nitrogen.  It  is  insoluble  in  water, 
but  very  easily  soluble  in  acids  or  alkalies.  It  is  not  dissolved  by  ammonia 
in  the  presence  of  ammonium  chloride.  Nitric  acid  precipitates  it  in 
the  cold,  but  not  when  warm.  It  may  be  coagulated  by  heat,  but  the 
coagulum  is  readily  soluble  in  acids.  Because  of  these  reactions  it  is 
considered  as  a  histone  by  Schulz. 

On  hydrolytic  cleavage  globin  (from  horse-blood)  yields,  accord- 
ing to  Abderhalden,2  the  ordinary  cleavage  products  of  the  proteins 
and  especially  leucine,  29  per  cent.  It  is  also  important  to  call  attention 
to  the  large  amount  of  histidine,  10.96  per  cent,  while  the  quantities  of 
arginine  and  lysine  were  only  5.42  and  4.28  per  cent  respectively. 

The  pigment  split  off  is  different,  depending  upon  the  conditions 
under  which  the  cleavage  takes  place.  If  the  decomposition  takes  place 
in  the  absence  of  oxygen,  a  coloring-matter  is  obtained  which  is  called 
by  Hoppe-Seyler  hcemochromogen,  by  other  investigators  (Stokes) 
reduced  hcematin.  In  the  presence  of  oxygen,  hsemochromogen  is  quickly 
oxidized  to  hsematin,  and  there  is  therefore  obtained  in  this  case  hcematin 
as  a  colored  decomposition  product.  As  hsemochromogen  is  easily 
converted  by  oxygen  into  hsematin,  so  this  latter  may  be  reconverted 
into  hsemochromogen  by  reducing  substances. 

Heemochromogen  was  discovered  by  Hoppe-Seyler.3  It  is,  accord- 
ing to  Hoppe-Seyler,  the  colored  atomic  group  of  hsemoglobin  and  of 
its  combinations  with  gases,  and  this  atomic  group  is  combined  with 
proteins  in  the  pigment.  The  characteristic  absorption  of  light  depends 
on  the  hsemochromogen,  and  it  is  also  this  atomic  group  which  binds,  in 
the  oxy hsemoglobin,  1  molecule  of  oxygen  and,  in  the  carbon-monoxide 
hsemoglobin,  1  molecule  of  carbon  monoxide  with  1  atom  of  iron.  Hsemo- 
chromogen is  produced  in  an  alkaline  solution  of  hsematin  by  the  action 
of  reducing  bodies.  By  the  reduction  of  hsematin  in  alcoholic  ammoniacal 
solution  by  means  of  hydrazine  v.  Zeynek4  was  able  to  obtain  the  solid 
brownish-red  ammonia  combination.  A  crystalline  combination  between 
pyridine   and   hsemochromogen    can  be   obtained  according  to  Kalmus 


1  Lawrow,  ibid.,  26;  Schulz,  ibid.,  24;  Preyer,  Die  Blutkristalle,  Jena,  1871. 

2  Zeitschr.  f.  physiol.  Chem.,  37;  with  Baumann,  ibid.,  51. 

3  Ibid.,  13. 

*  Zeitschr.  f.  physiol.,  Chem.,  25. 


290  THE  BLOOD. 

and  v.  Zeynek  1  from  haemoglobin  and  pyridine  by  boiling,  or  from 
haematin  and  haemin  and  pyridine  after  the  addition  of  hydrazin-hydrate. 

Haemochromogen  also  combines,  as  Hoppe-Seyler  first  showed,  with 
carbon  monoxide.  This  compound,  which  in  aqueous  solution  gives 
a  spectrum  similar  to  oxyhemoglobin,  has  been  obtained  by  Pregl2 
in  the  solid  condition  as  a  deep-violet  powder  which  is  insoluble  in 
absolute  alcohol.  In  opposition  to  haemoglobin  the  haemochromogen 
combines  with  oxygen  more  firmly  than  with  carbon  monoxide.  The 
assumption  of  Hoppe-Seyler,  that  this  compound  is  a  combination  of  1 
molecule  haemochromogen  and  therefore  contains  1  molecule  carbon 
monoxide  for  1  molecule  of  iron  has  been  experimentally  substantiated 
by  Hufner  and  Ruster  and  by  Pregl.3 

An  alkaline  haemochromogen  solution  has  a  beautiful  cherry-red 
color.  It  shows  two  absorption-bands,  first  described  by  Stokes  (spec- 
trum Plate,  6),  one  of  which  is  dark  and  whose  center  corresponds  to 
X  =  556.4  between  D  and  E,  and  a  second  broader  band,  less  dark,  which 
covers  the  Fraunhofer  lines  E  and  b.  The  middle  of  this  band  cor- 
responds to  X  =  526  to  530  according  to  Lewin,  Miethe  and  Stenger. 
In  acid  solution  haemochromogen  shows  four  bands,  which,  according 
to  Jaderholm,4  depend  on  a  mixture  of  haemochromogen  and  haemato- 
porphyrin  (see  below),  this  last  formed  by  a  partial  decomposition 
resulting  from  the  action  of  the  acid. 

Milroy,5  from  an  alcoholic  solution  of  haematin  containing  oxalic 
acid,  after  driving  out  the  air  by  means  of  hydrogen  gas,  gradually  obtained 
an  acid  solution  of  reduced  haematin  (haemochromogen)  by  means  of 
zinc  dust.     This  solution  showed  one  absorption-band  between  D  and  E. 

Haemochromogen  may  be  obtained  as  crystals  by  the  action  of  caustic 
soda  on  haemoglobin  at  100°  C.  in  the  absence  of  oxygen  (Hoppe-Seyler). 
By  the  decomposition  of  haemoglobin  by  acids  (of  course  in  the  absence 
of  air)  we  obtain  haemochromogen  contaminated  with  a  little  haemato- 
porphyrin.  An  alkaline  haemochromogen  solution  is  easily  obtained  by 
the  action  of  a  reducing  substance  (Stokes'  reduction  liquid)  on  an 
alkaline  haematin  solution.  An  ammoniacal  solution  of  haematin  on 
reduction  with  hydrazine  yields  haemochromogen  very  easily.  An  alco- 
holic, alkaline  hydrazine  solution  is  also  recommended  by  Riegler  6 
as  a  reagent  for  blood-pigments,  converting  them  into  haemochromogen. 

Haematin,  also  called  Oxyh^ematin,  is  sometimes  found  in  old  transu- 
dates.    It  is  formed  by  the  action  of  the  gastric  or  pancreatic  juices  on 

1  E.  Kalmue,  Zeitschr.  f.  Chem.,  70;  v.  Zeynek,  ibid.,  70. 

2  Ibid.,  44. 

•  Hufner  and  Kiister,  Arch.  f.  (Anat.  u.)  Physiol.,  1904,  Suppl.  Pregl,  1.  c. 

*  Nord.  Med.  Arkiv.,  16. 
5  Journ.  of  Physiol.,  32. 

8  Zeitschr.  f.  anal.  Chem.,  43. 


HiEMATIN.  291 

oxyhemoglobin,  and  is,  therefore,  found  in  tlio  feces  after  hemorrhage 
in  the  intestinal  canal,  and  also  after  a  meat  diet  and  food  rich  in  blood. 
It  is  stated  that  hsematin  may  occur  in  urine  after  poisoning  with  arseniu- 
reted  hydrogen.  As  shown  above,  the  haematin  is  formed  by  the  decom- 
position of  oxyhemoglobin,  or  at  least  of  haemoglobin,  in  the  presence 
of  oxygen. 

The  views  in  regard  to  the  composition  of  haematin  are  rather  con~ 
tradictory,  which  seems  to  be  due  to  the  fact  that  the  substance  haemin 
(see  below),  from  which  the  formula  of  haematin  is  derived,  has  a  some- 
what different  composition,  dependent  upon  various  conditions.  Accord- 
ing to  Hoppe-Seyler  haematin  has  the  formula  C34H34N4FeOs,  and 
from  the  recent  investigations  upon  haemin,  which  will  be  mentioned 
below,  this  formula  seems  to  be  now  generally  accepted.  According 
to  this  formula  1  atom  of  iron  occurs  with  every  4  atoms  of  nitrogen. 
According  to  Cloetta,  and  also  Rosenfeld,1  hsematin  has  the  formula 
CaoH34N3Fe03,  with  1  atom  of  iron  for  every  3  atoms  of  nitrogen. 

v.  Zeynek  has  prepared  a  hsematin  by  the  digestion  of  an  oxyhemoglobin 
solution  with  pepsin-hydrochloric  acid,  from  which  he  then  prepared  haemin. 
As  this  hsematin  of  v.  Zeynek  was  readily  convertible  into  haemin,  and  while  the 
ordinarily  prepared  haematin  from  haemin  cannot  be  retransformed  into  haemin, 
Kuster  considers  that  these  two  forms  of  haematin  are  not  identical.  The 
first  he  calls  a-haanatin  and  the  ordinary  which  is  a  polymeric  body,  he  calls 
/3-haematin.  That  a  retransformation  of  haemin  is  possible  from  ordinary  haematin 
is  still  admitted  by  Piloty  and  Ellinger.2 

Haematin  contains  at  least  three  hydroxy  1  groups,  one  of  which  acts 
as  hydroxyl  ion  and  seems  to  be  united  with  the  iron,  and  is  replaced 
in  the  haemin  formation  (see  below)  by  the  chlorine.  By  means  of  the 
two  others,  salts  with  metals  as  well  as  alkyl  derivatives  may  be  formed, 
which  latter  (as  haemin  derivatives)  have  been  especially  studied  by  Nencki 
and  Zaleski  and  Kuster.3  Haematin  dissolves  in  concentrated  sulphuric 
acid  and  is  converted  into  haematoporphyrin,  with  the  splitting  off  of 
iron.  On  heating  dry  hsematin  it  yields  an  abundance  of  pyrrol.  The 
products  produced  on  the  oxidation  and  reduction  of  haematin  and  the 
question  as  to  the  constitution  of  haematin  will  be  discussed  in  connec- 
tion with  haematoporphyrin. 

Haematin  is  amorphous,  dark  brown  or  bluish-black.  It  may  be 
heated  to  180°  C.  without  decomposition;   on  burning  it  leaves  a  residue 


1  Hoppe-Seyler,  Med.-chem.  Untersuch.,  p.  525;  Cloetta,  Arch.  f.  exp.  Path.  u. 
Pharm.,  36;  Rosenfeld,  ibid.,  40. 

2v.  Zeynek,  Zeitschr.  f.  physiol.  Chem.,  30  and  49;  Kuster.  ibid.,  66  and  Ber. 
d.  d.  chem.  Gesellsch.,  43;  Piloty,  Annal.  d.  Chem.  u.  Pharm.,  377;  Eppinger,  Unters. 
uber  den  Blutfarbstoff.  Dissert.  Munchen,  1907. 

5  Xencki  and  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  30;  Kuster,  Ber.  d.  d.  Chem. 
Gesellsch.  43  and  45,  and  Zeitschr.  f.  physiol.  Chem.,  82. 


292  THE  BLOOD. 

consisting  of  iron  oxide.  It  is  insoluble  in  water,  dilute  acids,  alcohol, 
ether,  and  chloroform,  but  it  dissolves  slightly  in  warm  glacial  acetic  acid. 
Haematin  dissolves  in  acidified  alcohol  or  ether.  It  easily  dissolves  in 
alkalies,  even  when  very  dilute.  The  alkaline  solutions  are  dichroic; 
in  thick  layers  they  appear  red  by  transmitted  light  and  in  thin  layers 
greenish.  The  alkaline  solutions  are  precipitated  by  lime-  and  baryta- 
water,  as  also  by  solutions  of  neutral  salts  of  the  alkaline  earths.  The 
acid  solutions  are  always  brown. 

An  acid  haematin  solution  (spectrum  Plate,  4),  absorbs  the  red  part 
of  the  spectrum  only  slightly  and  the  violet  parts  strongly.  The  solu- 
tion shows  a  rather  sharply  defined  band  between  C  and  D,  whose  posi- 
tion may  change  with  the  variety  of  acid  used  as  a  solvent.  Between 
D  and  F  a  second,  much  broader,  less  sharply  defined  band  occurs,  which 
by  proper  dilution  of  the  liquid  is  converted  into  two  bands.  The  one 
between  b  and  F,  lying  near  F,  is  darker  and  broader;  the  other,  between 
D  and  E,  lying  near  E,  is  lighter  and  narrower.  Also  by  proper  dilution 
a  fourth  very  faint  band  is  observed  between  D  and  E,  lying  near  D. 
Haematin  may  thus  in  acid  solution  show  four  absorption-bands;  ordi- 
narily one  sees,  distinctly,  only  the  bands  between  C  and  D  and  the  broad, 
dark  band — or  the  two  bands — between  D  and  F.  In  alkaline  solution, 
hsematin  (spectrum  Plate,  5),  shows  a  broad  absorption-band,  which 
lies  in  greatest  part  between  C  and  D,  but  reaches  a  little  over  the  line 
D  toward  the  right  in  the  space  between  D  and  E.  As  the  position  of 
the  haematin  bands  in  the  spectrum  is  quite  variable,  the  exact  wave- 
lengths corresponding  thereto  cannot  be  given  exactly. 

Haemin,  Haemin  Crystals,  or  Teichmann's  Crystals.  Haemin  is 
formed,  as  generally  admitted,  by  the  replacement  of  an  HO  group  by 
chlorine  in  the  haematin,  and  is  the  starting  point  in  the  preparation  of 
the  latter. 

The  statements  as  to  the  composition  of  haemin  differ  quite  considerably, 
and  various  haemins  have  been  accepted,  which  is  partly  due  to  the  fact, 
as  first  shown  by  Nencki  and  Zaleski,  that  haemin  combines  with  acid 
and  alkyl  radicals  and  can  also  give  addition  products  with  other 
bodies.  Thus  for  example  the  methylhaemins,  carefully  studied  by 
Kuster,  especially  monomethylhaemin,  is  produced  in  the  preparation 
of  haemin  according  to  Morner's  method  (see  below)  by  means  of  methyl 
alcohol.  These  behaviors  have  been  further  explained  by  the  work 
of  numerous  investigators,  especially  by  Kuster,  and  most  investigators 
generally  admit  that  only  one  haemin  exists  whose  general  formula  is 
C34H3304N4FeCl.  According  to  Piloty  the  formula  is  C34H3204N4FeCl 
while    Piettre    and    Vila  l     deny    this    formula    and    claim    to    have 

1  Nencki  and  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  30;  Nencki  and  Sieber,  Arch. 
f.  exp.  Path.  u.  Pharm.,  18  and  20,  and  Ber.  d.  d.   chem.   Gesellsch.,  18;   Schalfejeff 


H/EMTN.  293 

prepared  a  haemin  free  from  chlorine,  from  pure  crystalline  oxy- 
hemoglobin. 

Ha>min  crystals  form,  in  large  masses,  a  bluish-black  powder,  but  are 
so  small  that  they  can  be  seen  only  by  aid  of  the  microscope.  They 
consist  of  dark-brown  or  nearly  brownish-black  long,  rhombic,  or  spool- 
like crystals,  isolated  or  grouped  as  crosses,  rosettes,  or  stellar  forms. 
Cubical  crystals  may  also  occur,  according  to  Cloetta.  They  are 
insoluble  in  water,  dilute  acids  at  the  normal  temperature,  alcohol,  ether, 
and  chloroform.  They  are  slightly  soluble  in  glacial  acetic  acid  with  heat. 
They  dissolve  in  acidified  alcohol,  as  also  in  dilute  caustic  alkalies  or 
carbonates;  and  in  the  last  case  they  form,  besides  alkali  chlorides, 
soluble  haematin  alkali,  from  which  the  haematin  may  be  precipitated  by 
an  acid.  As  shown  by  Piloty  and  Eppinger  and  then  also  by  v.  Sie- 
wert,1  crystalline  haemin  can  be  reobtained  from  the  haematin. 

On  shaking  with  cold  aniline  and  treating  first  with  acetic  acid  and  then 
with  ether,  Kuster  obtained  a  product,  dehydrochloride  haemin,  which  was 
poor  in  the  elements  of  hydrochloric  acid,  and  which  again  took  up  HC1  and  was 
converted  into  haemin.  By  the  action  of  boiling  aniline,  hydrogen  is  driven  out 
and  a  combination  with  aniline,  without  loss  of  iron,  takes  place. 

The  principle  of  the  preparation  of  haemin  crystals  in  large  quan- 
tities is  as  follows:  The  washed  sediment  from  the  blood-corpuscles 
is  coagulated  with  alcohol  or  by  boiling  after  dilution  with  water  and 
the  careful  addition  of  acid.  The  strongly  pressed  but  not  dry  mass 
is  rubbed  with  90-95  per  cent  alcohol  which  has  been  previously  treated 
with  oxalic  acid  or  |-1  per  cent  concentrated  sulphuric  acid,  and  this 
is  allowed  to  stand  several  hours  at  the  temperature  of  the  room.  The 
filtrate  is  warmed  to  about  70°  C,  treated  with  hydrochloric  acid  (for 
each  liter  of  filtrate  add  10  cc.  25  per  cent  hydrochloric  acid  diluted 
with  alcohol — Morner),  and  allowed  to  stand  in  the  cold.  The  crystals, 
which  separate  in  one  or  two  days,  are  first  washed  with  alcohol  and  then 
"with  water.  On  dissolving  the  haemin  in  chloroform  containing  quinine 
and  treating  the  filtrate  with  alcoholic  hydrochloric  or  acetic  acid  we  can 
recrystallize  the  haemin  according  to  Schalfejefp.  By  adding  glacial 
acetic  acid  saturated  with  salt  to  a  solution  of  haematin  in  chloroform 
containing  quinine  Piloty  and  Eppinger  obtained  crystalline  haemin. 
For  particulars  as  to  the  various  methods  of  preparation  and  purification 
we  refer  the  reader  to  the  above-cited  works  of  Nencki  and  Sieber, 
Morner,  Nencki  and  Zaleski  (ScHALFEjEFF),and  especially  to  Kuster.2 

Haematin  is  obtained  on  dissolving  the  haemin  crystals  in  very  dilute 
caustic  alkali  and  precipitating  with  an  acid. 

with  Nencki  and  Zaleski,  1.  c;  Bialobrzeaki,  Arch,  des  scienc.  biol.  de  St.  P6tersbourg- 
5;  K.  Morner,  Nord.  Med.  Arkiv.  Festband,  1897,  Nos.  1  and  26,  and  Zeitschr.  f. 
physiol.  Chem.,  41;  Zaleski,  ibid.,  37;  Hetper  and  Marchlewski,  ibid.,  41  and  42; 
Kuster,  ibid.,  40  and  82  and  footnote  1,  page  292;  Piettre  and  Vila,  Compt.  Rend.,  141, 
p.  734;  Piloty,  1.  c. 

1  Piloty  and  Eppinger,  1.  c;  v.  Siewert,  Arch.  f.  exp.  Path.  u.  Pharm.,  58. 

J  Kuster,  Zeitschr.,  f .  physiol.  Chem.,  40. 


294  THE  BLOOD. 

In  preparing  haemin  crystals  in  small  quantities  proceed  in  the  fol- 
lowing manner:  The  blood  is  dried  after  the  addition  of  a  small  quantity 
of  common  salt,  or  the  dried  blood  may  be  rubbed  with  a  trace  of  the 
same.  The  dry  powder  is  placed  on  a  microscope  slide,  moistened 
with  glacial  acetic  acid,  and  then  covered  with  the  cover-glass.  Add, 
by  means  of  a  glass  rod,  more  glacial  acetic  acid  by  applying  the  drop 
at  the  edge  of  the  cover-glass  until  the  space  between  the  slide  and  the 
cover-glass  is  full.  Now  warm  over  a  very  small  flame,  with  the  pre- 
caution that  the  acetic  acid  does  not  boil  and  pass  with  the  powder  from 
under  the  cover-glass.  If  no  crystals  appear  after  the  first  warming 
and  cooling,  warm  again,  and  if  necessary  add  some  more  acetic  acid. 
After  cooling,  if  the  experiment  has  been  properly  performed,  a  number 
of  dark-brown  or  nearly  black  haemin  crystals  of  varying  forms  will 
be  seen. 

In  regard  to  the  preparation  and  properties  of  the  iodine-,  bromine-, 
and  acetone-haemin  we  refer  to  the  work  of  Strzyzowski,  Meruno- 
wicz  and  Zaleski.1 

By  the  action  of  acids  upon  haemochromogen,  haematin,  or  haemin,  a 
new  iron-free  pigment,  which  was  first  closely  studied  by  Hoppe-Seyler 
and  called  hcematoporphyrin,  is  produced.  According  to  the  method  of 
preparation,  hsematoporphyrins  having  different  solubilities,  and  whose 
relation  to  each  other  is  not  perfectly  clear,  are  produced,  but  all  show 
the  same  characteristic  absorption-spectrum.  The  best-studied  haema- 
toporphyrin  is  the  one  obtained  according  to  Nencki  and  Sieber's 
method,  by  the  action  of  glacial  acetic  acid  saturated  with  hydrobromic 
acid  upon  haemin  crystals,  best  at  the  temperature  of  the  body  (Nencki 
and  Zaleski).  Another  porphyrin  is  the  mesoporphyrin  obtained  by 
Nencki  and  Zaleski  2  by  the  reduction  of  haemin  in  glacial  acetic  acid 
by  hydriodic  acid  and  iodophosphonium. 

Haematoporphyrin,  C34H38N4O6,  which,  according  to  recent  molec- 
ular weight  determinations  must  perhaps  be  doubled  (Piloty)  occurs 
according  to  Mac  Munn  3  as  a  physiological  pigment  in  certain  animals. 
A  porphyrin  occurs,  as  shown  by  Garrod  and  Saillet,  as  a  normal  con- 
stituent in  human  urine,  although  only  as  traces  and  it  has  also  been 
observed  several  times  in  large  amounts  in  the  urine  after  the  use  of 
sulphonal  (see  Chapter  XIV).  This  urine  porphyrin  is  generally  con- 
sidered as  haematoporphyrin. 

In  the  production  of  haematoporphyrin  from  haemin  or  haematin  the  iron  is 
split   off.      Opinions   are  not   unanimous  in  regard  to  this  process.     According 

1  Strzyzowski,  Therap.  Monatsh.,  1901  and  1902;  Merunowicz  and  Zaleski,  Bull, 
de  1'Acad.  d.  Scienc.  de  Cracovie,  1907. 

2  Hoppe-Seyler,  Med.-chem.  Untersuch.,  528;  Nencki  and  Sieber,  Monatshefte  f. 
Chem.,  9,  and  Arch.  f.  exp.  Path.  u.  Pharm.,  18,  20,  and  24;  Nencki  and  Zaleski, 
Zeitschr.  f.  physiol.  Chem.,  30. 

3  Piloty,  Annal.  d.  Chem.  u.  Pharm.,  388;  MacMunn,  Journ.  of  Physiol.,  7. 


ILfflMATOPOKPHYRIN.  295 

to  Piloty  two  carboxyl  groups  axe  formed  with  the  taking  up  of  water  and 
these  occur  to  a   certain  extent   latent  in  lactam  combination  in  the  baemin. 

According  to  Kusteh,1  who  admits  of  two  already  funned  carboxylfl  in  the  ha-min, 
two  bydroxyls  are  produced  secondarily  in  the  fuematoporphyrin,  in  that  (by the 
action  of  the  glacial  acetic  acid  and  hydrobromie  acid)  primarily  an  attachment 
of  hydrobromic  acid  takes  place  and  then  from  this  as  intermediary  product, 
by  tiie  action  of  water,  bromine  is  split  off  ami  is  replaced  by  hydroxyl.  In  the 
formation  of  mesoporphyrin  the  procedure  is  still  different  because  among  others, 
mesoporphyrin  contains  2  oxygen  atoms  less  than  the  luematoporphyrin. 

On  the  gentle  reduction  of  hsemin  with  glacial  acetic  acid,  bydriodic  acid 
and  red  phosphorus,  Piloty  and  Fink  obtained  besides  mesoporphyrin  a  second 
body,  phonoporphyrin,  which  differs  from  the  mesoporphyrin  by  containing 
more  oxygen,  a  brown  color  and  almost  complete  insolubility  in  dilute  hydro- 
chloric acid.  It  is  not  reduced  to  mesoporphyrin  by  hydriodic  acid  but  yields 
hsematinic  acid  and  methyl-ethyl  maleic  imide  on  oxidation  with  chromic  acid. 
They  obtained  no  other  cleavage  products  from  hsemin  under  the  above  men- 
tioned experimental  conditions.  The  two  porphyrins  were  produced  in  about 
equal  quantities  and  they  formed  about  90  per  cent  of  the  calculated  cleavage 
products.  They  each  represent  one-half  of  the  ha?min,  whose  formula  corresponds 
to  CesH^XgOsFciCla  which  must  be  doubled.  As  these  two  porphyrins  yield 
methyl  ethylmaleic  imide,  while  this  is  not  the  case  with  either  the  haemin  or  the 
hsematoporphyrin,  it  is  believed  that  both  are  combined  together  in  the  haemin 
or  ha?matoporphyrin  with  that  part  of  their  molecules  which  allow  of  the  maleic 
imide  formation. 

By  the  action  of  glacial  acetic  acid,  and  hydriodic  acid  upon  hsemin  in  the 
cold  (room  temperature)  in  the  presence  of  iodophosphonium,  H.  Fischer  and 
Bartholomaus  have  obtained  a  beautiful  crystalline,  colorless  product,  por- 
phyrinogen, whose  formation  and  behavior  have  been  further  studied  by  Rose.2 
Porphyrinogen,  C.^H^NjO^  is  formed  from  hsemin,  the  meso-  and  the  hsemato- 
porphyrin in  acid,  and  from  the  meso-  or  hsematoporphyrin  also  in  alkaline 
reduction.  Porphyrinogen  can  be  transformed  into  mesoporphyrin  by  oxidative 
action  of  various  kinds.  Like  the  latter  it  yields  hsematinic  acid  as  well  as  methyl 
ethyl  maleic  imide  as  oxidation  products. 

Haematoporphyrin  is  closely  related  to  the  bile  pigment  bilirubin 
(see  Chapter  VII)  and  also  stands  in  close  relation  with  the  urinary 
pigment,  urobilin.  By  action  of  reducing  substances  several  investiga- 
tors (Hoppe-Seyler,  Nencki  and  Sieber,  Le  Nobel,  MacjMunn  and 
others)  have  obtained  pigments  similar  to  urobilin,  and  by  experiments 
with  rabbits,  Nencki  and  Rotschy3  have  proved  that  haematoporphyrin 
introduced  into  the  animal  body  may  in  part  be  transformed  into  a 
urobilin  substance. 

In  connection  with  the  question  of  the  behavior  of  haematoporphyrin  in  the 
animal  body,  the  poisonous  action  of  this  body,  as  discovered  by  Hausmann, 
and  which  manifests  itself  as  a  photobiological  sensibilisation,  is  of  interest. 
Hausmann  has  found  that  white  mice  that  have  had  fuematoporphyrin  injected 

1  Piloty,  1.  c;  Kiister,  Ber.  d.  d.  ehem.  Gesellsch.,  45. 

2  Piloty  and  Fink,  Ber.  d.  d.  chem.  Gesellsch.,  46,  2021;  H.  Fischer  and  Bartholo- 
maus, ibid.,  46,  511;   Rose,  Zeitschr.  f.  physiol.  Chem.,  84. 

3  Hoppe-Seyler,  Med.  Chem.  Unters.  p.  533;  Le  Xobel,  Pfluser's  Arch.,  40; 
Nencki  and  Sieber,  1.  c;  MacMunn,  Proc.  Roy.  Soc,  30,  and  Journ.  of  Physiol.,  10; 
Nencki  and  Rotschy,  Monatsh.  f.  chem.,  10. 


29G  THE  BLOOD. 

subcutaneously  and  then  exposed  to  bright  light  die  very  quickly  with  character- 
istic symptoms,  while  control  animals  kept  in  the  dark  show  no  symptoms  of  disease. 
H.  Fischer  and  Meyer-Betz  '  have  also  shown  that  in  this  regard  a  certain 
difference  exists  between  the  hsematoporphyrin  and  the  mesoporphyrin.  The 
perfectly  pure  crystalline  mesoporphyrin  does  not  show  the  photobiological 
action  which  occurs  with  crystalline  hamaatoporphyrin. 

Of  especial  interest  is  the  close  relationship  of  the  hsematoporphyrin 
to  certain  chlorophyll  derivatives,  especially  to  phylloporphyrin, 
C32H36N4O2.  Phylloporphyrin  is  similar  to  the  above-mentioned  meso- 
porphyrin, C34H^sN404,  and  the  absorption  spectrum  of  the  brom- 
porphyrins,  bromphylloporphyrin  and  brommesoporphyrin  as  prepared 
by  Schunck  and  Marchlewski,  seems  to  be  almost  identical.  Just  as 
from  mesoporphyrin,  with  sodium  chloride,  glacial  acetic  acid  and  an 
iron  salt  we  can  regenerate  a  product  very  similar  to  haemin  (Zaleski) 
so  Marchlewski  2  has  been  able  under  similar  conditions  to  prepare  from 
phylloporphyrin  a  pigment,  phyllohcemin,  which  contained  iron  and  was 
similar  to  hgemin.  A  comparison  of  the  cleavage  products  gives  still 
more  conclusive  and  important  proofs  of  the  close  relationship  of  the 
"blood  and  leaf  pigments. 

We  have  important  investigations  of  Kuster,3  Piloty,  Willstatter, 
H.  Fischer  4  and  their  collaborators  upon  the  constitution  of  hsemin  and 
hgematoporphyrin.  The  constitution  of  chlorophyll  has  been  explained 
by  the  pioneering  researches  of  Willstatter. 

On  the  oxidation  of  hsematin  in  glacial  acetic  acid  by  potassium 
dichromate  or  chromium  trioxide   Kuster  obtained  the    imide  of  the 

/CO— C.CH0.CH2.COOH 
tribasic  hcematinic  acid,  HN<^  ,  which  is  a  deriva- 

XCO— C.CH3 
tive  of    maleic  acid,  and  from  which  methylethylmaleic  acid  anhydride, 

.CO— C.CH2CH3 
0<f  ,  can   be   readily   obtained.     The   same   heematinic 

XCO— C.CH3 

1  Hausmann,  Bioch.  Zeitschr.,  30;  Hans  Fischer  and  Meyer-Betz.  Zeitschr.  f. 
physiol.  Chem.,  82. 

2  The  pertinent  literature  will  be  found  in  L.  Marchlewski,  Die  Chemie  der  Chlor- 
ophylle  und  ihre  Beziehung  zur  Chemie  des  Blutfarbstoffes,  1909  and  Ber.  d.  d.  chem. 
Gesellsch.,  45. 

3  Beitrage  zur  Kenntnis.  des  Hamatins.  Tubingen,  1896;  Ber.  d.  d.  chem.  Gesellsch., 
27,  30,  32,  35,  43,  and  15,  Annal.  d.  Chem.  u.  Pharm.,  315,  arid  Zeitschr.  f.  physiol. 
Chem.,  28,  40,  44,  54,  55,  61,  62,  and  82. 

*  The  work  of  Piloty  and  collaborators  may  be  found  in  Annal.  d.  Chem.  u.  Pharm., 
360,  37",  388,  390,  and  392  and  Ber.  d.  d.  chem.  Gesellsch.,  42,  43,  and  45.  In  regard 
to  the  work  of  Willstatter  and  the  literature  on  chlorophyll  (to  1911)  see  Willstatter 
in  Abderhalden's  Bioch.  Handlexicon,  Bd.  VI  and  Annal.  d.  Chem.  u.  Pharm.,  378; 
380,  382,  and  385.  Hans  Fischer  and  collaborators,  Zeitschr.  f.  physiol.  Chem.,  82, 
and  footnote  2,  p.  297,  and  the  literature  on  bilirubin  Chapter  VII. 


CONSTITUTION   OF  THE  BLOOD-PIGMENTS.  297 

acid  imide,  which  also  haemato-  and  mesoporphyrin  give,  were  obtained  by 
Marchlewski  on  the  oxidation  of  phylloporphvrin  and  by  Willstatter, 

XX)— C.C2H5 
besides  methylclhylmaleic   imide,   HX<^  ,  on  the  oxidation  of 

xCO— C.CH3 
certain  chlorophyll  derivatives.     The  sarne  two  products  were   obtained 
1  \   Ivuster  l  on  the  oxidation  of  mesoporphyrin  while  haemin  and  haema- 
toporphyrin  gave  no  methylethylmaleic  imide. 

It  has  bem  known  for  a  long  time  that  haemin  and  hacmatoporphyrin 
gave  an  abundance  of  pyrrol  on  heating,  and  that  phylloporphyrin  has  a 
similar  behavior  was  first  shown  by  Schunck  and  Marchlewski. 
That  at  least  one  pyrrol,  of  the  pyrrol  mixture,  the  so-called  hcemopyrrol 
is  common  to  both  the  blood  and  leaf  pigments  has  been  shown  by  the 
investigations  of  Marchlewski  and  his  collaborators,  and  from  haemo- 
pyrrol  Kuster  was  the  first  to  obtain  methylethylmaleic  imide  on  oxida- 
tion, showing  that  haemopyrrol  was  probably  a  dimethylethylpyrrol. 
This  behavior  has  been  further  developed  by  the  investigations  of 
Piloty  and  Willstatter  on  the  reduction  products  of  the  blood  and 
leaf  pigments  and  by  H.  Fischer  and  Bartholomaus  2  on  the  sub- 
stituted pyrrols. 

Willstatter  obtained  a  pyrrol  mixture  from  hsemin  and  haemato- 
porphyrin,  as  well  as  from  chlorophyll  derivatives,  by  reduction,  from 
which  he  isolated  three  different  pyrrols.  The  first,  which  he  calls  hcemo- 
pyrrol, was  perhaps  not  perfectly  pure,  consisted  at  least  in  great  part 
of  the  cryptopyrrol  (Fischer  and  Bartholomaus,  c-haemopyrrol  of 
Piloty   and   Stock)    which    is   identical  with  the  2,  4-dimethyl-3-ethyl 

H3C.C C.C2H5 

pvrrol  =  prepared  svnthetically  bv  Knorr  and  Hess.3 

HC— NH— C.CH3 
The  second  haemopyrrol,   which    he  calls  isohocmopyrrol   (  =  haemopyrrol 
of  Fischer  and  Bartholomaus,  B-haemopyrrol  of  Piloty  and  Stock) 
is    also    a     trisubstituted     pyrrol,    namely    2,  3-diinethyl-l-ethylpyrrol 

C2H5.O C.CH3 

HC— NH-C.CH3 

These  two  dimethylethylpyrrols  give  with  nitrous  acid  the  correspond- 

1  Ber.  d.  d.  chem.  Gesellsch.,  45. 

2  Piloty,  Annal.  d.  Chem.  u.  Pharm.,  377,  388,  390,    and    392;    Willstatter    and 

Asahina,  ibid.,  385.  In  this  article  will  be  found  on  pages  1S9  and  190  the  references 
to  the  literature  on  the  investigations  of  Marchlewski  and  others  on  ha?mopyrrol. 
H-lFischer  and  Bartholomaus,  Zeitschr.  f.  physiol.  Chem.,  77  and  80  and  Ber.  d.  d. 
chem.  Gesellsch.,  45. 

3  Piloty   and  Stock,  Annal.  d.  Chem.  u.  Pharm.,  392;    Knorr  and   Hess,  Ber.  d. 
d.  chem.  Gesellsch.,  44  and  45. 


29S  THE  BLOOD. 

ing  oxime   of    methylethylmaleic    imide.      The  third  pyrrol  found  by 
Willstatter,  which  he  calls  phyllopyrrol,  is  a  tetra-substituted  pyrrol, 

H3C.C C.C2H5 

namelv,  2,  3,  5  trimethyl-4-ethvlpyrrol  = 

H3C.C— NH— C.CH3 

The  statement  that  the  haemopyrrol  of  Willstatter  is  in  part  derived 
from  cryptopyrrol  is  not  correct,  and  must  be  changed  because  of  the 
investigations  of  Piloty  and  Stock,  who  find  that  the  haemopyrrol  of 
Willstatter  (and  Asahina)  undoubtedly  contains  cryptopyrrol,  but 
consists  chiefly  of  the  B-haemopyrrol,  consequently  isohaemopyrrol.  Ac- 
cording to  more  recent  investigations  of  Piloty  and  Stock  l  the  haemo- 
pyrrol question  is  even  more  complicated  than  was  expected. 

In  the  crude  pyrrol  obtained  by  the  reduction  of  the  blood  pigments 
several  other  pyrrol  bodies  have  been  found,  for  example  the  phonopyrrol 
of  Piloty  which  has  not  been  sufficiently  explained.  According  to 
Grabowski  and  Marchlewski2  as  well  as  to  Piloty  and  Stock,  the 
crude  haemopyrrol  contains  also  disubstituted  pyrrol,  namely,  jSi,  /3-methyl- 
ethylpyrrol.  On  fusing  haematoporphyrin  or  haematopyrrolidinic  acid 
(see  below)  with  caustic  alkali  we  obtain,  according  to  Piloty,  a  mixture 
of  pyrrols  among  which  we  will  mention  2,  3-dimethylpyrrol 

HC- C.CH3 

,  which  has  been  studied  by  Piloty  and  Wilke.3 
HC— NH— C.CH3 

By  the  reduction  of  haematoporphyrin  and  haemin  by  various  methods, 
Piloty  and  co-workers  have  obtained,  besides  haemopyrrol,  several 
acids  namely  haematopyrrolidinic  acid,  phonopyrrolcarboxylic  acid 
(isophonopyrrolcarboxylic  acid)  and  xanthopyrrolcarboxylic  acid.  The 
haematopyrrolidinic  acid  seems  from  the  most  recent  investigations 
not    to   be  a  unit  substance.     Piloty  obtained   from    it   phonopyrrolcar- 

H3C.C C.CH2.CH2.COOH 

boxylic   acid,    C9Hi3N02=  which    on 

H3C.C— NH— CH 
treatment  with  nitrous  acid  lost  a  methyl  group  and  was  converted  into 

H3C.C CCH2CH2.COOH. 

the  oxime  of  haematinic  acid,  The  acid 

HONC— NH— CO 
received   this    name    because,   according  to  Piloty,  it  yields  a  special 
dimethylethylpyrrol,  called  phonopyrrol  by  him.     The  question  as  to  the 
nature  of  xanthopyrrolcarboxylic  acid  and  its  occurrence  has  not  been 


1  Piloty  and  Stock,  Annal.  d.  Chem.  u.  Pharm.,  392  and  Ber.  d.  d.  chem.  Gesellsch., 
46,  1008. 

'-'  Grabowski  and  Marchlewski,  Zeitschr.  f.  physiol.  Chem.,  81. 
'  Piloty  and  Wilke,  Ber.  d.  d.  chem.  Gesellsch.,  45. 


PYRROLE   DERIVATIVES.  299 

answered;  still  there  does  not  seem  to  be  any  doubt  that  there  exists 
an  isophonopyrrolcarboxylic  acid,  which  can  be  obtained  from  the  blood 
as  from  the  bile  pigments.  From  the  mixture  of  acid  cleavage  products 
obtained  by  the  reduction  of  haemin  with  hydriodic  acid,  and  glacial  acetic 
acid  Piloty  and  Dormann  1  have  obtained  as  well  characterized  prod- 
ucts, phonopyrrolcarboxylic  acid  and  isophonopyrrolcarboxylic  acid  and 
also  xarithopyrrolcarboxylic  acid,  C10H15NO2  and  they  consider  the 
existence  of  this  acid  as  positively  proved.  The  melting-point  of  the 
crystalline  acid  was  108°,  the  picrate  143°,  and  the  oxime  208°.  The 
corresponding  values  for  isophonopyrrolcarboxylic  acid  was  122°,  146° 
and  210°  respectively.  An  isomeric  xanthopyrrolcarboxylic  acid,  called 
D-phonopyrrolcarboxylic  acid,  seems  also  to  occur. 

It  is  extremely  difficult  to  correlate  the  somewhat  contradictory 
statements  of  the  various  authors  in  this  subject  and  to  draw  quite 
positive  conclusions  from  these  statements.  It  is  nevertheless  positive 
that  from  the  haemopyrrol  mixture  the  three  pyrrols,  cryptopyrrol, 
isohaemopyrrol  and  phyllopyrrol  can  be  obtained  and  also  that  there 
are  two  haemopyrrolcarboxylic  acids  (phonopyrrol-  and  isophonopyrrol- 
carboxylic acids),  of  which  one  possibly  is  related  to  the  crypto- 
pyrrol and  the  other  to  the  isohaemopyrrol.  On  account  of  the  uncer- 
tainty of  the  experimental  foundation  it  is  difficult  to  enter  into  a 
discussion  of  the  variously  proposed  hypothetical  constitutional  formulae 
for  the  derivatives  of  the  blood  pigments.  The  same  is  true  for  the 
disputed  question  as  to  the  form  of  binding  of  the  iron  in  haematin  and 
in  hsemin.  It  is  generally  admitted  that  the  iron  here  is  trivalent.  The 
views  are  different  in  regard  to  the  valence  of  the  iron  in  haemoglobin, 
namely,  Maxchot  considers  that  haemoglobin  is  a  ferric  combination 
while  Kuster  2  on  the  contrary  considers  it  a  ferrous  combination. 

Haematoporphyrin  gives  with  hydrochloric  acid  a  compound  which 
crystallizes  in  long  brownish-red  needles.  If  the  solution  in  hydrochloric 
acid  is  nearly  neutralized  with  caustic  soda  and  then  treated  with  sodium 
acetate,  the  pigment  separates  out  as  amorphous,  brown  flakes  not 
readily  soluble  in  amyl  alcohol,  ether,  or  chloroform,  but  readily  soluble 
in  ethyl  alcohol,  alkalies,  and  dilute  mineral  acids.  The  compound 
with  sodium  crystallizes  as  small  tufts  of  brown  crystals  and  several 
other  salts  of  haematoporphyrin  are  known  such  as  the  methyl  and  ethyl 
esters.  The  acid  alcoholic  solutions  have  a  beautiful  purple  color, 
which  become  violet-blue  on  the  addition  of  large  quantities  of  acid. 
The  alkaline  solution  has  a  beautiful  red  color,  especially  when  too  much 
alkali  is  not  present. 


1  Piloty  and  Dormann,  Ber.  d.  d.  chem.  Gesellsch.,  46. 

2  Manchot,  Zeitschr.  f.  physiol.  Chem.,  70;  Kuster,  ibid.,  71. 


300  THE  BLOOD. 

An  alcoholic  solution  of  heematoporphyrin,  acidulated  with  hydro- 
chloric or  sulphuric  acid,  shows  two  absorption-bands  (spectrum  Plate, 
7),  one  of  which  is  fainter  and  narrower  and  lies  between  C  and  D,  near 
D.  The  other  is  much  darker,  sharper,  and  broader,  and  lies  midway 
between  D  and  E.  An  absorption  extends  from  these  bands  toward 
the  red,  terminating  with  a  dark  edge,  which  may  be  considered  as  a 
third  band  between  the  other  two. 

A  dilute  alkaline  solution  shows  four  bands,  namely,  a  band  between 
C  and  D;  a  second,  broader  band  surrounding  D  and  with  the  greater 
part  between  D  and  E;  a  third,  between  D  and  E,  nearly  at  E;  and 
lastly,  a  fourth,  broad  and  dark  band  between  b  and  F.  On  the  addi- 
tion of  an  alkaline  zinc-chloride  solution  the  spectrum  changes  more 
or  less  rapidly,1  and  finally  a  spectrum  is  obtained  with  only  two  bands, 
one  of  which  surrounds  D  and  the  other  lies  between  D  and  E.  If  an 
acid  haematoporphyrin  solution  is  shaken  with  chloroform,  a  part  of  the 
pigment  is  taken  up  by  the  chloroform,  and  this  solution  often  shows  a 
five-banded  spectrum  with  two  bands  between  C  and  D.  The  position 
of  the  hsematoporphyrin  bands  in  the  spectrum  differ  with  the  various 
methods  of  preparation  and  other  conditions,  so  that  they  do  not  cor- 
respond to  the  same  wave  length.  These  facts  coincide  well  with  the 
recent  investigations  of  A.  Schulz;2  according  to  which  the  appearance 
of  the  spectrum  is  not  only  dependent  upon  the  reaction  but  also  upon 
the  character  of  the  solvent  and  the  method  of  preparation. 

In  regard  to  the  preparation  of  hsematoporphyrin,  see  Hoppe-Seyler- 
Thierfelder's  Handbuch,  8.  AufL,  and  the  works  cited  on  page  294. 

Mesoporphyrin,  C34H38N4O4,  has  the  same  spectrum  as  haematoporphyrin. 
It  has  two  oxygen  atoms  less,  and  further  differs  from  it  in  that  on  oxidation  it  yields 
hsematinic  acid  as  well  as  methylethylmaleic  imide,  and  does  not  show  the 
above-mentioned  biological  action  of  haematoporphyrin. 

Haematinogen  is  a  ferruginous  pigment  so  named  by  Freund,3  which  he 
obtained  by  carefully  extracting  blood  with  alcohol  containing  hydrochloric 
acid.  It  is  closely  related  to  haematin,  but  is  not  sufficiently  characteristic  and  is 
not  considered  as  a  cleavage  product. 

A  question  of  great  interest  is  whether  it  is  possible  to  produce  the 
blood-pigment  from  its  cleavage  products.  In  this  respect  certain  recent 
investigations  are  interesting.  Zaleski  obtained  from  mesoporphyrin 
hydrocloride  dissolved  in  80  per  cent  acetic  acid  saturated  with  NaCl 
and  heated  to  50°-70°,  a  hsemin-like  pigment  by  the  addition  of  a  solu- 
tion of  iron  in  acetic  acid,  and  this  pigment  had  a  spectrum  in  acid 
solution  very  similar  to  that  of  haematin,  although  not  identical  with  it. 

1  See  Hammarsten,  Skand.  Arch.  f.  Physiol.,  3,  and  Garrod,  Journ.  of  Physiol.,  13. 

2  Arch.  f.  (Anat.  u.)  Physiol.,  1904,  Suppl. 

3  Wien.  klin.  Wochenschx.,  1903. 


KLEMATOIDIN.  301 

Zaleski  considers  this  pigment  as  a  hydrogcnized  haemin.  A  regenera- 
tion of  haematin  from  haematoporphyrin  has  been  performed  by  Laid- 
law.  If  ha'matoporphyrin  is  dissolved  in  dilute  ammonia  and  warmed 
with  Stokes'  solution  and  hydrazine  hydrate,  iron  is  taken  up  again 
and  haemochromogen  is  produced,  which  is  changed  into  hsematin  by 
shaking  with  air.  According  to  Ham  and  Balean,1  it  is  possible  to  pro- 
duce haemoglobin  from  haemochromogen  and  globin,  and  it  is  indeed 
possible  that  other  proteins  can  replace  globin  in  this  formation. 

Haematoidin,  thus  called  by  Virchow,  is  a  pigment  which  crystallizes 
in  orange-colored  rhombic  plates,  and  which  occurs  in  old  blood  extrav- 
asations, and  whose  origin  from  the  blood-coloring  matters  seems  to 
be  established  (Langhans,  Cordua,  Quincke,  and  others2).  A  solu- 
tion of  haematoidin  shows  no  absorption-bands,  but  only  a  strong  absorp- 
tion from  the  violet  to  the  green  (Ewald  3).  According  to  most  observers, 
haematoidin  is  identical  with  the  bile-pigment  bilirubin.  It  is  not 
identical  with  the  crystallizable  lutein  from  the  corpora  lutea  of  the  ovaries 
of  the  cow  (Piccolo  and  Lieben,4  Kuhne  and  Ewald). 

In  the  detection  of  the  above-described  blood-coloring  matters  the 
spectroscope  is  the  only  entirely  trustworthy  means  of  investigation. 
If  it  is  only  necessary  to  test  for  blood  in  general  and  not  to  determine 
definitely  whether  the  coloring-matter  is  haemoglobin,  methaemoglobin 
or  haematin,  then  the  preparation  of  haemin  crystals  is  an  absolutely 
positive  test.  In  regard  to  the  detection  of  blood  in  urine,  see  Chapter 
XIV,  and  for  the  detection  of  blood  in  intestinal  contents,  in  pathological 
fluids  and  in  chemico-legal  cases  we  must  refer  the  reader  to  more  extended 
text-books. 

The  methods  proposed  for  the  quantitative  estimation  of  the  blood- 
coloring  matters  are  partly  chemical  and  partly  physical. 

Among  the  chemical  methods  to  be  mentioned  is  the  incineration  of  the 
blood  and  the  determination  of  the  amount  of  iron  contained  in  the  ash  from 
which  the  amount  of  haemoglobin  may  be  calculated.  We  must  refer  to  works 
on  chemical  methods  of  investigation  in  regard  to  these  methods. 

The  physical  methods  consist  either  of  colorimetric  or  of  spectroscopic 
investigations. 

The  principle  of  Hoppe-Seyler's  colorimetric  method  is  that  a  measured 
quantity  of  blood  is  diluted  with  an  exactly  measured  quantity  of  water 
until  the  diluted  blood  solution  has  the  same  color  as  a  pure  oxy haemo- 
globin solution  of  a  known  strength.  The  amount  of  coloring-matter 
present  in  the  undiluted  blood  may  be  easily  calculated  from  the  degree 
of  dilution.     In  the  colorimetric  testing  we  use  a  glass  vessel  with  parallel 

1  Zaleski,  Zeitsehr.  f.  physiol.  Chem.,  43;  Laidlaw,  Journ.  of  Physiol.,  31;  Ham 
and  Balean,  ibid.,  32. 

2  A  comprehensive  review  of  the  literature  pertaining  to  haematoidin  may  be  found 
in  Stadelmann,  Der  Icterus,  etc.,  Stuttgart,  1891,  pp.  3  and  45. 

»  Zeitsehr.  f.  Biologie,  22,  475. 

4  Cit.  from  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4.  Aufl.,  1878. 


302  THE  BLOOD. 

sides  containing  a  layer  of  liquid  1  cm.  thick  (Hoppe-Seyler's  hsematinom- 
eter).  The  use  of  Hoppe-Seyler's  colorimetric  double  pipette  is  more 
advantageous.  Other  good  forms  of  apparatus  have  been  constructed 
by  Giacosa  and  Zangermeister.1  Instead  of  an  oxyhemoglobin  solu- 
tion we  now  generally  use  a  carbon-monoxide  haemoglobin  solution  as 
a  standard  liquid  because  it  may  be  kept  for  a  long  time.  The  blood 
solution  in  this  case  is  saturated  with  carbon  monoxide.2 

The  quantitative  estimation  of  the  blood-coloring  matters  by  means 
of  the  spectroscope  may  be  done  in  different  ways,  but  at  the  present 
time  the  spectrophotometric  method  is  chiefly  used,  and  this  seems  to  be 
the  most  reliable.  This  method  is  based  on  the  fact  that  the  extinction 
coefficient  of  a  colored  liquid  for  a  certain  region  of  the  spectrum  is  directly 
proportional  to  the  concentration,  so  that  C  :  E  =  Ci  :  E\,  when  C  and 
C\  represent  the  different  concentrations  and  E  and  E\  the  corresponding 

C     C. 
coefficients  of  extinction.     From  the  equation  —=-=r7  it  follows  that  for 

hi    tii\ 

one  and  the  same  pigment  this  relation,  which  is  called  the  absorption 
ratio,  must  be  constant.  If  the  absorption  ratio  is  represented  by  A, 
the  determined  extinction  coefficient  by  E,  and  the  concentration  (the 
amount  of  coloring-matter  in  grams  in  1  cc.)  by  C,  then  C  =  AE. 

Different  forms  of  apparatus  have  been  constructed  (Vierordt  and 
Hufner3)  for  the  determination  of  the  extinction  coefficient,  which  is 
equal  to  the  negative  logarithm  of  those  rays  of  light  which  remain 
after  the  passage  of  the  light  through  a  layer  1  cm.  thick  of  an  absorbing 
liquid.  In  regard  to  this  apparatus  the  reader  is  referred  to  other  text- 
books. 

For  purposes  of  control  the  extinction  coefficients  are  determined  in  two  dif- 
ferent regions  of  the  spectrum.  Hufner  has  selected  (a)  the  region  between  the 
two  absorption-bands  of  oxyhemobglobin,  especially  between  the  wave-lengths 
554  nn  and  565  n^  and  (b)  the  region  of  the  second  band,  especially  the  inter- 
val between  the  wave-lengths  531.5  mm  and  542.5  mm-  The  constants  or  the 
absorption  ratio  for  these  two  regions  of  the  spectrum  are  designated  by  Hufner 
by  A  and  A'.  Before  the  determination  the  blood  must  be  diluted  with  water, 
and  if  the  proportion  of  dilution  of  the  blood  be  represented  by  V,  then  the  con- 
centration or  the  amount  of  coloring-matter  in  100  parts  of  the  undiluted  blood 
is 

C  =  100.  V.  A.  E  and 

C  =  100.  V.A'.E'. 

The  absorption  ratio  or  the  constants  in  the  two  above-mentioned  regions 
of  the  spectrum  have  been  determined  for  oxyhemoglobin,  haemoglobin,  carbon- 
monoxide  hemoglobin,  and  methemoglobin,  as  follows: 

Oxyhemoglobin A0  =0.002070  and  A'0  =0.001312 

Hemoglobin Ar  =0.001354  and  A'r  =0.001778 

Carbon-monoxide  hemoglobin.    .A<-  =0.001383  and  A'c  =0.001263 

Methemoglobin Am  =0.002077  and  A'm  =  0.001754 

1  F.  Hoppe-Seyler,  Zeitschr.  f.  physiol.  Chem.,  16;  G.  Hoppe-Seyler,  ibid.,  21; 
Winternitz,  ibid.;  Giacosa,  Maly's  Jahresber.,  26;  Zangermeister,  Zeitschr.  f.  Biol- 
ogic, 88. 

-  See  Haldane.  Journ  of  Physiol.,  26. 

3  See  Vierordt.  Die  Anwondung  des  Spektralapparates  zu  Photometrie,  etc.    (Tubin- 
gen, 187  3),  and  Hufner.  Arch.  f.  (Anat.  u.)  Physiol.,  1894,  and   Zeitschr.  f.  physiol 
Chern  ,  3,  v.  Noorden,  ibid  .  4;  Otto,  Pfluger's  Arch,  31  and  36. 


QUANTITATIVE  ESTIMATION  OF  BLOOD-PIGMENTS.  303 

From  what   has   been  said  above  about    the    absorption    behavior) 

the  concentration  and  the  extinction   coefficient  it  follows  that  the  quo- 

E' 
tient  of  the  extinction  coefficient  —  measured  at  two  different  parts  of 

E 

the   spectrum,  independently   of   the    concentration,   is   a    characteristic 

constant  for  the  respective  pigments.     According  to   Hufner's  figures 

this   quotient  for   oxyhemoglobin   is   1.58,   for   haemoglobin    0.76,    for 

carbon-monoxide  haemoglobin  1.10  and  for  methemoglobin  1.19.     But- 

terfield  l   who  has  made  a  thorough  investigation  on  this,   finds  the 

figure   1.58  for  normal   and  pathological   human   blood  as  well  as  for 

crystalline  human,  horse  and  ox  oxyhemoglobin. 

The  quantity  of  each  coloring-matter  may  be  determined  in  a  mixture 
of  two  blood-coloring  matters  by  this  method;  this  is  of  special  impor- 
tance in  the  determination  of  the  quantity  of  oxyhemoglobin  and 
haemoglobin  present  in  blood  at  the  same  time. 

In  order  to  facilitate  these  determinations,  Hufner  2  has  worked 
out  tables  which  give  the  relation  between  the  two  pigments  existing 
in  a  solution  containing  oxyhemoglobin  and  another  pigment  (hemo- 
globin, methemoglobin,  or  carbon-monoxide  hemoglobin),  and  thus 
allowing  of  the  calculation  of  the  absolute  quantity  of  each  pigment. 

Among  the  many  apparati  constructed  for  clinical  purposes  fcr  the 
quantitative  estimation  of  hemoglobin,  Fleischl's  hcemometer,  which  has 
undergone  numerous  modifications,  Henoccue's  hwmatoscope,  and 
Sahli's  hcemometer,  are  to  be  specially  mentioned.  In  regard  to  these 
apparati  we  must  refer  to  larger  hand-books  and  text-books  on  clinical 
methods. 

Many  other  pigments  are  found  besides  the  often-occurring  hemoglobin 
in  the  blood  of  invertebrates.  In  a  few  Arachnide,  Crustacea,  Gasteropoda? 
and  Cephalopoda1  a  body  analogous  to  hemoglobin,  containing  copper,  hcemo- 
cyanin,  has  been  found  by  P'redericq.  By  the  taking  up  of  loosely  bound  oxygen 
this  body  is  converted  into  blue  oxyhecmocyanin,  and  by  the  escape  of  the  oxygen 
becomes  colorless  again.  According  to  Henze  1  gram  haunocyanin  combines 
with  about  0.4  cc.  oxvgen.  It  is  crvstalline  and  has  the  following  composition: 
C  53.66;  H  7.33;  N  16.09;  S  0.86;  Cu  0.38;  O  21.67  per  cent.  On  hydrolytic 
cleavage  with  hydrochloric  acid  Henze  found  the  following  division  of  the  nitro- 
gen in  ha>mocyanin:  Of  the  total  nitrogen  5.78  per  cent  was  split  off  as  ammonia, 
2.67  per  cent  as  humus  nitrogen,  27.65  per  cent  as  diamino  nitrogen,  and  63.39 
per  cent  as  monamino  nitrogen.  He  found  no  arginine  in  the  cleavage  products, 
but  could  detect  histidine.  lysine,  tyrosine,  and  glutamic  acid.  A  coloring- 
matter  called  chlorocruorin  by  Lankester  is  found  in  certain  Chaetopodffi. 
Hamcrylhrin,  so  called  by  Krukenberg  but  first  observed  by  Schwalhe.  is  a 
red  coloring-matter  from  certain  Gephyrea.  Besides  luemocyanin  we  find  in  the 
blood  of  certain  Crustacea  the  red  coloring-matter  tetronerythrin  (Halliburton), 
which  is  also  widely  spread  in  the  animal  kingdom.     Echinochrom,  so  named 

1  Zetischr.  f.  physiol.  Chem.,'62. 

2  Arch.  f.  (Anat.  u.)  Physiol.,  1900. 


304  THE  BLOOD. 

by  MacMunn,1  is  a  brown  coloring-matter  occurring  in  the  perivisceral  fluid  of 
a  variety  of  echinoderms.  According  to  Henze  2  the  Ascidia  contain  a  brown 
pigment  which  contains  vanadium  but  does  not  hold  any  oxygen  in  a  dissociable 
form. 

The  quantitative  constitution  of  the  red  blood-corpuscles.  The  amount 
of  water  varies  in  different  varieties  of  blood-corpuscles  between  570- 
644  p.  m.,  with  a  corresponding  amount,  430-356  p.  m.,  of  solids.  The 
chief  mass,  about  tV-jV  °f  the  dried  substance  consists  of  haemoglobin 
(in  human  and  mammalian  blood). 

According  to  the  analyses  of  Hoppe-Seyler  3  and  his  pupils,  the  red 
corpuscles  contain  in  1000  parts  of  the  dried  substance : 

Haemoglobin.  Protein.  Lecithin.  Cholesterin. 

Human  blood 868-944  122-51  7.2-3.5             2.5 

Dog's        "    865  126  5.9                3.6 

Goose's     "    627  364  4.6                4.8 

Snake's     "    467  525 

Abderhalden  found  the  following  composition  for  the  blood- 
corpuscles  from  the  domestic  animals  investigated  by  him:  Water, 
591.9-644.3  p.  m.;  solids  408.1-335.7  p.  m.;  haemoglobin,  303.3-331.9 
p.  m.;  protein,  5.32  (dog)-7.85  p.  m.  (sheep);  cholesterin,  0.388  (horse) 
-3.593  p.  m.  (sheep) ;  and  lecithin,  2.296  (dog)-4.855  p.  m. 

Of  special  interest  is  the  varying  proportion  of  the  haemoglobin  to  the 
protein  in  the  nucleated  and  in  the  non-nucleated  blood-corpuscles.  These 
last  are  much  richer  in  haemoglobin  and  poorer  in  protein  than  the 
former. 

The  amount  of  mineral  bodies  in  various  species  of  animals  is  different. 
According  to  Bunge  and  Abderhalden  the  red  corpuslces  from  the  pig, 
horse,  and  rabbit  contain  no  soda,  while  those  from  man,  the  ox,  sheep, 
goat,  dog,  and  cat  are  relatively  rich  in  soda.  In  the  five  last-mentioned 
species  the  amount  of  soda  was  2.135-2.856  p.  m.  The  quantity  of  potash 
was  0.257  (dog) -0.744  p.  m.  (sheep).  In  the  horse,  pig,  and  rabbit 
the  quantity  of  potash  was  3.326  (horse)-5.229  p.  m.  (rabbit).  Human 
blood-corpuscles  contain,  according  to  Wanach,  about  five  times  as 
much  potash  as  soda,  on  an  average  3.99  p.  m.  potash  and  0.75  p.  m. 
soda.     The   nucleated  erythrocytes   of  the   frog,   toad,   and  turtle   also 


1  Fredericq,  Extrait  des  Bulletins  de  l'Acad.  Roy.  de  Belgique  (2),  46,  1878;  Lan- 
kester,  Journ.  of  Anat.  and  Physiol.,  2  and  4;  Henze,  Zeitschr.  f.  physiol.  Chem., 
33  and  43;  Krukenberg,  see  Vergl.  physiol.  Studien  Reihe  1,  Abt.  3,  Heidelberg, 
1880;  Halliburton,  Journal  of  Physiol.,  6;  MacMunn.  Quart.  Journ.  Microsc.  Science, 
1885. 

2  Zeitschr.  f.  physiol.  Chem.,  72  and  79. 

3  Med.-chem.  Untersuch.,  390  and  393. 


WHITE  BLOOD-CORPUSCLES.  305 

contain,  according  to  Bottazzi  and  Cappelli,1  considerably  more 
potassium  than  sodium.  Lime  is  claimed  to  be  absent  in  the  blood- 
corpuscles,  but  according  to  Hamburger2  this  is  not  true  for  at  least 
ox-blood,  and  magnesia  occurs  only  in  small  amounts:  0.016  (sheep) 
-0.150  p.  m.  (pig).  The  blood-corpuscles  of  all  animals  investigated 
contain  chlorine,  0.460-1.949  p.  m.  (both  in  horse),  generally  1  to  2 
p.  m.,  and  also  phosphoric  acid.  The  amount  of  inorganic  phosphoric 
acid  shows  great  variation:  0.275  (sheep)-1.916  p.  m.  (horse).  All 
of  the  above  figures  are  calculated  on  the  fresh,  moist  blood-corpuscles. 

By  quantitative  determinations  of  the  swelling  and  shrinking  of  the  cells 
under  the  influence  of  NaCl  solutions  of  various  concentration,  or  of  serum  of 
various  dilutions,  Hamburger  has  attempted  to  determine  for  the  erythrocytes, 
as  well  as  the  leucocytes,  the  percentage  relationship  between  the  two  chief  con- 
stituents of  the  cells  (the  frame  and  the  intracellular  fluid).  He  found  that  the 
volume  of  the  frame-substance  for  both  varieties  of  blood-corpuscles  of  the  horse 
was  equal  to  53-56.1  per  cent.  The  volume  for  the  red  blood-corpuscles  was 
for  the  rabbit  48.7-51;  hen,  52.4-57.7,  and  for  the  frog,  72-76.4  per  cent. 
Koeppe  has  raised  objections  to  these  determinations.3 

The  White  Blood- corpuscles  and  the  Blood-plates. 

The  White  Blood-corpuscles,  also  called  Leucocytes  or  Lymphoid 
Cells,  are  of  different  kinds,  and  ordinarily  we  differentiate  between 
the  small  forms  poor  in  protoplasm,  called  lymphocytes,  and  the  larger, 
granular,  often  more  nucleated  forms,  called  leucocytes.  The  poly- 
nuclear  leucocytes  occur  in  greater  abundance  in  the  blood  than  the 
lymphocytes.  In  human  and  mammalian  blood,  most  of  the  white 
blood-corpuscles  are  larger  than  the  red  blood-corpuscles.  They  also 
have  a  lower  specific  gravity  than  the  red  corpuscles,  move  in  the  circulat- 
ing blood  nearer  to  the  walls  of  the  blood-vessels,  and  also  have  a  slower 
motion. 

The  number  of  white  blood-corpuscles  varies  not  only  in  the  different 
blood-vessels,  but  also  under  different  physiological  conditions.  On 
an  average  there  is  only  1  white  corpuscle  for  350-500  red  corpuscles. 
According  to  the  investigations  of  Alex.  Schmidt4  and  his  pupils, 
the  leucocytes  are  destroyed  in  great  part  on  the  discharge  of  the  blood 
before  and  during  coagulation,  so  that  discharged  blood  is  much  poorer 
in  leucocytes  than  the  circulating  blood.  The  correctness  of  this  state- 
ment has  been  denied  by  other  investigators. 

1  Bunge,  Zeitschr.  f.  Biologie,  12,  and  Abderhalden,  Zeitschr.  f.  physiol.  Chem., 
23  and  25;  Wanach,  Maly's  Jahresber.,  18,  88;  Bottazzi  and  Cappelli,  Arch.  Ital. 
de  Biologie,  32. 

2  Zeitschr.  f.  physik.  Chem.  69. 

3  Hamburger,  Arch.  f.  (Anat.  u  )  Physiol.,  1898;  Koeppe,  ibid.,  1899  and  1900. 

4  Pfluger's  Arch.,  11  and  Kriiger,  Arch.  f.  exp.  Path.  u.  Pharm.,  51. 


306  THE  BLOOD. 

From  a  histological  standpoint  we  generally,  as  above  indicated, 
discriminate  between  the  different  kinds  of  colorless  blood-corpuscles. 
Chemically  considered,  however,  there  is  no  known  essential  difference 
between  them,  and  what  little  we  do  know  chemically  is  chiefly  in  con- 
nection with  the  leucocytes.  With  regard  to  their  importance  in  the 
coagulation  of  fibrin,  Alex.  Schmidt  and  his  pupils  distinguish  between 
the  leucocytes  which  are  destroyed  in  the  coagulation  and  those  which 
are  not.  The  last  mentioned  give  with  alkalies  or  common-salt  solutions 
a  slimy  mass;  the  first  do  not  show  such  behavior. 

The  protoplasm  of  the  leucocytes  has,  during  life,  amoeboid  move- 
ments which  serve  partly  to  make  possible  the  wandering  of  the  cells, 
and  partly 'to  aid  in  the  absorption  of  smaller  grains  or  foreign  bodies 
and  make  the  phagocytosis  possible.  The  action  of  various  agents 
such  as  hyper-  and  hypotonic '  salt  solutions,  of  foreign  ions,  such  as 
iodine,  bromine,  and  salts  of  the  alkaline  earths  upon  the  chemotaxis 
and  the  phagocytic  activity  of  the  leucocytes  has  been  thoroughly 
studied  by  Hamburger  and  de  Haan,1  and  among  other  things  they  have 
shown  that  the  Ca  causes  an  accellerating  influence  upon  phagocytosis 
which  is  peculiar  for  Ca  and  does  not  depend  upon  its  properties  as  a 
divalent  ion.  Because  of  the  contractibility  of  the  leucocytes,  the 
occurrence  of  myosin  in  them  has  been  admitted  even  without  any 
special  proof  therefore.  We  know  nothing  positively  whether  in  the 
leucocytes,  or  in  the  cells,  in  general,  globulins  occur  with  traces 
of  albumins,  because  cell  constituents  which  used  to  be  called  globulins 
have  on  more  careful  investigation  been  found  to  be  nucleoalbumins 
or  nucleoproteins.  The  substance  observed  by  Halliburton,2  and 
occurring  in  all  cells,  which  coagulates  at  47  to  50°  C,  is  considered  as 
a  true  globulin.  Alex.  Schmidt  claims  to  have  found  serglobulin  in 
equine-blood  leucocytes  which  have  been  washed  with  ice-cold  water. 

The  proteins  of  the  leucocytes  as  well  as  the  cells  in  general  are  prin- 
cipally compound  proteins.  For  the  present  it  is  impossible  to  state  to 
what  extent  the  nucleoalbumins  occur  in  leucocytes  or  cells,  because  in  the 
past  no  careful  differentiation  was  made  between  the  nucleoalbumins 
and  nucleoproteins.  The  nucleoproteins  are  without  any  doubt  the 
principal  constituents  of  the  protoplasm  of  the  white  blood-corpuscles, 
and  one  of  these  it  seems  is  identical  with  the  so-called  hyaline  substance 
of  Rovida,  which  yields  a  slimy  mass  when  treated  with  alkalies  or 
NaCl  solutions  and  which  occur  in  pus-cells. 

On  digesting  the  leucocytes  with  water,  a  solution  of  a  protein  body 


1  Bioch.  Zeitschr.,  24  and  20. 

2  See  Halliburton,  On  the  chem.  Physiol,  of  the  animal  cell.     King's  College,  London, 
Physiol.  Labor.     Collected  papers,  1893. 


LEUCOCYTE-.  307 

ia  obtained  which  ran  be  precipitated  by  acetic  acid  and  which  forms  the 
chief  mass  of  the  leucoeytes.  This  substance,  which  is  undoubtedly 
concerned  in  the  coagulation  of  the  blood,  has  been  described  under 
different  names,  such  as  tissue  fibrinogen  (Wooldridge)  cytoglobin  and 
prdglobulin  (Alex.  Schmidt)  or  nucleohistone  (Kossel  and  Lilienfeld  ') 
and  consists,  chiefly  at  least,  of  nucleoprotein.  The  ordinary  view  that 
this  is  nucleohistone  does  not  seem  to  be  correct,  according  to  the  invest- 
igations of  Bang,2  and  further  proof  is  necessary. 

Besides  these  constituents  of  the  protoplasm  of  the  leucocytes  we 
must  also  include  lecithin  and  especially  phosphatides,  cholesterin,  glu- 
cotkiorHc  acid  (in  pus-corpuscles,  Mandel  and  Levene3),  purine  bodies 
derived  from  the  nuclein  substances  and  glycogen.  According  to  Hoppe- 
Seyler  glycogen  is  a  constant  constituent  of  all  cells  having  amoeboid 
movement,  and  he  found  it  in  the  colorless  blood-corpuscles  but  not  in 
the  non-mobile  pus-cells.  Nevertheless  glycogen  has  also  been  found 
in  pus-cells  by  Salomon4  and  by  others.  The  glycogen  found  by 
Huppert,  Czerny,  Dastre,5  and  others  in  blood  and  lymph  probably 
originated  from  the  leucocytes.  Enzymes  also  occur  in  the  leucocytes 
and  the  proteolytic  enzymes  are  of  special  importance.  According  to 
Opie  and  Barker  two  proteolytic  enzymes  occur  in  the  leucocytes, 
one  of  which  is  active  in  alkaline  solution  and  occurs  in  the  polynuclear 
cells  while  the  other  is  active  in  acid  solution  and  occurs  in  the  large 
mononuclear  cells.  According  to  Fiessinger  and  Marie,  the  leucocytes 
contain  a  proteolytic  enzyme  which  forms  peptone,  leucine  and  tyrosine 
from  protein  and  which  is  probably  identical  with  the  proteolytic  enzyme 
discovered  earlier  by  Achalme  in  pus.  It  acts  best  in  faintly  alkaline 
solution,  but  also  in  weak  acid  reaction,  and  is  destroyed  at  75-80°  C. 
It  occurs  in  the  polynuclear  leucocytes  but  principally  in  those  which  have 
a  medullary  origin,  while  it  is  absent  in  the  leucocytes  of  the  lymph  series. 
The  lipase  occurring  in  pus  and  in  blood  seems,  according  to  the  above 
experimenters,  to  originate  in  the  lymphocytes.     Tschernoruzki  6   has 


1  See  Wooldridge,  Die  Gerinnung  des  Blutes  (published  by  M.  v.  Frey,  Leipzig,  1S91); 
A.  Schmidt,  Zur  Blutlehre,  Leipzig,  1892;  Lilienfeld,  Zeitsehr.  f.  physiol.  Chem.,  18. 
2 1.  Bang,  Studier  over  Xukleoproteider,  Kristiania,  1902. 

3  Bioehem.  Zeitsehr.,  4. 

4  In  regard  to  the  literature  on  Cdycogen,  see  Chapter  VII. 

5  Huppert,  Centralbl.  f.  Physiol.,  6,  394;  Czerny,  Arch.  f.  exp.  Path.  u.  Pharm., 
31;  Dastre,  Compt.  Rend.,  120,  and  Arch,  de  Physiol.  (5),  7.  See  also  Hirschberg, 
Zeitsehr.  f.  klin.  Med..  64. 

6  In  regard  to  the  enzymes  see  Erbcn,  Jochmann  and  E.  Midler,  Jochmann  and 
Lockemann,  Hofmeister*s  Beitriige,  11,  which  contains  the  literature.  Opie.  Journ. 
of  exper.  Medicine,  8;  with  Barker,  ibid.,  9;  Fiessinger,  and  Marie,  Journ.  de  physiol. 
et  de  pathol.  generate,  11,  which  also  contains  the  literature  and  Compt.  rend'  soc. 
biol.,  66,  6";  Tschernoruzki,  Zeitsehr.  f.  physiol.  Chem.,  73. 


308  THE  BLOOD. 

also  shown  the  presence  of  amylase  (diastase),  catalase,  nuclease  and  per- 
oxidase in  the  polynuclear  leucocytes. 

The  blood-plates  (Bizzozero),  hsematoblasts  (Hayem),  whose  nature, 
preformed  occurrence,  and  physiological  importance  have  been  much 
questioned,  are  pale,  colorless,  gummy  disks,  round  or  somewhat  oval 
in  shape,  generally  with  a  diameter  one-half  or  one-third  that  of  the 
blood-corpuscles.  In  mammalia  their  number,  according  to  Aynaud, 
is  on  an  average  500,000  in  1  c.mm.  They  change  their  shape  readily, 
attack  foreign  bodies  and  agglutinate  under  conditions  which  Aynaud 
has  carefully  studied.  Human  blood-plates  consist,  according  to  Deetjen,1 
of  a  nucleus  and  a  hyaline  protoplasm.  They  are  very  sensitive  toward 
alkalies  and  much  more  so  than  the  plates  from  other  mammalia.  They 
are  destroyed  in  a  concentration  of  hydroxyl  ions,  CoH  =  iXlO~5  and 
in  a  concentration  of  H  ions,  CH  =  2X10-4. 

According  to  the  researches  of  Kossel  and  of  Lilienfeld  2  the  blood- 
plates  consist  of  a  chemical  combination  between  protein  and  nuclein, 
and  hence  they  are  also  called  nuclein-plates  by  Lilienfeld,  and  are 
considered  as  derivatives  of  the  cell  nucleus.  It  seems  certain  that  the 
blood-plates  have  some  connection  with  the  coagulation  of  blood.  The 
views  on  this  question,  especially  in  regard  to  the  manner  in  which  these 
plates  act  in  coagulation,  are  unfortunately  very  divergent. 

HI.    THE   BLOOD  AS  A  MLXTURE  OF   PLASMA  AND   BLOOD-CORPUSCLES. 

The  blood  in  itself  is  a  thick,  sticky,  light  or  dark  red  liquid,  opaque 
even  in  thin  layers,  having  a  salty  taste  and  a  faint  odor  differing  in 
different  kinds  of  animals.  On  the  addition  of  sulphuric  acid  to  the 
blood  the  odor  is  more  pronounced.  In  adult  human  beings  the  specific 
gravity  ranges  between  1.045  and  1.075.  It  has  an  average  of  1.058 
for  grown  men  and  a  little  less  for  women.  Lloyd  Jones  found  that  the 
specific  gravity  is  highest  at  birth  and  lowest  in  children  until  about 
two  years  old,  and  in  pregnant  women.  The  determinations  of  Lloyd 
Jones,  Hammerschlag,3  and  others  show  that  the  variation  of  the  specific 
gravity,  dependent  upon  age  and  sex,  corresponds  to  the  variation  in 
the  quantity  of  haemoglobin. 

The  determination    of    the    specific    gravity  is  accurately  obtained 


1  Aynaud,  Maly's  Jahresb.,  39;  Deetjen,  Zeitschr.  f.  physiol.  Chem.,  63. 

2  In  regard  to  the  literature  of  the  blood-plates,  see  Lilienfeld,  Arch.  f.  (Anat.  u.) 
Physiol..  1892,  and  "Leukocyten  und  Blutgerrinnung,"  Verhandl.  d.  physiol.  Gesellsch. 
zu  Berlin,  1892;  and  also  Mosen,  Arch.  f.  (Anat.  u.)  Physiol.,  1893,  and  Maly's  Jahres- 
ber.,  30  and  31. 

3  Lloyd  Jones,  Journ.  of  Physiol.,  8;  Hammerschlag,  Wien.  klin.  Wochenschrift, 
1880,  and  Zeitschr.  f.  klin.  Med.,  20. 


ALKALINITY  OF  THE  BLOOD.  309 

by  means  of  the  pyknometer.  For  clinical  purposes,  where  only  small 
amounts  are  available,  it  is  best  to  proceed  by  the  method  as  suggested 
by  Hammerschlag.  Prepare  a  mixture  of  chloroform  and  benzene  of 
about  1.050  sp.  gr.  and  add  a  drop  of  the  blood  to  this  mixture.  If  the 
drop  rises  to  the  surface  then  add  benzene,  and  if  it  sinks  add  chloroform. 
Continue  this  until  the  drop  of  blood  suspends  itself  midway  and  then 
determine  the  specific  gravity  of  the  mixture  by  means  of  an  areometer. 
This  method  is  not  strictly  accurate  and  must  be  performed  quickly. 
In  regard  to  the  necessary  details  refer  to  Zuntz  and  A.  Levy.1 

The  reaction  of  the  blood  is  alkaline  toward  litmus,  and  various  bodies 
such  as  alkali  carbonates,  the  phosphates,  alkali-protein  combinations, 
the  amino-acids  and  carbon  dioxide  all  take  part  in  bringing  about  the 
normal  reaction.  According  to  Henderson  2  the  normal  reaction  is 
also  partly  brought  about  by  ammonia  formation  and  partly  by  the 
phosphates,  in  that  the  kidneys  secrete  acid  salts  (phosphates)  and  return 
alkali  to  the  blood  and  regulate  the  reaction  of  the  blood. 

In  considering  the  alkalinity  of  the  blood  we  must,  as  previously 
remarked,  differentiate  between  the  amount  of  titratable  alkali  in  the  blood 
and  the  true  alkalinity,  i.e.,  the  amount  of  hydroxyl  or  hydrogen  ions 
in  the  blood. 

We  have  a  large  number  of  determinations  of  the  quantity  of  titratable 
alkali,  calculated  as  Na2C03,  in  fresh  as  well  as  defibrinated  blood  of 
animals  and  man,  and  in  the  latter  case  under  healthy  and  diseased  con- 
ditions. As  these  determinations  have  been  carried  out  with  dif- 
ferent methods  which  were  not  without  error  they  cannot  be  given  any 
great  importance.  The  results  found  generally  vary  between  3  and  6 
p.  m.  Na2CC>3  and  for  man  the  figures  below  3.3  p.  m.  and  above  5.3  p.  m. 
are  considered  as  pathological.  The  alkaline  reaction  diminishes  out- 
side of  the  body,  and  indeed  the  more  quickly  the  greater  the  original 
alkalinity  of  the  blood.  This  depends  on  the  formation  of  acid  in  the 
blood,  in  which  the  red-blood  corpuscles  seem  to  take  part  in  some  way  or 
another.  After  excessive  muscular  activity  the  alkalinity  is  diminished 
(Peiper,  Cohnstein),  and  it  is  also  decreased  after  the  continuous 
ingestion  of  acids  (Lassar,  Freudberg,3)  and  others. 


1  Zuntz,  Pfliiger's  Arch.,  66;  Levy,  Proceed.  Roy.  Soc,  71. 

2  Amer.  Journ.  of  Physiol.,  21,  and  Journ.  of  biol.  Chem.,  9;  see  also  Robertson, 
ibid.,  6  and  7. 

3  Peiper,  Virchow's  Arch..  116;  Cohnstein,  ibid.,  130,  which  also  cites  the  works 
of  Minkowski,  Zuntz,  and  Geppert;  Freudberg,  ibid.,  125  (literature);  in  regard  to  the 
methods  for  the  estimation  of  the  alkalinity  see,  besides  the  above-mentioned  authors, 
v.  Jaksch,  Klin.  Diagnostik;  v.  Limbeck,  Wien.  med.  Blatter,  18;  Wright,  The  Lancet, 
1897;  Biernacki,  Beitrage  zur  Pneumatologie,  etc.,  Zeitschr.  f.  klin.  Med..  31  and 
32;    Hamburger,  Eine  Methode  zur  Trennung,   etc.,   Arch.,   f.    (Anat,   u.)   Physiol., 


310  THE  BLOOD. 

The  methods  for  the  determination  of  the  true  reaction  of  animal 
fluids,  also  the  blood,  have  been  given  in  Chapter  I.  For  the  true 
alkalinity  of  the  blood,  as  first  shown  by  Hober  and  especially  by 
Hasselbalch  and  Lundsgaard,  the  carbon  dioxide  is  of  the  greatest 
importance  in  that  with  an  increasing  carbon-dioxide  tension  the  con- 
centration of  the  H  ions  increase.  Thus  Hasselbalch  and  Lunds- 
gaard J  found  that  a  rise  in  the  carbon-dioxide  tension  of  30-50  mm., 
that  is  a  rise  of  20  mm.,  increased  the  concentration  of  the  H  ions  about 
36  per  cent. 

For  the  determination  of  the  true  reaction  the  temperature  at  which 
the  measurement  is  made  is  of  the  greatest  importance.  As  the  dis- 
sociation constant  of  water  strongly  rises  with  the  temperature,  the 
HO  ion  concentration  of  the  blood  must  rise  with  the  temperature,  and 
we  can  believe  that  the  alkalinity  of  the  blood  at  body  temperature 
must  be  2-3  times  greater  than  when  measured  at  18°  and  that  this 
alkalinity  increases  15-20  per  cent  when  the  normal  temperature  of  the 
body  (38°)  rises  to  that  of  a  high  fever  (42°). 

The  true  alkalinity  of  the  blood  is  somewhat  variable  under  different 
conditions.  In  this  connection  it  must  be  remarked  that  also  age  and 
other  conditions  have  an  action  upon  the  alkalinity.  As  the  determin- 
ations are  made  with  different,  and  not  always  exact  methods,  and  some- 
times without  consideration  of  the  action  of  carbon  dioxide  and  tem- 
perature, it  is  extremely  difficult  to  give  satisfactory  average  results. 
Under  these  circumstances  it  is  perhaps  sufficient  to  refer  to  the  figures 
given  in  Chapter  I  (page  76). 

The  alkali  of  the  blood  as  above  mentioned  exists  in  part  as  alkaline 
salts,  carbonate  and  phosphate,  and  partly  in  combination  with  protein 
or  hsemoglobin.  The  first  are  often  spoken  of  as  readily  diffusible  alkalies, 
while  the  others  are  not  or  are  only  diffusible  with  difficulty  (see  page 
268).  The  quantity  of  the  first,  in  human  blood,  is  about  one-fifth  of  the 
total  alkali  (Brandenburg).  The  readily  as  well  as  the  difficultly 
diffusible  alkali  is  divided  between  the  blood-corpuscles  and  plasma,  and 
the  blood-corpuscles  seem  to  be  richer  in  difficultly  diffusible  alkali  than 
the  plasma  or  serum.  This  division  may  be  changed  by  the  influence  of 
even  very  small  amounts  of  acid,  even  of  carbonic  acid,  and  also,  as  shown 
by  Zuntz,   Loewy  and  Zuntz,   Hamburger,   Limbeck,   and  Gurber,2 

189*1.  See  also  Maly'fl  Jahresber.,  2d,  30,  and  81;  Salaskin  and  Pupkin,  Zeitschr.  f. 
physiol.  Chem.,  42,  and  O.  Folin,  ibid.,  43;  Laitinen,  Hammarsten's  Festschr.,  1903; 
Westenrijk,  Arch.  f.  exp.  Path.  u.  Pharm.  Suppl.,  190S,  Schmiedeberg-Festschrift. 

1  The  literature  may  be  found  in  Sorenssen,  Messung  und  Bedeutung  der  Wasser- 
Btoff-ionkonzentrationen,  Ergbn.  d.  Physiol,  12;  Hasselbalch  and  Lundsgaard,  Bioch. 
Zeitsrhr.,  38,  and  Skand.  Arch.  f.  Physiol.,  27;  Hasselbalch,  Bioch.  Zeitschr.,  30;  Lunds- 
gaard, ibid.,  41. 

2  Zuntz,   in  Hermann's  Handbuch  der  Physiol.,  4,  Abt.  2;    Loewy  and  Zuntz, 


BLOOD-CORPUSCLES  AND  GASES.     VISCOSITY.  3U 

by  the  influence  of  the  respiratory  exchange  of  gas.  The  blood-corpuscles 
give  up  a  part  of  the  alkali  united  with  protein  to  the  serum  by  the  action 
of  carbon  dioxide,  hence  the  serum  becomes  more  alkaline.  The  equilib- 
rium of  the  osmotic  tension  in  the  blood-corpuscles  and  in  the  serum 
is  thus  disturbed;  the  blood-corpuscles  swell  up  because  they  take  up 
water  from  the  serum,  and  this  then  becomes  more  concentrated  and  richer 
in  alkali,  protein,  and  sugar.  Under  the  influence  of  oxygen,  the  cor- 
puscles take  their  original  form  again  and  the  above  changes  are  reversed. 
The  blood-corpuscles  for  this  reason  are  less  biconcave  in  their  small 
diameter  in  venous  than  in  arterial  blood  (Hamburger). 

These  conditions  have  been  further  studied  by  v.  Koranyi  and 
Bence,1  and  they  have  investigated  the  relation  between  the  changes  of 
the  volume  of  the  blood-corpuscles  and  the  electrical  conductivity,  the 
refractivity  of  the  serum  and  the  viscosity  of  the  blood.  The  refrac- 
tion coefficient  of  the  serum  is  highest  with  an  increase  in  the  amount  of 
carbon  dioxide,  while  it  is  lowest  when  the  blood  is  rich  in  oxygen  and 
poor  in  carbon  dioxide.  They  consider  this  as  an  action  of  acid,  as  a 
similar  rise  is  observed  after  the  addition  of  acid,  while  after  the  addition 
of  alkali  a  fall  in  the  refraction  coefficient  of  the  serum  takes  place,  and 
these  same  changes  can  be  brought  about  by  CO2  or  by  a  current  of  oxygen. 
With  an  increase  in  the  amount  of  carbon  dioxide,  the  conductivity 
of  the  blood  diminishes;  the  viscosity  is,  on  the  other  hand,  highest 
when  the  blood  is  richest  in  carbon  dioxide.  If  the  CO2  is  driven  off 
by  O  the  viscosity  diminishes  to  a  minimum,  and  on  leading  in  more 
oxygen  it  rises  again.  The  changes  in  viscosity  of  the  blood  runs  parallel 
with  the  volume  changes  of  the  blood-corpuscles,  and  changes  in  the 
viscosity,  which  can  be  brought  about  by  the  removal  of  carbon  dioxide, 
cause  a  change  in  the  electric  charge  of  the  blood-corpuscles  (v.  Koranyi 
and  Bence).  The  viscosity  of  the  blood  is  a  variable  quantity  which, 
besides  the  gas  content  of  the  blood,  is  also  dependent  upon  many  other 
circumstances  (Adam  2)  and  which  is  different  at  various  ages  and  under 
unequal  physiological  and  pathological  conditions. 

The  color  of  the  blood  is  red — light  scarlet-red  in  the  arteries  and  dark 
bluish-red  in  the  veins.  Blood  free  from  oxygen  is  dichroic,  dark  red 
by  reflected  light  and  green  by  transmitted  light.  The  blood-coloring 
matters  occur  in  the  blood-corpuscles.     For  this  reason  blood  is  opaque 

Pfliiger's  Arch.,  58;  Hamburger,  Arch.  f.  (Anat.  u.)  Physiol.,  1894  and  1898,  and 
Zeitschr.  f.  Biologie,  28  and  35;  v.  Limbeck,  Arch.  f.  exp.  Path.  u.  Pharm.,  35;  Giirber, 
Sitzungsber.  d.  phys.  med.  Gesellsch.  zu  Wiirzburg,  1895. 

1  Pfliiger's  Arch.,  110. 

-  In  regard  to  the  viscosity  of  the  blood  and  the  literature  of  the  subject,  see  R. 
Hober  in  Oppenheimer's  Handb.  der  Bioch.,  2,  p.  12-18.  See  also  Adam  Zeitschr 
f.  klin.  Med.,  68. 


312  THE  BLOOD. 

in  thin  layers.  If  the  haemoglobin  is  removed  from  the  stroma  and 
dissolved  by  the  blood  liquid  by  any  of  the  above-mentioned  means 
(see  page  273),  the  blood  becomes  transparent  and  has  then  a  "  lake 
color." 1  Less  light  is  now  reflected  from  its  interior,  and  this  laky 
blood  is  therefore  darker  in  thicker  layers.  On  the  addition  of  salt 
solutions  to  the  blood-corpuscles  they  shrink,  more  light  is  reflected, 
and  the  coJor  appears  lighter.  A  great  abundance  of  red  corpuscles 
makes  the  blood  darker,  while  by  diluting  with  serum  or  by  a  greater 
abundance  of  white  corpuscles  the  blood  becomes  lighter  in  appearance. 
The  different  colors  of  arterial  and  of  venous  blood  depend  on  the  vary- 
ing quantities  of  gas  contained  in  these  two  varieties  of  blood,  or,  bet- 
ter, on  the  different  amounts  of  oxyhemoglobin  and  haemoglobin  they 

contain. 

The  most  striking  property  of  blood  consists  in  its.  coagulating  within 
a  shorter  or  longer  time,  but  as  a  rule  very  shortly  after  leaving  the  veins. 
Different  kinds  of  blood  coagulate  with  varying  rapidity;  in  human 
blood  the  first  marked  sign  of  coagulation  is  seen  in  two  to  three  minutes, 
and  within  seven  to  eight  minutes  the  blood  is  thoroughly  converted  into 
a  gelatinous  mass.  If  the  blood  is  allowed  to  coagulate  slowly,  the  red 
corpuscles  have  time  to  settle  more  or  less  before  the  coagulation,  and 
the  blood-clot  then  shows  an  upper  yellowish-gray  or  reddish-gray  layer 
consisting  of  fibrin  enclosing  chiefly  colorless  corpuscles.  This  layer 
has  been  called  crusta  inflammatoria  or  phlogistica,  because  it  has  been 
especially  observed  in  inflammatory  processes  and  is  considered  one 
of  the  characteristics  of  them.  This  crustaf  or  "  huffy  coat,'"  is  not 
characteristic  of  any  special  disease,  and  it  occurs  chiefly  when  the  blood 
coagulates  slowly  or  when  the  blood-corpuscles  settle  more  quickly 
than  usual.  A  buffy  coat  is  often  observed  in  the  slowly  coagulating 
equine  blood.  The  blood  from  the  capillaries  is  not  supposed  to  have  the 
power  of  coagulating. 

Coagulation  is  retarded  by  cooling,  by  diminishing  the  oxygen,  and  by 
increasing  the  amount  of  carbon  dioxide,  which  is  the  reason  that  venous 
blood  and  to  a  much  higher  degree  blood  after  asphyxiation  coagulates 
more  slowly  than  arterial  blood.  The  coagulation  may  be  retarded  or 
prevented  by  the  addition  of  acids,  alkalies,  or  ammonia,  even  in  small 
quantities;  by  concentrated  solutions  of  neutral  alkali  salts  and  alkaline 
earths,  alkali  oxalates  and  fluorides;  also  by  egg-albumin,  solutions  of 
sugar  or  gum,  glycerin,  or  much  water;  also  by  receiving  the  blood  in 
oil.  Coagulation  may  be  prevented  by  the  injection  of  a  proteose  solu- 
tion or  of  an  infusion  of  the  leech  into  the  circulating  blood,  but  this 

1  R.  Du  Bois-Reymond  presents  objections  to  the  general  use  of  the  above  terms, 
in  Centralbl.  f.  Physiol.,  19,  p.  65. 


COAGULATION  OF  THE  BLOOD.  313 

infusion  also  acts  in  the  same  way  on  blood  just  drawn.  Coagulation 
is  also  hindered  by  snake  poison  (cobra-poison),  and  bacterial  toxines. 
The  coagulation  may  be  facilitated  by  raising  the  temperature;  by  con- 
tact with  foreign  bodies,  to  which  the  blood  adheres;  by  stirring  or  beat- 
ing it;  by  admission  of  air;  by  diluting  with  very  small  amounts  of 
water;  by  the  addition  of  platinum-black  or  finely  powdered  carbon; 
by  the  addition  of  laky  blood,  which  does  not  act  by  the  presence  of 
dissolved  blood-coloring  matters,  but  by  the  stromata  of  the  blood-corpus- 
cles; and  also  by  the  addition  of  the  leucocytes  from  the  lymphatic 
glands,  or  of  a  watery  saline  extract  of  the  lymphatic  glands,  testicles,  or 
thymus  and  various  other  organs  (Delezenne,  Wright,  Arthus,1  and 
others) . 

An  important  question  to  answer  is  why  the  blood  remains  fluid 
in  the  circulation,  while  it  quickly  coagulates  when  it  leaves  the  circula- 
tion. The  reason  why  blood  coagulates  on  leaving  the  body  is  therefore 
to  be  sought  for  in  the  influence  which  the  walls  of  the  living  and  unin- 
jured blood-vessels  exert  upon  it.  These  views  are  derived  from  the 
observations  of  many  investigators.  From  the  observations  of  Hewson, 
Lister,  and  Fredericq  it  is  known  that  when  a  vein  full  of  blood  is 
ligatured  at  the  two  ends  and  removed  from  the  body,  the  blood  may  remain 
fluid  for  a  long  time.  Brucke  2  allowed  the  heart  removed  from  a  tortoise 
to  beat  at  0°  C,  and  found  that  the  blood  remained  uncoagulated  for 
some  days.  The  blood  from  another  heart  quickly  coagulated  when 
collected  over  mercury.  In  a  dead  heart,  as  also  in  a  dead  blood-vessel, 
the  blood  soon  coagulates,  and  also  when  the  walls  of  the  vessel  are  changed 
by  pathological  processes. 

What  then  is  the  influence  which  the  walls  of  the  vessels  exert  on 
the  liquidity  of  the  circulating  blood?  Freund  found  that  the  blood 
remains  fluid  when  collected  by  means  of  a  greased  canula  under  oil  or 
in  a  vessel  smeared  with  vaseline.  If  the  blood  collected  in  a  greased 
vessel  be  beaten  with  a  glass  rod  previously  oiled,  it  does  not  coagulate, 
but  it  quickly  coagulates  on  beating  it  with  an  unoiled  glass  rod  or  when 
it  is  poured  into  a  vessel  not  greased.  The  non-coagulability  of  blood 
collected  under  oil  was  confirmed  later  by  Haycraft  and  Carlier. 
Freund  found  on  further  investigation  that  the  evaporation  of  the 
upper  layers  of  blood  or  their  contamination  with  small  quantities  of  dust 
causes  a  coagulation  even  in  a  vessel  treated  with  vaseline.     According 


1  Delezenne,  Arch,  de  Physiol.  (5),  8;  Wright,  Journ.  of  Physiol.,  28;  Arthus, 
Journ.  de  Physiol,  et  Pathol.,  4. 

2  Hewson's  works,  edited  by  Gulliver,  London,  1876,  cited  from  Gamgee,  Text- 
book of  Physiol.  Chem.,  1,  1880;  Lister,  cited  from  Gamgee,  ibid.;  Fredericq,  Recher- 
ches  sur  la  constitution  du  plasma  sanguin,  Gand,  1878;  Brucke,  Virchow's  Arch.  12. 


314  THE  BLOOD. 

to  Frevnd  1  it  is  this  adhesion  between  the  blood  and  a  foreign  substance 
— and  the  diseased  walls  of  the  vessel  also  act  as  as  such — that  gives  the 
impulse  toward  coagulation,  while  the  lack  of  adhesion  prevents  the 
blood  from  coagulating.  Bordet  and  Gengou  2  have  also  shown  that 
the  plasma  obtained  by  centrifuging  blood  collected  in  a  paraffined 
vessel,  and  perfectly  free  from  form-elements,  can  be  kept  without  coagulat- 
ing in  a  paraffined  vessel,  and  that  it  does  coagulate  on  being  transferred 
to  an  unparaffined  vessel.  The  adhesion  of  the  plasma  to  a  foreign 
body  may  also,  in  the  absence  of  form-elements,  give  the  impulse  to 
coagulation.  That  this  adhesion  of  the  form-elements  is  of  great  impor- 
tance cannot  be  denied  and  is  also  generally  accepted.  By  this  adhesion 
the  form-elements  undergo  certain  changes  which  seem  to  stand  in  a 
certain  relation  to  the  coagulation  of  the  blood. 

The  views  in  regard  to  these  changes  are,  unfortunately,  very  diver- 
gent. According  to  Alex.  Schmidt3  and  the  Dorpat  school  an  abun- 
dant destruction  of  the  leucocytes,  especially  polynuclear  leucocytes, 
takes  place  in  coagulation,  and  important  constituents  for  the  coagula- 
tion of  the  fibrin  pass  into  the  plasma.  A  direct  relation  between  the 
destruction  of  leucocytes  and  coagulation  is  denied  by  many  investigators, 
while  according  to  other  experimenters  the  essential  factor  is  not  a 
destruction  of  the  leucocytes,  but  an  elimination  of  constituents  from 
the  cells  into  the  plasma.  This  process  is  called  plasmoschisis  by  Lowit.4 
The  passage  of  cell  constituents  into  the  plasma  before  coagulation  must 
not  necessarily  be  considered  as  a  phenomenon  of  death,  as  it  may  just 
as  well  be  a  secretory  process  (Arthus,  Morawitz,  Dastre  5). 
1 '  Great  importance  has  also  been  ascribed  to  the  blood-plates  in  coagula- 
tion as  certain  investigators  (Bizzero,  Lilienfeld,  Schwalbe,  Mora- 
witz, Burker,  Deetjen,  Le  Sourd  and  Pagniez)  found  that  they 
induce,  accelerate  or  make  coagulation  possible.  According  to  Vinci  and 
Chistoni  they  are  not  necessary  as  they  are  absent  in  the  blood  of 
birds,  which  coagulates  rapidly,  and  also  in  the  lymph,  of  the  dog,  rabbit 
and  cat.  They  may  nevertheless  accelerate  coagulation  and  they  are 
necessary  for  the  contraction  of  the  clot.     According  to  Aynaud  they 

1  Freund,  Wien.  med.  Jahrb.,  1886;  Haycraft  and  Carlier,  Journ.  of  Anat.  and 
Physiol.,  22. 

1  Annal.  de  l'Institute  Pasteur,  17. 

3  Pfliiger's  Arch.,  11.  The  works  of  Alex.  Schmidt  are  found  in  Arch.  f.  Anat. 
und  Physiol.,  1861,  1862;  Pfluger's  Arch.,  6,  9,  11,  13.  See  especially  Alex.  Schmidt, 
Zur  Hlutlehre  (Leipzig,  1892),  which  also  gives  the  work  of  his  pupils,  and  Weitere 
Beitnige  zur  Blutlehre,  1895. 

4  Wien.  Sitzungsber.,  89  and  90,  and  Prager  med.  Wochenschr.,  1889,  referred 
to  in  Centralbl.  f.  d.  med.  Wissensch.,  28,  265. 

6  Morawitz,  Hofmeister's  Beitriige,  5;  Arthus,  Compt.  rend.  soc.  biolog.,  55; 
Dastre,  ibid.,  55. 


FORM-ELEMENTS  AND  COAGULATION.  315 

arr  nol  necessary  for  the  contraction  of  the  clot  nor  for  the  coagulation 
ae  a  whole,  and  they  are  absent  in  the  lymph  and  serous  fluids.  Accord- 
ing to  Petrone  '  they  indeed  have  a  function  in  retarding  coagulation. 

WoOLDBTOGl  2  takes  a  very  peculiar  position  in  regard  to  this  question:  he 
considers  the  form-elements  as  only  of  secondary  importance  in  coagulation. 
Afl  he  has  found,  a  peptone-plasma  which  has  been  freed  from  all  form-con- 
stituents by  means  of  centrifugal  force  yields  abundant  fibrin  when  it  is  not 
separated  from  a  substance  which  precipitates  on  cooling.  This  substance, 
which  WOOLDRIDGE  has  called  A-fibrinogen,  seems  to  all  appearances  to  be  a 
nucleoproteid.  which,  according  to  the  unanimous  view  of  several  investigators, 
originates  from  the  form-elements  of  the  blood,  either  the  blood-plates  or  the 
leucocytes  and  the  generally  accepted  view  as  to  the  great  importance  of  the 
form-elements  in  the  coagulation  of  the  blood  is  not  really  contrary  to  Wool- 
dridge's  experiments. 

There  is  great  diversity  of  opinion  in  regard  to  those  bodies  which 
are  eliminated  from  the  form-elements  of  the  blood  before  and  during 
coagulation. 

According  to  Alex.  Schmidt  the  leucocytes,  like  all  cells,  contain 
two  chief  groups  of  constituents,  one  of  which  accelerates  coagulation, 
while  the  other  retards  or  hinders  it.  The  first  may  be  extracted  from  the 
cells  by  alcohol,  while  the  other  cannot  be  extracted.  Blood-plasma 
contains  only  traces  of  thrombin,  according  to  Schmidt,  but  does  con- 
tain its  antecedent,  prothrombin.  The  bodies  which  accelerate  coagu- 
lation are  neither  thrombin  nor  prothrombin,  but  they  act  in  this  wise 
in  that  they  split  off  thrombin  from  the  prothrombin.  On  this  account 
they  are  called  zymoplastic  substances  by  Alex.  Schmidt.  The  nature 
of  these  bodies  is  unknown,  and  Schmidt  has  given  no  opinion  as  to 
their  relation  to  the  lime  salts,  which  have  been  found  to  have  zymoplastic 
activity  by  other  investigators. 

The  constituents  of  the  cells  which  hinder  coagulation  and  which  are  insoluble 
in  alcohol-ether  are  compound  proteins,  and  have  been  called  eytoglobin  and 
preglobidin  by  Schmidt.  The  retarding  action  of  these  bodies  may  be  sup-, 
pressed  by  the  addition  of  zymoplastic  substances,  and  the  yield  of  fibrin  on  coagula- 
tion in  this  case  is  much  greater  than  in  the  absence  of  the  compound  protein 
retarding  coagulation.  This  last  supplies  the  material  from  which  the  fibrin  is 
produced.  The  process  is,  according  to  Schmidt,  as  follows:  The  preglobulin 
first  splits,  yielding  serglobulin,  then  from  this  the  fibrinogen  is  derived,  and  from 
this  latter  the  fibrin  is  produced.  The  object  of  the  thrombin  is  two-fold.  The 
thrombin  first  splits  the  fibrinogen  from  the  paraglobulin,  and  then  converts  the 


»See  footnote  2,  p.  308.  Also  Schwalbe,  ntcrs.  z.  Blutgerinnung,  etc.,  Braun- 
schweig, 1900;  Morawitz,  Deutsch.  Arch.  f.  klin.  Med..  79,  and  Hofmeister's  Beitrage, 
4  and  5;  Biirker,  Pflliger's  Arch.,  102,  and  Centralbl.  f.  Physiol.,  21;  Deetjen,  1.  c; 
Le  Sourd  and  Pagniez,  Journ.  de  Physiol.,  11;  Vinci  and  Chistoni,  Chem.  Centralbl., 
1909,  2,  838,  and  Maly's  Jahresb.,  39;  Aynaud,  Maly's  Jahresb.,  39;  165;  Petrone, 
Mary's  Jahresber.,  31,  p.  170. 

2  Die  Gerinnung  des  Blutes  (published  by  M.  v.  Frey,  Leipzig,  1891). 


316  THE  BLOOD. 

fibrinogen  into  fibrin.     The  assumption  that  fibrinogen  can  be  split  from  para- 
globulin  has  not  sufficient  foundation  and  is  even  improbable. 

According  to  Schmidt  the  retarding  action  of  the  cells  is  prominent 
during  life,  while  the  accelerating  action  is  especially  pronounced  out- 
side of  the  body  or  by  coming  in  contact  with  foreign  bodies.  The  paren- 
chymous  masses  of  the  organs  and  tissues,  through  which  the  blood  flows 
in  the  capillaries,  are  those  cell-masses  which  serve  to  keep  the  blood 
fluid  during  life. 

Lilienfeld  has  given  further  proof  as  to  the  occurrence,  in  the  form- 
elements  of  the  blood,  of  bodies  which  accelerate  or  retard  the  coagula- 
tion. According  to  this  author  the  nature  of  these  bodies  is  very  markedly 
different  from  Schmidt's  idea.  While,  according  to  Schmidt,  the  coagula- 
tion accelerators  are  bodies  soluble  in  alcohol,  and  the  compound  proteins 
exhausted  with  alcohol  act  only  retardingly  on  coagulation,  Lilienfeld 
states  that  the  substance  which  acts  acceleratingly  and  retardingly 
on  coagulation  are  contained  in  a  nucleoprotein,  namely,  nucleohistone. 
Nucleohistone  readily  splits  into  leuconuclein  and  histone,  the  first 
of  which  acts  as  a  coagulation-excitant,  while  the  other,  introduced 
into  the  blood-vascular  system,  either  intravascular  or  extravascular,  robs 
the  blood  of  its  property  of  coagulating.  Introduced  into  the  circulatory 
system  the  nucleohistone  splits  into  its  two  components.  It  therefore 
causes  extensive  coagulation  on  one  side  and  makes  the  remainder  of 
the  blood  uncoagulable  en  the  other.  This  theory  as  well  as  that  of 
Schmidt  is  not  based  upon  sufficiently  demonstrated  facts. 

Brucke  showed  long  ago  that  fibrin  left  an  ash  containing  calcium 
phosphate.  The  fact  that  calcium  salts  may  facilitate  or  even  cause  a 
coagulation,  in  liquids  poor  in  ferment,  has  been  known  for  several  years, 
through  the  researches  of  Hammarsten,  Green,  Ringer  and  Sains- 
bury.  The  necessity  of  the  lime  salts  for  the  coagulation  of  blood  and 
plasma  was  first  shown  positively  by  the  important  investigations  of 
Arthus  and  Pages.  Recent  investigations  of  Sabbatani  1  have  also 
shown  the  importance  of  calcium  salts  or  the  free  calcium  ions  for 
coagulation  without  explaining  the  mode  of  their  action. 

According  to  the  generally  accepted  tow  of  Arthus  and  Pages  the  soluble 
lime  salts  precipitable  by  oxalate  are  necessary  requisites  for  the  fermentive 
transformation  of  fibrinogen,  because  thrombin  remains  inactive  in  the  absence 
of  soluble  lime  salts.  This  view  is  untenable,  as  shown  by  the  researches  of 
Alex.  Schmidt,  Pekelharing,  and  Hammarsten.2  Thrombin  acts  as  well  in 
the  absence  as  in  the  presence  of  precipitable  lime  salts. 

1  Hammarsten,  Nova  Acta,  reg.  Soc.  Scient.  Upsala,  (3),  10,  1879;  Green,  Journ. 
of  Physiol.,  8;  Ringer  and  Hainsbury,  ibid.,  11  and  12;  Arthus  et  Pages  and  Arthus, 
see  footnote  4,  p.  251;  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  22;  Sabbatani, 
rited,  Centralbl.  f.  Physiol.,  16,  665. 

2  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  22,  where  the  other  investigators  are 
cited. 


COAGULATION   OF  THE   BLOOD.  317 

According  to  Pekelhalixg  '  thrombin  is  the  lime  compound  of 
prothrombin,  and  the  process  of  coagulation  consists,  according  to  him, 
in  the  thrombin  transferring  the  lime  to  the  fibrinogen,  which  is  thereby 
converted  into  an  insoluble  lime  compound,  fibrin.  Among  the  objec- 
tions to  this  theory  can  be  mentioned,  the  fact  that  fibrin  has  not 
been  obtained  absolutely  free  from  lime,  but  still  so  poor  in  lime 
(Hammarsten  2)  that  if  the  lime  belongs  to  the  fibrin,  its  molecule 
must  be  more  than  ten  times  greater  than  the  haemoglobin  molecule, 
which  is  not  probable.  These  as  well  as  many  other  observations 
indicate  that  the  lime  is  carried  down  by  the  fibrinogen  only  as  a 
contamination. 

If,  as  it  seems,  the  lime  is  not  of  importance  in  the  transformation 
of  fibrinogen  into  fibrin  in  the  presence  of  thrombin,  still  this  does  not 
contradict  the  above-mentioned  observations  of  Arthus  and  Pages  that 
the  lime  salts  are  necessary  for  coagulation  of  blood  and  plasma.  It 
is  very  probable  that  the  lime  salts,  as  admitted  by  Pekelharing,  are 
a  requisite  for  the  transformation  of  prothrombin  into  thrombin. 

If  we  attempt  to  summarize  the  more  or  less  contradictory  investi- 
gations and  views  as  given  in  the  preceding  pages,  we  can  consider  the 
following  facts  as  conclusive:  In  the  first  place,  two  bodies,  the  fibrin- 
ogen and  the  thrombin,  are  necessary  for  the  coagulation.  The  fibrinogen 
exists  preformed  in  the  plasma.  The  thrombin,  on  the  contrary,  does 
not  occur  in  living  blood,  at  least  not  in  appreciable  amounts  as  such, 
but  is  formed  from  another  substance,  the  prothrombin.  The  presence 
of  calcium  salts  is  necessary  for  the  formation  of  this  thrombin,  while 
the  calcium  salts  are  not  necessary  for  the  enzymotic  transformation 
of  fibrinogen  into  fibrin.  Besides  the  calcium  salts  also  other  substances, 
the  zymoplastic  active  substances,  are  active  in  the  formation  of  thrombin 
from  its  mother-substance,  and  these  zymoplastic  substances  stand  in 
some  relation  to  the  form-elements  of  the  blood. 

The  formation  of  thrombin  and  the  relation  of  the  form-elements 
therewith  are  still  unexplained  and  disputed  questions. 

It  is  a  question  whether  the  mother-substance  of  thrombin  exists  in 
the  plasma  of  the  circulating  blood  or  whether  it  is  a  body  eliminated 
from  the  form-elements  before  coagulation.  We  have  two  opposing 
views  on  this  question,  namely,  those  of  Alex.  Schmidt  and  of  Pekel- 
haring. According  to  Schmidt  prothrombin  occurs  preformed  in  the 
circulating  plasma,  and  it  is  transformed  into  thrombin  by  the  zymo- 
plastic substances  which  pass  out  from  the  form-elements.  Pekel- 
haring, on  the  contrary,  holds  the  view  that  the  plasma  does  not  contain 

1  See  footnote  4,  p.  256,  and  especially  Virchow's  Festschrift,  1,  1891. 

2  Zeitschr.  f.  physiol  Chem.,  28. 


318  THE  BLOOD. 

appreciable  amounts  of  prothrombin.  This  body,  according  to  him, 
passes  before  coagulation  from  the  form-elements  into  the  plasma,  and 
is  there  converted  into  thrombin  by  the  calcium  salts.  The  observa- 
tion that  uncoagulated  leech-plasma  does  not  coagulate  on  the  addition 
of  calcium  salts,  while  it  does  coagulate  on  the  addition  of  prothrombin 
solutions,  seems  to  support  this  view;  still  it  is  not  quite  conclusive. 
Leech-extract  contains  a  body,  hirudin,  which,  seems  to  be  an  anti- 
body toward  thrombin  and  quantitatively  neutralizes  it.  On  the  addi- 
tion of  prothrombin,  new  thrombin  may  be  formed,  which  may  act 
if  the  hirudin  is  not  present  in  too  great  an  excess. 

Other  observations  which  dispute  the  occurrence  of  prothrombin  in 
the  circulating  plasma  can  be  explained  in  various  ways  and  it  is  the 
general  view  at  present  that  the  prothrombin  is  a  preformed  constituent 
of  the  plasma. : 

Although  the  opinions  are  rather  united  as  to  the  occurrence  of  at 
least  three  bodies,  fibrinogen,  prothrombin  (thrombogen)  and  lime  salts 
in  the  plasma,  still  the  question  arises  how  the  thrombin  is  formed  from 
the  thrombogen.  The  zymoplastic  substances  must  be  here  considered, 
and  the  starting-point  in  these  new  investigations  is  the  accelerat- 
ing action  upon  coagulation,  of  different  tissue  extracts,  an  action  which 
has  been  known  for  a  long  time  and  was  especially  studied  by  Dele- 
zenne  on  the  plasma  from  bird's  blood.  Unfortunately  we  are  not  in 
accord  as  to  the  nature  and  manner  of  action  of  the  active  constituents 
of  these  extracts.  According  to  Morawitz  the  active  body  is  not 
thrombin,  but  another  substance  called  thrombokinase,  besides  lime- 
salts,  which  are  necessary  for  the  transformation  of  prothrombin  (throm- 
bogen according  to  Morawitz).  The  production  of  thrombokinase  is, 
according  to  Morawitz,  a  general  property  of  the  protoplasm,  and  also 
occurs  in  the  leucocytes  (and  blood-plates).  Three  substances  are  nec- 
essary, according  to  his  view,  for  the  formation  of  thrombin,  namely: 
thrombogen,  thrombokinase  and  lime  salts.  Thrombogen  is,  he  claims, 
not  quite  identical  with  the  prothrombin  (other  investigators),  which 
he  calls  a-prothrombin,  but  is  a  mother-substance  of  it.  The  process 
of  thrombin  formation  can  be  given  as  follows:  The  kinase  first  trans- 
forms the  thrombogen  into  a-prothrombin,  which  latter  then  is  converted 
into  thrombin  (a)  by  the  lime  salts. 

'Arthus,  Journ.  de  Physiol,  et  Pathol.,  3  and  4,  and  Compt.  rend.  soc.  biol.,  56. 
The  works  of  Morawitz  may  be  found  in  Hofmeister's  Beitrage,  4  and  5,  Deutsch. 
Arch.  f.  klin.  Med.,  79  and  80,  and  in  Oppenheimer's  Handb.  der  Bioch.,  2;  Fuld, 
Centralbl.  f.  Physiol.,  17,  p.  529;  with  Spiro,  Hofmeister's  Beitrage,  5;  Schittenhelm 
and  Bodong,  Arch.  f.  exp.  Path.  u.  Pharm.,  54;  Bordet  and  Gengou,  Annal.  Institut 
Pasteur,  18.  For  more  recent  literature  see  Loeb,  Biochem.  Centralbl.,  6,  p.  907. 
P.  Xolf,  Arch   internat.  de  Physiol.,  6,  1908. 


COAGULATION   OF  THE   BLOOD.  319 

The  thrombokinase  does  not  occur  to  any  appreciable  extent  in 
the  circulating  blood,  but  is  supplied  by  the  form-elements.  The 
accelerating  action  upon  coagulation  of  tissues  or  parts  of  tissues  depends, 
as  above  stated,  upon  their  content  of  kinase;  but  it  also  in  part  depends 
upon  the  fact  that  the  tissue  fluids  excite  the  secretory  activity  of  the  form- 
elements. 

Fuld  l  has  arrived  at  about  the  same  results  independently  of  Mora- 
witz, but  he  has  selected  other  names.  The  three  substances,  throm- 
bogen,  kinase,  and  thrombin  are  called  by  him  plasmozym,  cytozym, 
and  holozym.  The  principal  reason  why  circulating  blood  remains  fluid  is, 
according  to  Fuld,  because  the  cytozym  is  only  slowly  formed  therein 
and  the  ferment  (holozym)  produced  thereby  is  quickly  changed  into  an 
inactive  form.  Another  reason  is  that  the  blood  contains  an  antibody 
for  the  fibrin  ferment.  The  assumption  of  Alexander  Schmidt  that 
the  blood  contains  substances  retarding  coagulation  (anti-thrombins) 
has  recently  also  received  support  by  the  observations  of  Fuld  and 
Spiro,  Morawitz,  Loeb,  Nolf,  Pugliese,  Howell  2  and  others.  Accord- 
ing to  Howell  the  non-coagulability  of  circulating  blood  depends  on 
the  fact  that  the  antithrombin  prevents  the  activation  of  the  prothrombin 
into  thrombin. 

According  to  the  theory  of  Morawitz,  Fuld  and  Spiro,  which  is  the 
most  accepted,  of  those  substances  necessary  for  coagulation,  only 
the  thrombokinase  (the  cytozym)  is  absent  in  the  circulating  blood, 
and  this  is  the  reason  why  the  circulating  blood  remains  fluid.  The 
reason  why  the  plasma  does  not  contain  any  thrombokinase  lies  in  the 
fact  that  the  healthy  endothelium  of  the  vessels  does  mot  have  any  irritat- 
ing action  upon  the  form-elements,  and  therefore  no  mentionable  quan- 
tity of  kinase  is  given  off  under  these  circumstances.  Such  an  elimina- 
tion occurs  first  outside  of  the  blood  vessels,  and  indeed  very  quickly 
in  contact  with  foreign  bodies.  The  formation  of  thrombin  from  the 
thrombogen  takes  place  in  an  unknown  manner  by  the  action  of  the 
kinase  only  in  the  presence  of  lime  salts  (in  the  plasma),  and  this  throm- 
bin then  transforms  the  fibrinogen  into  fibrin. 

A  serum  poor  in  ferment  and  having  a  weak  action  can  be  reactivated  by  the 
addition  of  acid  or  alkali  (Alex.  Schmidt,  Morawitz),  and  in  this  action,  accord- 
ing to  Morawitz,  a  thrombin  (/3)  is  produced  which  is  somewhat  different  from 
a-thrombin.  The  ^-thrombin  is  produced  from  a  special  ^-prothrombin  which 
never  occurs  in  the  plasma,  but  only  in  the  serum.  Fuld  explains  this  by 
affirming  that  the  a-thrombin  is  changed  in  the  serum  into  metazym  (/?-pro- 

^entralbl.  f.  Physiol.,  17.     See  also  Fuld  and  Spiro,  Hofmeister's  Beitrage,  5. 

2  Fuld  and  Spiro,  1.  c; 'Morawitz,  1.  c;  Loeb,  Hofmeister's  Be;trage,  5;  Xolf,  Arch, 
internat.  de  Physiol.,  6;  Pugliese,  Biochem.  Centralbl.,  5,  p.  930;  Howell,  Amer. 
Journ.  of  Physiol.,  29. 


320  THE  BLOOD. 

thrombin),  which  is  then  transformed  by  the  alkali  or  acid  into  neozym  (=/3- 
thrombin).  Nevertheless  it  is  a  fact  that  the  quantity  of  thrombin  in  the  serum 
diminishes  after  coagulation,  and  that  the  thrombin  action  is  considerably  increased 
by  the  addition  of  alkali  or  acid  as  well  as  by  zymoplastic  substances.  The  above 
view  as  to  the  occurrence  of  different  thrombins  has  not  sufficient  basis,  and 
Pekelharing  x  has  also  raised  objections  thereto. 

The  theories  of  Morawitz,  Fuld  and  Spiro  at  least  stand  in  accord 
with  several  known  facts  but  do  not  take  sufficient  account  of  the  action 
of  the  zymoplastic  substances  of  Alex.  Schmidt.  Thrombokinase  is 
precipitated,  by  alcohol  and  is  not  thermostabile,  while  the  zymoplastic 
substances,  of  Schmidt  are  thermostabile  and  soluble  in  alcohol.  The 
thrombokinase  cannot  therefore  be  identical  with  these  zymoplastic 
substances,  and  hence  this  theory  does  not  explain  the  action  of  these 
latter.  Further,  the  mode  of  action  of  tissue  extracts  is  unexplained, 
and  is  a  much  disputed  subject.  It  can  be  said  that  these  two  views  are 
in  the  main  opposed  to  each  other.  According  to  one  (Alex.  Schmidt, 
Arthtjs,  Morawitz  and  others)  they  do  not  act  like  fibrin  ferment, 
but  have  an  indirect  action.  According  to  the  other  (Pekelharing, 
Huiskamp,  Delezenne  and  Loeb  2)  they  are  thrombin,  or  at  least  bodies 
having  an  analogous  action. 

Cramer  and  Pringle  3  have  made  the  important  observation  that  a 
carefully  prepared  oxalate  plasma  when  filtered  through  a  Berkefeld 
filter  does  not  coagulate  on  adding  calcium  chloride,  while  the  unfiltered 
but  centrifuged  plasma  does  coagulate.  The  reason  for  this  lies  in  the 
fact  that  centrifuged  plasma  contains  blood-plates,  which  are  absent 
in  the  filtered  plasma.  By  means  of  these  blood-plates,  which  yield 
thrombokinase,  the  coagulation  is  produced  on  the  addition  of  calcium 
chloride.  The  points  in  Nolf's  theory  of  coagulation  that  are  difficult 
to  understand  as  well  as  the  observations  of  Freund  (page  313)  and  of 
Bordet  and   Gengou    (page   314)    are   explained  by  this  observation. 

L.  Loeb,4  who  has  carried  out  complete  investigations  on  the  coagulation  of 
blood,  especially  of  Crustaceae,  has  arrived  at  the  following  view:  The  coagula- 
tion in  the  Crustacea?  can,  according  to  him,  be  of  two  kinds.  It  may  in  part 
be  an  agglutination  of  the  amcebocytes  and  in  part  a  fibrin  formation  from  a  fibrino- 
gen of  the  plasma.  This  latter  coagulation  is  essentially  the  same  as  occurs  in 
vertebrates.  The  substance  acting  here  as  the  excitant  for  the  coagulation  is 
also  active  in  the  absence  of  lime  salts,  and  behaves  therefore  like  a  thrombin. 
The  tissues  contain  constituents  which  accelerate  coagulation,  which  Loeb  calls 
coagnlins,  which  are  not  identical  with  the  coagulins  of  the  clot  or  the  blood  serum, 

1  Bioch.  Zeitschr.,  11. 

2  Huiskamp.  Zeitschr.  f.  physiol.  Chem.,  34,  39;  Delezenne,  Arch.  de.  physiol., 
1897;  Loeb,  Biochem.  Centralbl.,  6,  pages  829  and  889. 

•  Quarterly  Journ.  of  exp.  Physiol.,  6. 

4  Medical  News,  New  York,  1903,  and  Virchow's  Arch.,  176;  Hofmeister's  Beitrage, 
5,  6,  8.  9,  and  Biochem.  Centralbl.,  6,  pages  829  and  889. 


COAGULATION  OF  THE  BLOOD.  321 

and  these  have  also,  although  only  in  the  presence  of  lime  salts  (if  the  author 
understands  Loeb),  a  direct  coagulating  action  upon  fibrinogen.  According  to 
Loeb  the  tissue  coagulina  do  not  act  as  kinases  in  the  invertebrates,  and  he  also 
finds  it  improbable  that  they  would  act  as  kinases  in  the  vertebrates.  Under 
favorable  conditions  the  combined  blood  and  tissue  coagulins  are  more  active 
than  the  Bum  of  the  individual  action.  That  this  is  due  to  an  activation  by  a 
kinase,  which  is  a  possible  explanation,  has,  in  Loeb's  opinion,  not  been  proved. 

The  coagulins  of  the  blood  are,  as  above  stated,  according  to  Loeb,  different 
from  the  tissue  coagulins.  The  latter  are  for  different  classes  of  animals  so 
adapted  that  they  bring  about  a  quicker  coagulation  in  the  blood  of  certain  classes 
of  animals  than  do  the  other  class.  The  erythrocytes  of  mammalia  (cat,  dog,  ral )1  tit ) 
contain,  on  the  contrary,  according  to  Loeb  and  Fleisher  '  coagulins  of  such  a 
specific  adaptability  that  it  is  possible  to  differentiate  between  the  blood  corpuscles 
of  different  kinds  of  mammalia  or,  if  the  erythrocytes  are  known,  to  detect  an 
unknown  plasma. 

Opinions  are  strikingly  at  variance  in  regard  to  the  mode  cf  action 
of  the  tissue  constituents  which  accelerate  coagulation,  and  their  nature 
also  is  entirely  unknown,  hence  great  confusion  exists  on  the  whole  in 
this  subject. 

If  we  accept  the  fact  that  thrombokinase  does  not  occur  in  the  plasma, 
but  is  produced  under  the  influence  of  a  foreign  body  acting  as  an  excitant, 
it  is  rather  difficult  to  understand  why  the  plasma  obtained  from  blood 
collected  in  a  paraffined  vessel  and  quickly  and  strongly  centrifuged, 
and  which  is  perfectly  free  from  form-elements,  should  remain  fluid  for 
a  long  time  in  a  paraffined  vessel  while  it  coagulates  in  an  ordinary  glass 
vessel.  Nolf  has  tried  by  his  theory  to  explain  this  difficulty,  as  well 
as  the  action  of  the  alcohol-soluble  zymoplastic  substances  (Alex. 
Schmidt). 

According  to  Nolf2  the  following  bodies  take  direct  part  in  the 
coagulation  of  the  blood,  namely:  Fibrinogen,  thrombogen  (formerly 
called  hepatothrombin  by  him)  thrombozym  (  =  thrombokinase  of  Mora- 
witz)  and  lime  salts.  The  coagulation  of  the  blood,  according  to  him, 
is  a  different  process  from  the  coagulation  of  a  fibrinogen  solution  by 
thrombin.  While  in  this  last  case  the  thrombin  is  the  substance  exciting 
coagulation,  in  the  other  case  the  thrombin  is  a  product  of  the  coagu- 
lation, as  suggested  by  Wooldridge.  In  the  coagulation  of  the  plasma, 
according  to  Nolf  we  have  a  mutual  precipitation  of  the  three  above- 
mentioned  colloids — fibrinogen,  thrombogen  and  thrombozym,  all  three 
of  wliich  are  contained  in  the  fibrin  clot.  This  latter  has  correspondingly 
no  constant  composition,  but  varies  according  to  the  relative  propor- 
tions of  these  three  colloids.  In  the  presence  of  only  a  little  fibrinogen 
thrombin  is  produced  from  the  three  colloids  (in  the  presence  of  lime 
salts) ;  in  the  presence  of  abundance  of  fibrinogen,  on  the  contrary,  fibrin 


1  Loeb  and  Fleisher,  Bioch.  Zeitchr.  28. 

2  Arch,  internat.  de  Physiol.,  6,  Fasc,  1,  2,  and  3  and  7  and  9. 


322  THE  BLOOD. 

is  formed.  Thrombin  is  a  fibrin  incompletely  saturated  with  fibrinogen, 
and  in  the  coagulation  of  fibrinogen  with  thrombin  the  still  unsatisfied 
affinities  of  the  latter  are  saturated.  ("La  thrombine  d'A.  Schmidt  n'est- 
pas  autre  chose  que  de  la  fibrine  insuffisamment  pourvue  de  fibrinogene. 
Dans  la  coagulation  du  fibrinogene  par  la  thrombine  les  affinites  restees 
libres  de  celle-ci  peuvent  s'assouvir;  le  compose  moins  sature  se  trans- 
forme  en  un  compose  plus  satureV')  The  formation  of  fibrin  from 
fibrinogen  is  not,  according  to  Nolf,  an  enzymotic  process,  and  the 
thrombin  is  only  a  residue  of  the  fibrin  remaining  in  solution. 

In  Nolf's  opinion  the  thrombogen  is  probably  formed  in  the  liver 
and  found  to  a  large  extent  in  all  plasma.  The  thrombozym  is  secreted 
by  the  leucocytes  and  the  endothelial  cells,  and  in  opposition  to  Mora- 
witz  is  not  secreted  by  other  cells.  It  is  also  a  normal  constituent  of  the 
blood-plasma  circulating  in  the  living  body.  Most  tissues,  on  the  con- 
trary, contain  no  thrombozym.  The  tissue  extracts,  Nolf  believes, 
also  contain  no  substances  absolutely  necessary  for  the  coagulation, 
but  only  bodies  which  can  have  a  powerful  accelerating  action,  the 
thromboplastic  substances  which  are  mixed  with  the  thrombokinase  of 
Morawitz.  The  circulating  blood-plasma  contains  all  the  bodies  directly 
necessary  in  the  coagulation,  namely,  fibrinogen,  thrombogen,  throm- 
bozym and  lime  salts.  Besides  these  it  also  contains  a  substance  that 
inhibits  coagulation,  antithrombin,  which  is  formed  in  the  liver.  There 
exists,  if  the  author  understands  the  work  of  Nolf,  a  labile  equilibrium 
between  the  various  constituents  of  the  plasma,  and  this  equilibrium  is 
destroyed  in  coagulation.  The  first  impulse  to  coagulation  is  given  by 
the  thromboplastic  substances. 

Nolf  considers  as  thromboplastic  active  any  influence  of  a  physical 
or  chemical  nature  which,  be  it  produced  by  the  walls  of  the  vessel,  a 
suspended  body,  a  solvent  or  a  dissolved  body,  a  colloid  or  crystalloid, 
a  molecule  or  an  ion,  makes  the  combination  of  the  three  above  colloids 
possible.  To  the  thromboplastic  agents  belong  the  walls  of  a  glass 
vessel,  finely  powdered  glass,  the  precipitates  of  calcium  oxalate  or 
calcium  fluoride,  also  living  protoplasm,  aqueous  tissue  extracts,  the 
alcohol  soluble  zymoplastic  substances  of  Alex.  Schmidt,  and  other 
substances.  All  these  agents  in  some  way  or  other  may  serve  as  points 
of  precipitation.  That  a  plasma  free  from  form-elements  coagulates 
for  example  on  contact  with  the  walls  of  a  glass  vessel  depends  upon  the 
fact  that  the  inhibitory  action  of  the  antithrombin  is  retarded  by  the 
thromboplastic  action  of  the  foreign  surface.  Unfortunately  we  are 
ox  1  certain  as  to  how  this  thromboplastic  action  is  brought  about. 

An  important  side  of  Nolf's  theory  of  coagulation  is  also  the  fibrinol- 
ysis whifli  is  brought  about  by  the  thrombin.  The  proteolytic  action 
of  the  thrombin  is  due  only  to  the  thrombozym  contained  therein,  and 


COAGULATION  OF  THE  BLOOD.  323 

it  has  a  proteolytic  action  only  upon  fibrin  and  not  upon  fibrinogen. 
According  to  'Nolf,  coagulation  is  merely  a  preparation  for  the  prote- 
olysis, ;ui<l  is  a  nutrition  phenomenon,  and  in  addition  is  of  special 
importance,  In  arresting  hemorrhage.  In  order  to  prevent  a  rapid 
fibrinolysis,  the  plasma  also  contains  one  or  more  antifibrinolytic  sub- 
stances,  which  are  secreted  by  the  liver. 

What  has  been  given  contains  the  chief  points  in  Nolf's  theory 
of  coagulation,  and  it  is  impossible  in  a  text-book  to  enter  more  into 
detail  in  regard  to  his  remarkable  investigations  or  the  foundations 
on  which  he  bases  his  theory  and  the  objections  which  can  be  raised 
against  it. 

Recently  other  investigators  as  Rettger  and  Howell  have  raised 
objections  to  the  view  that  the  coagulation  of  the  blood  is  an  enzymotic 
process.  Stromberg  1  also  leans  toward  such  a  conception  and  they 
all  raise  the  objection  that  the  quantity  of  fibrin  increases  with  the  quan- 
tity of  thrombin.  This  behavior,  which  has  been  known  for  a  long  time, 
is  of  such  a  complicated  nature,  that  no  positive  conclusions  can  be  drawn 
therefrom. 

The  belief  of  Mellanbt  2  that  the  plasma  originally  only  contains  one 
globulin,  fibrinogen,  from  which  by  enzymotic  cleavage  the  fibrin  and  serglobulin 
are  formed,  is  untenable  and  is  based  upon  the  imperfect  methods  of  preparing 
fibrinogen  that  he  used. 

From  the  above  description  of  the  various  theories  of  coagulation 
it  at  least  follows  that  in  the  study  of  the  coagulation  of  the  blood  there 
are  many  contradictor}'  statements  and  observations,  and  so  many  obscure 
points,  that  for  the  present  it  is  impossible  to  give  a  clear,  comprehensive 
summary  of  the  different  views  and  to  deduce  a  theory  of  the  process 
of  coagulation  which  would  embrace  all  the  factors.  In  spite  of  this 
confusion  and  all  contradictions,  still  we  are  sure  that  certain  bodies  such 
as  fibrinogen  and  thrombin,  even  though  this  latter  be  an  enzyme  or  a 
colloid  combination,  are  directly  concerned  in  the  formation  of  fibrin, 
while  other  bodies  act  indirectly  as  accelerators  or  inhibitors  of  coagulation. 

The  bodies  accelerating  coagulation,  with  the  exception  of  gelatin, 
whose  action  in  this  regard  has  not  been  positively  proved,  have  been 
mentioned  several  times  above.  The  mode  of  action  of  the  bodies  retard- 
ing coagulation  is  not  clear  and  is  much  disputed.  Their  action  may, 
it  seems,  also  be  more  of  a  direct  or  indirect  kind.  Thus,  for  example, 
the  oxalate  and  fluoride  may  prevent  the  formation  of  thrombin  by 
precipitation  of  the  lime.     The  cobra-poison  seems  to  prevent  the  forma- 

1  Rettger,  Amer.  Journ.  of  Physiol.,  24;  Howell,  ibid.,  26;  Stromberg,  Bioch. 
Zeitschr.,  37. 

2  Journ.  of  Phvsiol.,  38. 


324  THE  BLOOD. 

tion  of  thrombin  by  the  action  upon  the  thrombokinase ;  the  hirudin  l 
may,  it  is  generally  believed,  as  antithrombin  make  the  thrombin  inactive, 
and  the  normal  constituents  of  the  plasma  retarding  coagulation  perhaps 
act  in  a  similar  manner.  In  other  cases  the  retarding  bodies  act  indirectly, 
for  they  may,  like  the  proteoses  and  others,  cause  the  body  to  produce 
special  bodies  which  stand  in  close  relation  to  intravascular  coagulation. 

Intravascular  Coagulation.  It  has  been  shown  by  Alex.  Schmidt 
and  his  students,  as  also  by  Wooldridge,  Wright,2  and  others,  that  an 
intravascular  coagulation  may  be  brought  about  by  the  intravenous 
injection  into  the  circulating  blood  of  a  large  quantity  of  a  thrombin 
solution,  as  also  by  the  injection  of  leucocytes  or  tissue  fibrinogen  (impure 
nucleoprotein) .  Intravascular  coagulation  may  also  be  brought  about 
under  other  conditions,  such  as  after  the  injection  of  snake-poison 
(Martin3  and  others)  or  certain  of  the  protein-like  colloid  substances, 
synthetically  prepared  according  to  Grimaux's  method  (Halliburton 
and  Pickering  4).  If  too  little  of  the  above-mentioned  bodies  be  injected, 
then  we  observe  only  a  marked  retarding  tendency  in  the  coagulation 
of  the  blood.  According  to  Wooldridge  it  can  generally  be  maintained 
that  after  a  short  stage  of  accelerated  coagulability,  which  may  lead  to  a 
total  or  partial  intravascular  coagulation,  a  second  stage  of  a  diminished 
or  even  arrested  coagulability  of  the  blood  follows.  The  first  stage  is 
designated  (Wooldridge)  as  the  positive  and  the  other  as  the  negative 
phase  of  coagulation.  These  statements  have  been  confirmed  by  several 
investigators. 

There  is  no  doubt  that  the  positive  phase  is  brought  about  by  an 
abundant  introduction  of  thrombin,  or  by  a  rapid  and  abundant  for- 
mation of  the  same.  The  explanation  of  the  production  of  the  negative 
phase,  which  can  easily  be  brought  about  by  pepsin  proteoses,  by  various 
bodies  such  as  extracts  of  crabs'  muscles  and  other  organs,  eel-serum, 
enzymes,  bacterial  toxines,  certain  snake-poisons,  etc.,  has  been  attempted 
in  different  ways.  The  best  studied  is  the  action  of  proteoses,  but  no 
conclusive  results  have  been  obtained  thus  far.  The  assertion  of  Pick 
and  Spiro  that  the  action  of  the  proteoses  does  not  depend  upon  the 
proteoses  themselves,  but  upon  a  contaminating  substance,  the  protozym, 
is  claimed  to'  be  incorrect  by  Underhill,  while  the  recent  investigations 
of  Popielski  indicate  that  this  is  correct.     The  bodies  retarding  coagu- 


1  The  action  of  hirudin  is  somewhat  doubtful.     See  Schittenhelm  and  Bodong,  1.  e. 

2  A  study  of  the  Intravascular  Coagulation,  etc.,  Proceed,  of  the  Roy.  Irish  Acad. 
(3),  2.  See  also  Wright,  Lecture  on  Tissue  or  Cell  Fibrinogen,  The  Lancet,  1892; 
and  Wooldridge's  Method  of  Producing  Immunity,  etc.,  Brit.  Med.  Journ.,  Sept.,  1891. 

3  Journ.  of  Physiol.,  15. 
*  Uriel,  18. 


RETARDATION  OF  COAGULATION.  325 

lation,  obtained  by  Conradi  l  in  autolysis,  which  are  probably  antithrom- 
bins,  seem  to  act  in  a  different  way  from  the  proteoses,  and  cannot  for 
the  present  be  made  use  of  in  explaining  this  question. 

There  are  a  large  number  of  researches  on  the  action  of  proteoses 
and  of  other  similar  retarding  substances  by  a  great  number  of  different 
investigators,  especially  by  Gley  and  Pachon,  Spiro,  Morawitz,  Nolf, 
Delezenne,  Doyon  and  collaborators.2  We  can  say  with  certainty 
that  the  action  is  indirect,  and  that  the  liver  is  important  for  the  process. 
The  non-coagulability  of  "  peptone-blood  "  seems  to  be  due  to  several 
reasons,  but  it  has  not  been  thoroughly  explained.  On  the  one  hand 
such  blood  contains  an  antithrombin,  and  on  the  other  it  seems  as  if  the 
formation  of  thrombin  is  not  sufficient,  although  the  plasma  contains 
the  necessary  conditions  for  the  thrombin  formation,  as  it  coagulates  as 
a  rule  on  dilution  with  water  or  the  addition  of  a  little  acid.  This  last 
behavior  speaks,  according  to  Mellanby,3  for  the  assumption  that  the 
liver,  because  of  the  proteose  injection,  gives  up  an  excess  of  alkali  to  the 
blood  thus  preventing  the  coagulation  of  the  peptone-blood.  Opinions 
in  regard  to  the  occurrence  of  an  antithrombin  in  the  peptone- 
plasma  seem  to  be  unanimous,  and  we  have  gained  considerable 
experience  in  regard  to  the  formation  of  this  antithrombin.  According 
to  Nolf,  the  peptones  (more  correctly  the  proteoses)  cause  an  altera- 
tion in  the  leucocytes  and  the  walls  of  the  vessels,  and  a  substance  is 
secreted  which  brings  about,  in  the  liver,  the  formation  of  antithrombin. 
According  to  Delezenne  the  proteoses  bring  about  a  destruction  of 
leucocytes,  and  thereby  a  substance  accelerating  coagulation  and  another 
having  a  retarding  action  is  set  free.  The  first  is  destroyed  by  the  liver, 
and  hence  the  action  of  the  retarding  substance  (the  antithrombin)  is 
obtained.  Doyon  and  co-workers  have  also  shown  that  the  isolated 
washed  liver  on  transfusing  normal  arterial  blood,  gives  off  a  thermo- 
stable antithrombin,  which  behaves  like  a  nucleoprotein.  That  the  liver 
takes  part  in  the  retardation  of  coagulation  is  positively  known. 


1  Pick  and  Spiro,  Zeitschr.  f.  physiol.  Chem.,  31;  Underhill,  Amer.  Journ.  of 
Physiol.,  9;  Popielski,  Arch.  f.  expt.  Path.  u.  Pharm.,  Suppl.  1908,  Schmiedeberg's 
Festschrift;  Conradi,  Hofmeister's  Beitrage,  1. 

2  Grosjean,  Travaux  du  laboratoire  de  L.  Fredericq,  4,  Liege,  1892;  Ledoux,  ibid., 
5,  1896;  Nolf,  Bull.  l'Acad.  roy.  de  Belgique,  1902  and  1905,  and  Biochem.  Centralbl., 
3;  and  footnote  1,  p.  318;  Spiro  and  Ellinger,  Zeitschr.  f.  physiol.  Chem.,  23;  Fuld 
and  Spiro,  1.  c;  Morawitz,  1.  c.  The  works  of  the  above-mentioned  French  investi- 
gators can  be  found  in  Compt.  rend.  soc.  biol.,  46,  47,  48,  50,  and  51,  and  Arch.  d. 
Physiol.  (5),  7,  8,  9,  and  10;  see  also  especially  Delezenne,  Arch.  d.  Physiol.  (5),  10; 
Compt.  rend.  soc.  biol.,  51,  and  Compt.  Rend.,  130;  Doyon,  Compt.  rend.  soc.  biol., 
68,  with  Morel  and  Policard,  ibid,  70. 

3  Journ.  of  Physiol.,  38. 


326  THE  BLOOD. 

The  reason  of  the  slow  coagulation  of  the  blood  in  hcemophilia  is  not 
well  known.  Recent  investigations  of  Morawitz  and  Lossen,  Sahli, 
Nolf  and  Henry  x  make  it  very  probable  that  the  thrombokinase  plays 
an  important  part.  According  to  Sahli  the  quantity  of  kinase  is  dimin- 
ished, while  according  to  Nolf  and  Henry,  it  is  qualitatively  changed 
so  that  it  is  less  active.  Both  cases  explain  the  repeatedly  observed 
relation  of  the  vessel-walls  to  haemophilia  as,  according  to  Nolf,  the 
thrombokinase  (his  thrombozym)  is  also  secreted  by  the  endothelial  cells. 

The  non-coagulability  of  cadaver  blood  depends  usually,  according  to  Mora- 
witz,2 upon  the  fact  that  it  contains  no  fibrinogen,  due  to  a  fibrinolysis. 

The  gases  of  the  blood  will  be  treated  in  Chapter  XVI  (on  respiration) 

IV.     THE   QUANTITATIVE   COMPOSITION   OF   THE  BLOOD. 

The  quantitative  analyses  of  the  blood  are  of  little  value.  We  must 
ascertain  on  one  side  the  relation  of  the  plasma  and  blood-corpuscles  to 
each  other,  and  on  the  other  the  constitution  of  each  of  these  two  chief 
constituents.  The  difficulties  which  stand  in  the  way  of  such  a  task, 
especially  in  regard  to  the  living,  non-coagulated  blood,  have  not  been 
removed.  Since  the  constitution  of  the  blood  may  differ  not  only  in 
different  vascular  regions,  but  also  in  the  same  region  under  different 
circumstances,  which  renders  a  number  of  blood  analyses  necessary,  it 
can  hardly  appear  remarkable  that  our  knowledge  of  the  constitution  of 
the  blood  is  still  relatively  limited. 

The  relative  volume  of  blood-corpuscles  and  serum  in  blood  has  been 
determined  by  various  methods.  Of  these  methods  that  of  L.  and  M. 
Bleibtreu,3  against  which  important  objections  have  been  raised  by 
several  investigators,  such  as  Eykman,  Biernacki  and  Hedin,4  must 
be  especially  mentioned.  In  regard  to  this  as  well  as  to  the  method 
of  St.  Bugarsky  and  Tangl,  which  is  based  upon  a  difference  in  the 
electrical  conductivity  of  the  blood  and  the  plasma,  and  Stewart's5 
colorimetric  method,  we  must  refer  to  the  original  publications. 

For  clinical  purposes  the  relative  volume  of  corpuscles  in  the  blood 
may  be  determined  by  the  use  of  a  small  centrifuge  called  a  hcematocrit, 
constructed  by  Blix  and  described  and  tested  by  Hedin.  A  measured 
quantity  of  blood  is  mixed  with  a  known  volume  (best  an  equal  volume) 

1  Morawitz  and  Lossen,  Deutsch.  Arch.  f.  klin.  Med.,  94;  Sahli,  ibid.,  99;  Nolf 
and  Henry,  Revue  de  medicine,  29,  1909. 

2  Hofmeister's  Beitrage,  8. 

3  Pfliiger's  Arch.,  51,  55,  and  60. 

4  Biernacki,  Zeitschr.  f.  physio! .  Chem.,  19;  Eykman,  Pfliiger's  Arch..  60;  Hedin, 
ibid.,  and  Skand.  Arch.  f.  Physiol.,  5. 

6  Bugarsky  and  Tangl,  Centralbl.  f.  Physiol.,  11;  Stewart,  Journ.  of  Physiol.,  24. 


QUANTITATIVE  COMPOSITION  OF  THE  BLOOD.  327 

of  a  fluid  which  prevents  coagulation.  This  mixture  is  introduced  into 
a  tube  and  then  centrifuged.  According  to  Hedin  it  is  best  to  treat  the 
blood,  which  is  kept  fluid  by  1  p.  m.  oxalate,  with  an  equal  volume  of 
a  9  p.  m.  NaCl  solution.  After  complete  centrifugalization,  the  layer  of 
blood-corpuscles  is  read  off  on  the  graduated  tube  and  the  volume  of 
blood-corpuscles  (or  more  correctly  the  layer  of  blood-corpuscles)  in  100 
vols,  of  the  blood  calculated  therefrom.  By  means  of  comparative  counts, 
Hedin  and  Daland  have  found  that  an  approximately  constant  relation 
exists  between  the  volume  of  the  layer  of  blood-corpuscles  and  the  number 
of  red  corpuscles  under  physiological  conditions,  so  that  the  number  of 
corpuscles  may  be  calculated  from  the  volume.  Daland  x  has  shown 
that  such  a  calculation  gives  approximate  results  also  in  disease,  when 
the  size  of  the  blood-corpuscles  does  not  essentially  deviate  from  the 
normal.  In  certain  diseases,  such  as  pernicious  anaemia,  this  method 
gives  such  inaccurate  results  that  it  cannot  be  used. 

Koppe  2  has  showTi  that  in  centrifuging  blood  very  rapidly,  more 
than  5000  turnes  per  minute,  the  blood-corpuscles  may  be  so  completely 
separated  that  all  intermediate  fluid  is  removed.  Because  of  the  absence 
of  this  intermediate  fluid  the  refraction  is  changed;  the  outer  layers  of 
the  erythrocytes  containing  fat  become  transparent,  and  the  column 
of  blood-corpuscles  becomes  transparent  and  laky.  If  the  volume  of 
the  separated  column  of  blood-corpuscles  is  determined  and  the  number 
of  red  blood-corpuscles  counted,  the  absolute  volume  of  these  latter 
can  be  determined  by  this  method. 

In  determining  the  relation  between  the  weight  of  blood-corpuscles  and 
the  weight  of  blood-fluid,  we  generally  proceed  in  the  following  manner: 

If  any  substance  is  found  in  the  blood  which  belongs  exclusively  to 
the  plasma  and  does  not  occur  in  the  blood-corpuscles,  then  the  amount  of 
plasma  contained  in  the  blood  may  be  calculated  if  we  determine  the 
amount  of  this  substance  in  100  parts  of  the  plasma  or  serum  respectively 
on  the  one  side,  and  in  100  parts  of  the  blood  on  the  other.  If  we  repre- 
sent the  amount  of  this  substance  in  the  plasma  by  p  and  that  in  the 
blood  by  b,  then  the  amount  of  x  in  the  plasma  from  100  parts  of  blood  is 

100.6 

x  = . 

P 

Such  a  substance,  which  occurs  only  in  the  plasma,  is  fibrin  according 
to  Hoppe-Seyler,  sodium  according  to  Bunge  (in  certain  kinds  of  blood). 
The  experimenters  just  named  have  tried  to  determine  the  amount  of  the 
plasma  and  blood-corpuscles,  respectively,  in  different  kinds  of  blood, 
starting  from  the  above-mentioned  substances. 

Another  method  suggested  by  Hoppe-Seyler  is  to  determine  the 
total  amount  of  haemoglobin  and  proteins  in  a  portion  of  blood,  and  on 
the  other  hand  the  amount  of  haemoglobin  and  proteins  in  the  blood- 
corpuscles  (from  an  equal  portion  of  the  same  blood)  which  have  been 
sufficiently  washed  with  common-salt  solution  by  centrifugal  force.  The 
figure  obtained,  as  a  difference  between  these  two  determinations,  corre- 
sponds to  the  amount  of  proteins  which  was  contained  in  the   serum  of 

1  Hedin,  Skand.  Arch.  f.  Physiol.,  2,   134  and  361,  and  5;    Pflliger's  Arch.,  t>0; 
Daland,  Fortschritte,  d.  Med.,  9. 

2  Pfluger's  Arch.,  107. 


328 


THE  BLOOD. 


Pig-blood. 


32 


Water l272.20518.36 

46.54 


162.89 

142.20 

8.35 


Solids 
Haemoglobin.  .  .  . 

Protein 

Sugar 

Cholesterin 

Lecithin.  ...... 

Fat 

Fatty  acids .     ... 

Phosphoric  ac!d  \ 

as  nuclein  J 

Soda 

Potash [? 

Iron  oxide J. 696 

Lime. . 

Magnesia.  . .  . 
Chlorine. ... 
Phosphoric   acid 


Inorganic  P2O5 0.7194 


38.26 
0.684 
0.231 
0.805 
1.104 
0.027    ,0.448 

0  0455  0.0123 

'2.401 
0.152   j 

'0.06891 

0.0233 

2.048 

0.1114 

0.0296 


0.213 

1.504 


57 


0.0656 

0.642 

0.8956 


Ox-b'o  >d. 


192.65  616.25 

132.85,58.249 

103.10     — 

20.89  48.901 

—      0.708 


1.100 
1.220 


0.835 
1.129 
0.625 


0.0178.0.0089 

0.7266 
0.2351 
0.544 


2.9084 
0.1719 


0.0056 
0.5901 
0.2392 
0.1140 


0.0805 
0.0300 
2.4889 
0.1646 
0.0571 


Horse-blood.    Dog-blood 


-3   ~f- 


30 


243.86  551.14 

153.84    51.15 

125.8 

20.05 


0.26, 
1.93 

0.02, 
0.05 

1.32 
0.59; 

0.04 
0.18 
0.98, 

0.761 


42.65 
0.90 
0.31 
1.05 
0.50 
0.36 

0.01 
2.62 
0.15 

0.07 
0.03 
2.20 
0.15 
0.05 


o  w*  1 

s 


277.71  514.30 


165.10 

145.6   i 

2.36 

0.56 
1.02 


0.05 

1.27 
0.11 
0.71 

0.03 
0.60 
0.67 
0.54 


Bull-blood. 


42.89 

34.05 
0.74 
0.37 
0.98 
0.91 
0.70 

0.01 

2.39 
0.14 

0.06 
0.03 
2.31 
0.14 
0.05 


206.81  608.03 
127.50    57.66 
106.40      — 
15.38;   46.41 

—  !0.679 
0.610  0.599 
0.953  1.244 

—  12.357 

—  0.494 

0.01940.0089 


0.839 
0.233 
0.562 

0.009 
0.628 
0.236 
0.133 


i2.873 
0.174 

0.073 
0.027 
2.453 
[0.156 
0.041 


Sh?ep-blood. 


200.03  624.16 

118.82    56.63 

102.80     — 

12.80,   46.56 

—      0.708 


1.147 
1.329 


0.891 
1.088 
■      0.859 

—  0.4908 

0.0235  0.0109 

0.760  2.917 

0.236  10.172 

0.545  I     — 

—  !0.089 
0.006  0.027 
0.575  !2.516 
0.228  |0.163 
0.088  10.057 


Goat-blood. 


SUM 

5 


E<N 


Water 

Solids 

Hamoglobin.  .  .  . 

Protein 

Sugar 

Cholesterin 

Lecithin 

Fat 

Fatty  acids 

Phosphoric  acid  \ 
as  nuclein        / 

Soda 

Potash 

Iron  oxide 

Lime 

Magnesia 

Chlorine 

Phosphoric  acid. . 
Inorganic  PiOs. .  . 


211.35 

135.86 

112.50 

18.76 

0.601 
1.339 


0.028 

0.755 
0.236 
0.547 

0.014 
0.514 
0.243 
0.097 


592.54 
60.25 

50.96 
0.822 
0.698 
1.127 
0.0407 
0.398 

0.0117 

2.824 
0.160 

0.078 
0.026 
2.409 
0.154 
0.045 


Cat-blood. 


270.90 
163.11 
143.2 
11.62 

0.556 
1.354 


0.063 

1.174 
0.112 
0.694 

0.035 
0.455 
0.697 
0.515 


-3 

Esi 
3 '-2 


524.17 
41.35 

33.16 
0.860 
0.339 
0.971 
0.446 
0.282 

0.009 

2.512 
0.148 

0.062 
0.024 
2.360 
0.133 
0.040 


Rabbit-blood. 


235.74 

136.37 

123.50 

4.55 

0.268 
1.722 


0.040 

1.946 
0.615 

0.029 
0.460 
0.835 
0.645 


Sr>: 

3?N 


518.18 
46.71 

33.63 
1.036 
0.343 
1.105 
0.749 
0.507 

0.015 

2.789 
0.162 

0.072 
0.028 
2.438 
0.151 
0.040 


Human  Blood, 
Man. 


'a 
B<N 

o  o-< 
o  oO 

s 


349.69 
163.33 


i  Organic 
y  bodies 
I  159.59 


Inorg. 
3.74 

0.24 

1.59 


Eo 


439.02 
47.96 


43.82 


4.14 

1.66 
0.15 


1.72 


Human  Blood, 
Woman. 


o  on 

2 


272.56 
123.68 


120.13 


3.55 

0.65 
1.41 


0.36 


551.99 
51.77 


46.70 


5.07 

-1.92 
0.20 


0.14 


the  first  portion  of  blood.  If  we  now  determine  the  proteins  in  a  special 
portion  of  serum  of  the  same  blood,  then  the  amount  of  serum  in  the 
blood  is  easily  determined.  The  usefulness  of  this  method  has  been 
confirmed  by  Bunge  by  the  control  experiments  with  sodium  determina- 
t  ions.  If  the  amount  of  serum  and  blood-corpuscles  in  the  blood  is  known, 
and  we  then  determine  the  amount  of  the  different  blood-constituents  in 
the  blood-serum  on  one  side  and  of  the  total  blood  on  the  other,  the  dis- 
tribution of  these  different  blood-constituents  in  the  two  chief  components 


SUGAR   IN  THE  BLOOD.  329 

of  the  blood,  plasma  and  blood-corpuscles  may  be  ascertained.  In  the 
table  on  page  328  are  given  analyses  of  the  blood  of  various  animals  by 
Abderhalden  1  according  to  Hoppe-Seyler's  and  Bunge's  methods. 
The  analyses  of  human  blood  by  C.  Schmidt  2  are  older  and  were  made 
according  to  another  method,  hence  the  results  for  the  weights  of  the 
corpuscles  are  perhaps  a  little  too  high.  All  the  results  are  in  parts  per 
1000  parts  of  blood. 

The  relation  between  blood-corpuscles  and  plasma  may  vary  con- 
siderably under  different  circumstances  even  in  the  same  species  of  animal. 
In  animals,  in  most  cases  considerably  more  plasma  is  found,  some- 
times two-thirds  of  the  weight  of  the  blood.3  For  human  blood  Arronet 
has  found  478.8  p.  m.  blood-corpuscles  and  521.2  p.m.  serum  (in  defibrinated 
blood)  as  an  average  of  nine  determinations.  Schneider4  found  349.6 
and  650.4  p.  m.  respectively  in  women. 

The  sugar  was  considered  as  occurring  only  in  the  serum  and  not  with 
the  blood-corpuscles.  According  to  the  investigations  of  Rona  and 
Michaelis  the  blood-corpuscles  of  the  dog  contain  considerable  amounts 
of  sugar;  and  the  quantity  of  sugar  in  the  blood,  in  the  blood-corpuscles 
as  well  as  in  the  plasma,  is  increased  in  man  with  diabetes  mellitus. 
Hollinger  5  also  found  that  in  man,  with  normal  quantity  of  sugar 
in  the  blood,  the  sugar  was  distributed  almost  equally  between  the 
blood-corpuscles  and  the  plasma. 

The  amount  of  sugar  in  the  blood-corpuscles,  which  was  shown  by 
Lepixe  and  Boulud  before  Michaelis  and  Rona,  has  been  the  sub- 
ject of  numerous  investigations  by  Bang  and  his  pupils,  Lyttkens  and 
Sandgren  on  the  one  hand  and  by  Rona,  Michaelis,  Takahashi, 
Frank  and  others  on  the  other  hand  6.  The  results  of  these  investiga- 
tions are  so  contradictory  that  it  is  hardly  possible  for  the  present  to 
draw  any  positive  conclusions.  It  seems  to  follow  from  them,  nevertheless, 
that  the  dog  blood-corpuscles  always  contain  sugar,  while  for  the  corpuscles 
of  the  rabbit  and  man  the  conditions  are  somewhat  doubtful  and  may 
be  variable  (Frank  and  Bretschneider).  According  to  Lyttkens 
and  Sandgren   the  blood-corpuscles  of  man  contain  as  maximum  0.06 

1  Zeitschr.  f.  physiol.  Chem.,  23  and  85. 

1  Cited  and  in  part  recalculated  from  v.  Gorup-Besanez,  Lehrb.  d.  physiol  Chem., 
4.  Aufl.,  345. 

'See  Sacharjin  in  Hoppe-Seyler's  Physiol.  Chem.,  447;  Otto,  Pfliiger's  Arch., 
35;   Bunge.  Zeitschr.  f.  Biol.,  12;   L.  and  M.  Bleibtreu,  Pfliiger's  Arch.,  51. 

4  Arronet,  Maly's  Jahresber.,  17;  Schneider,  Centralbl,  f.  Physiol..  5,  362. 

5  Rona  and  Michaelis,  Bioch.  Zeitschr.  16  and  18;  Hollinger,  ibid..  17. 

6  Lupine  and  Boulud,  Bioch.  Zeitschr.,  32;  Lyttkens  and  Sandgren,  ibid.,  26,  31. 
36:  Rona  with  Doblin,  ibid.,  31,  with  Michaelis,  ibid.,  37,  with  Takahashi,  ibid.,  30; 
Takahashi,  ibid.,  37;  E.  Frank,  Zeitschr.  f.  physiol.  Chem.,  70,  with  Bretschneider, 
ibid.,  71  and  76;  see  also  Oppler,  ibid.,  64  and  75. 


330  THE   BLOOD. 

p.  m.  sugar.  The  blood-corpuscles  of  the  ox,  sheep,  horse,  pig,  cat  and 
guinea-pig  do  not  contain  any  sugar  according  to  these  last-mentioned 
investigators.  On  the  contrary  the  blood-plasma  as  well  as  the  blood- 
corpuscles  contain  a  non-fermentable  reducing  substance.  The  quan- 
tity of  this  in  the  human  blood-corpuscles  is  0.6  p.  m.  according  to 
Lyttkens  and  Sandgren  and  in  the  blood-corpuscles  of  different 
animals  an  average  of  0.44-0.8  p.  m.  calculated  as  glucose.  The  quan- 
tity of  the  non-fermentable  bodies  in  the  blood-plasma  of  the  animals 
investigated  by  them  was  0.3  to  0.5  p.  m. 

The  quantity  of  glucose  in  the  blood  cannot  be  exactly  determined. 
As  the  blood  also  contains  other  reducing  substances  besides  glucose  the 
total  reduction  naturally  cannot  be  used  as  an  exact  value  for  the  glucose 
content;  and  it  must  also  be  added  that  the  different  methods  do  not 
give  uniform  results.  Thus  on  using  the  methods  of  Knapp  and  Bang, 
which  give  the  total  reduction,  higher  values  are  obtained  than  with 
Allihn's  or  Bertrand's  methods,  in  which  the  quantity  of  precipitated 
cuprous  oxide  is  determined.  The  polarization  method  cannot  give 
exact  results  because  of  the  presence  of  other  optically  active  substances 
and  objections  can  also  be  raised  against  the  fermentation  method.1 
On  using  this  last  method  Otto  2  first  observed,  and  was  substantiated 
later  by  others,  namely  Bang  and  his  co-workers,  that  the  blood  contained 
non-fermentable  bodies  which  reduced  Knapp's  (and  also  Bang's)  solu- 
tion. The  remaining  reduction  "rest  reduction"  after  the  fermenta- 
tion cannot  be  detected  according  to  Bertrand's  titration  method. 

The  nature  of  this  reducing  but  not  fermentable  substance  occurring 
in  the  plasma  as  well  as  in  the  blood-corpuscles  is  not  known.  The 
assumption  of  Jacobsen,  Bing,  and  Henri ques3  that  this  question- 
able substance  is  jecorin  or  lecithin  sugar  does  not  have  sufficient  founda- 
tion, and  the  question  of  the  identity  with  jecorin  is  doubtful  and  is  con- 
nected with  the  question  as  to  the  existence  of  jecorin  at  all.  The 
conjugated  glucuronic  acids  have  also  been  considered  and  according 
to  the  investigations  of  Mayer,  Lepine  and  Boulud  4  they  occur  in 
blood  and  originate  in  the  form-elements.  For  these  assumptions  we 
do  not  have  sufficient  support,  and  especially  we  have  no  explanation 


1  In  regard  to  methods  see  Bang,  Der  Blutzucker,  Wiesbaden,  1913  which  also 
describes  a  new  method  suggested  by  him  for  the  determination  of  sugar  in  very  small 
amounts  of  blood. 

2  Pfluger's  Arch.,  35. 

3  Jacobson,  Centralbl.  f.  physiol.  6;  Bing,  Skand.  Arch.  f.  physiol.,  9;  Henriques, 
Zeitechr.  f.  physiol  Chem.,  23.     See  also  P.  Mayer,  Bioch.  Zeitschr.,  1  and  4. 

4  Mayer,  Zeitschr.  f.  physiol.  Chem.,  32;  Lepine  and  Boulud,  Compt.  Rend.,  133, 
135,  136,  138,  141  and  Journ.  de  Physiol.,  7  (cited  from  Bioch.  Centralbl.,  4,  page  421). 


SUGAR  IN  THE  BLOOD.  331 

for  the  total  rest  reduction.  Frank  and  Bretschneider  l  have,  never- 
theless, shown  that  the  reducing  substance  or  mixture  that  occurs  in  the 
blood-corpuscles,  and  which  does  not  reduce  Bertrand's  solution,  but 
does  reduce  Bang's  solution,  yields  a  reduceable  sugar  on  boiling  with  acid 
which  now  reduces  Bertrand's  solution.  The  corresponding  substance 
in  the  blood-plasma  has  a  similar  behavior.  If,  as  in  the  experiments 
of  Frank  and  Bretschneider,  the  extent  of  reduction  after  acid  hydrol- 
ysis is  about  the  same  as  the  original  substance  (titrated  according  to 
Bang)  we  cannot  here  be  dealing  with  dextrins  and  the  nature  of  this 
body  in  question  (or  mixture)  is  quite  unknown. 

In  close  relation  to  what  has  been  given  above  is  the  question  of 
"  sucre  immediat  "  and  the  "  sucre  virtuel  "  of  Lupine  and  Boulud.2 
They  designate  as  "  sucre  immediat  "  the  reduction,  calculated  as 
sugar,  of  the  blood  immediately  after  leaving  the  blood  vessels  and  as 
"  sucre  virtuel  "  the  increase  in  the  reducing  power  brought  on  in  part 
by  allowing  the  blood  to  stand  after  leaving  the  body,  in  part  by  the 
action  of  invertase  or  emulsin  at  39°  C.  and  in  part  by  boiling  with  hydro- 
fluoric acid.  The  quantity  of  "sucre  virtuel "  in  clogs  amounts  to  an  average 
of  70  per  cent  of  the  "sucre  immediat."  The  nature  of  the  "sucre  virtuel" 
is  not  well  known ;  from  what  was  said  above  we  are  probably  dealing  here 
to  all  appearances  with  very  different  bodies. 

From  what  has  been  presented  above  it  can  be  understood  why  the 
exact  sugar  content  of  the  blood  is  not  known.  In  consideration  of  the 
above  mentioned  difficulties  and  sources  cf  error  attempts  have  been  made 
to  determine  the  sugar  content  of  the  blood  and  we  will  give  the  results 
of  some  of  these. 

The  quantity  of  actual  sugar  in  the  blood,  amounts  according  to  Lytt- 
kens  and  Sandgren,  in  man  to  0.63,  in  sheep  0.64,  pig  0.82,  ox  0.86, 
horse  0.98,  rabbit  2.22,  guinea-pig  2.48  and  in  the  cat  2.91  p.  m.  Small 
animals  with  an  active  metabolism  contain  more  sugar  in  the  blood 
than  larger  animals.  According  to  Frank  the  amount  of  sugar  in  the 
blood-plasma  of  man  lies  between  0.8  and  1.1  p.  m.  and  according  to 
Frank  and  Cobliner3  it  is  1.19-1.26  p.  m.  in  new-born. 

The  amount  of  blood  sugar  seems  to  be  almost  independent  of  the 
character  of  the  food.  After  feeding  with  large  amounts  of  sugar  or  dex- 
trin, Bleile,  nevertheless,  has  observed  a  considerable  increase  in  the 
sugar.  The  amount  of  sugar  is  not  only  somewhat  different  with 
various  animals  but  it  also  varies  with  the  same  animal  under  different 


1  Zeitschr.  f.  physiol.  Chem.,  71  and  76. 

2  Compt.  Rend.,  137,  144,  147,  and  Journ  de  Physiol,  et  d.  Path.,  11  and  13. 

3  Lyttkens  and  Sandgren,  Bioch.  Zeitschr.,  36;    Frank  and  Cobliner,  Zetischr.  f. 
physiol.  Chem.,  70. 


332  THE  BLOOD. 

external  conditions.  When  it  amounts  to  more  than  3  p.  m.,  according 
to  a  statement  of  CI.  Bernard,1  sugar  appears  in  the  urine  and  a  gly- 
cosuria occurs,  a  view  that  has  not  been  substantiated.  On  the  one 
hand  a  glycosuria  may  occur  at  a  lower  sugar  content  in  the  blood  and 
on  the  other  hand  a  glycosuria  may  be  absent  for  a  time  with  a  higher 
sugar  content.  An  increase  in  the  sugar  content  occurs,  as  first  shown 
by  Bernard  and  subsequently  proved  by  others,  after  drawing  blood. 
In  this  case  not  alone  is  the  quantity  of  sugar  increased  but  also  the  other 
reducing  substances.  According  to  certain  investigators  the  quantity 
of  these  latter  is  especially  increased  (Henriques,  N.  Anderson,  Lyttkens 
andSANDGREN,  Lepine  and  Boulud2). 

Bernard3  has  shown  that  the  quantity  of  sugar  in  the  blood 
diminishes  more  or  less  rapidly  on  leaving  the  veins.  Lepine,  associated 
with  Barral,  has  specially  studied  this  decrease  in  the  quantity  of 
sugar,  and  calls  it  glycolysis.  Lepine  and  Barral,  as  well  as  Arthus, 
have  shown  that  this  glycolysis  takes  place  in  the  complete  absence  of 
micro-organisms.  It  seems  to  be  due  to  a  soluble  glycolytic  enzyme  whose 
activity  is  destroyed  by  heating  to  54°  C.  This  enzyme  is  derived, 
according  to  the  above  investigators,  from  the  leucocytes  and,  accord- 
ing to  Arthus  as  well  as  to  Doyon  and  Morel4  it  occurs  only  in  the 
serum  but  not  in  the  plasma.  According  to  Lepine,5  it  has  some  con- 
nection with  the  pancreas.  The  glycolysis  is,  according  to  Rohmann 
and  Spitzer  and  Sieber,  an  oxidation  which  is  produced,  according 
to  the  two  last-mentioned  investigators,  by  an  oxidation  ferment.  Accord- 
ing to  Rona  and  Doblin  it  takes  place  in  an  atmosphere  of  hydrogen, 
which  does  not  speak  for  the  above  view.  The  recent  investigations  of 
Slosse,  of  Embden  and  collaborators  Kraske,  Kondo  and  K.  v.  Noorden  6 


1  Bleile,  Arch.  f.  (Anat.  u.)  Physiol.,  1879;  Bernard,  Lecons  sur  le  diabete. 

2  Henriques,  Zeitschr.  f.  physiol.  Chem.,  23,  N.  Anderson,  Bioch.  Zeitschr.,  12; 
Lyttkens  and  Sandgren,  ibid.,  26;   Lepine  and  Boulud,  Journ.  de  Physiol.,  13. 

8  Lecons  sur  le  diabete,  Paris,  1877. 

4  Arthus,  Arch,  de  Physiol.  (5),  3;  Doyon  and  Morel,  Compt.  rend  soc.  biol.,  55. 

5  In  regard  to  the  numerous  memoirs  of  L6pine  and  Lepine  and  Barral,  see  Lyon 
medical.,  62  and  63  ;  Compt  Rend.  110,  112,  113,  120  and  139;  Lepine,  Le  ferment 
glycolytique  et  la  pathog6nie  du  diabete  (Paris,  1891),  and  Revue  analytique  et 
critique  des  travaux,  etc.,  in  Arch,  de  m6d.  exper.  (Paris,  1892);  Revue  de  m^decine 
1895;  Etat  actuel  de  la  question  de  la  glycolyse,  Semaine  m^dicale,  1911;  Arthus, 
Arch,  de  Physiol  (5),  3,  4;  Nasse  and  Framm,  Pfliiger's  Arch.,  63,  Paderi,  Maly's 
Jahresber.,  26;  see  also  Cremer,  Physiologie  des  Glykogens  in  Ergebnisse  d.  Physiol., 
1,  Abt.  1. 

•  Rohmann  and  Spitzer,  Ber.  d.  d.  chem.  Gesellsch.,  28;  Spitzer,  Pfliiger's  Arch., 
60  and  67;  Sieber,  Zeitschr.  f.  physiol  Chem.,  39  and  44;  Rona  and  Doblin,  Bioch. 
Zeitschr.,  32;  Slosse,  Arch,  internat.  de  Physiol.,  11;  Kraske,  Kondo,  and  v.  Noorden 
Bioch.  Zeitschr.,  45. 


LACTIC  ACID   FORMATION   FROM  SUGAR.  333 

speak  positively  for  the  statement  that  in  glycolysis  a  formation  of  lactic 
acid  from  the  sugar  occurs. 

That  a  formation  of  lactic  acid  from  glucose,  and  indeed  by  means  of 
the  leucocytes,  takes  place  in  glycolysis  was  shown  by  Levene  and  Meyer 
before  Embden  and  collaborators.  On  continuing  these  investigations 
Levene  and  Meyer  found  that  fructose  as  well  as  mannose  and  galactose 
under  the  same  conditions  with  leucocytes,  yield  d-lactic  acid  while  with 
the  investigated  pentoses,  arabinose  and  xylose,  this  is  not  the  case.  Accord- 
ing to  Embden  and  co-workers,  this  formation  of  lactic  acid  takes  place 
probably  with  glyceric  aldehyde,  and  perhaps  also  with  small  amounts 
of  dioxyacetone,  as  intermediary  steps,  and  a  formation  of  lactic  acid  from 
glyceric  aldehyde  (and  dioxyacetone)  can  in  fact,  as  A.  Loeb  and  Gries- 
bach  l  have  shown,  be  brought  about  by  enzymotic  means  by  the  form- 
elements  of  the  blood.  It  seems  as  if  several  enzymes  were  active  in  the 
formation  of  lactic  acid  from  glucose.  According  to  Loeb  those  varieties 
of  blood  which  show  no  glycolysis  with  the  formation  of  lactic  acid,  or 
none  worth  mentioning,  can  form  lactic  acid  from  glyceric  aldehyde 
and  according  to  Griesbach  in  this  last -mentioned  process  an  enzyme 
is  active  which  is  soluble  in  water  and  resistant  toward  the  haemolysis 
of  the  blood  with  water,  while  the  action  of  the  blood  upon  glucose  is 
destroyed  in  the  destruction  of  the  form-elements  by  haemolysis.  In 
regard  to  the  formation  of  lactic  acid  from  methyl  glyoxal  see  page  584. 
According  to  Lepine  and  Boulud  a  double  process  takes  place  in  the 
glycolysis.  On  one  side  the  sugar  is  destroyed  and  on  the  other  side 
a  re-formation  of  sugar  from  the  "sucre  virtuel"  takes  place.  Hereby  the 
actual  glycolysis  may  be  greater  than  the  visible,  and  the  mentioned 
investigators  have  therefore  suggested  a  method  for  determining  the 
extent  of  the  actual  glycolysis.2 

The  quantity  of  urea,  which,  according  to  Schondorff,  is  equally 
divided  between  the  blood-corpuscles  and  the  plasma,  is  greater  on  tak- 
ing food  than  in  starvation  (Grehant  and  Quinquatid,  Schondorff) 
and  varies  between  0.2  and  1.5  p.  m.  In  dogs  Schondorff  found  in 
starvation  a  minimum  of  0.348  p.  m.  and  a  maximum  of  1.529  p.  m.  at 
the  point  of  highest  urea  formation.  Gottlieb  obtained  much  lower 
results  by  another  direct  method,  namely,  in  starvation  0.1-0.2,  and 
after  meat  feeding  0.28-0.56  p.  m.,  Folin  and  Denis  found  0.3-0.77 
p.  m.  in  the  Mood  of  the  cat.     In  man  v.  Jaksch  3  found  0.5-0.6  p.  m. 

1  Levene  and  Meyer,  Journ.  of  biol.  Chem.,  11  and  14;  A.  Loeb,  Bioch.  Zeitschr. 
49  and  50;  Griesbach,  ibid.,  50. 

2  Lepine  and  Boulud,  Journ.  de  Physiol.,  et  de  Path.  gene>ale,  13. 

8  Grehant  et  Quinquaud,  Journ.  de  l'anatomie  et  de  la  physiol.,  20,  and  Compt. 
Rend.,  98;  Schondorff,  Pfliiger's  Arch.,  54  and  63;  Gottlieb,  Arch.  f.  exp.  Path.  u. 
Pharm.,  42;  Folin  and  Denis,  Journ.  of  biol.  Chem.,  11  and  12;  v.  Jaksch,  Leyden- 
Fetschr.,  I,  1901. 


33i  THE  BLOOD. 

urea  in  normal  blood.  The  quantity  of  urea  is  somewhat  increased  in 
fever,  and  in  general  in  augmented  protein  metabolism  the  increased 
urea  formation  is  dependent  upon  this.  A  more  important  increase  in  the 
quantity  of  urea  in  the  blood  occurs  in  a  retarded  elimination  of  urea, 
as  in  cholera,  also  in  cholera  infantum,  and  in  infections  of  the  kidneys 
and  urinary  passages.  After  ligaturing  the  ureters  or  after  extirpation 
of  the  kidneys  of  animals,  an  accumulation  of  urea  takes  place  in  the 
blood. 

v.  Schroder  first  showed  that  the  blood  of  the  shark  was  very  rich 
in  urea,  and  the  quantity  indeed  amounted  to  26  p.  m.  Baglioni  l 
has  recently  shown  that  this  large  quantity  of  urea  is  of  the  greatest 
importance,  as  the  presence  of  urea  in  these  animals  is  a  necessary  life- 
condition  for  the  heart  and  very  probably  for  all  organs  and  tissues. 

The  blood  also  contains  traces  of  ammonia.  According  to  Horodyn- 
ski,  Salaskin,  and  Zaleski,2  the  quantity  in  arterial  dog-blood  was 
0.41  milligram  in  100  grams  of  blood.  According  to  Winterberg,3  the 
blood  from  healthy  persons  contains  on  an  average  0.90  milligram  per 
100  cc.3  The  quantity  of  uric  acid  may  be  0.1  p.  m.  in  bird's 
blood  (v.  Schroder4).  Uric  acid  has  only  recently  been  positively 
detected  under  normal  conditions,  while  it  has  been  found,  earlier, 
in  the  blood  in  gout,  croupous  pneumonia,  and  certain  other  diseased 
conditions.  Folin  and  Denis  5  have  determined  the  uric  acid  in  the 
blood  of  certain  animals  as  well  as  in  man  by  a  colorimetric  method 
suggested  by  Folin.  Normal  human  blood  contains  not  less  than  1 
to  2-2.5  milligrams  uric  acid  per  100  grm.;  in  gout  they  found  5.5 
milligrams  as  maximum.  They  also  determined  the  quantity  of  total 
non-protein  nitrogen  and  urea  nitrogen  in  human  blood.  In  normal 
blood  the  first  was  equal  to  22-26  milligrams  and  the  last  equal  to  11-13 
( =  24-28  urea)  milligrams  in  100  grams  of  blood.  In  disease  great  varia- 
tions were  found.  Lactic  acid  was  first  found  in  human  blood  by 
Solomon  and  then  by  Gaglio,  Berlinerblau,  and  Irisawa.  The 
quantity  of  lactic  acid  may  vary  considerably.  Berlinerblau  found 
0.71  p.  m.  as  maximum,  in  dog's  blood.  Saito  and  Katsuyama  6  found 
on  an  average  0.269  p.  m.  in  hen's  blood,  and  after  carbon-monoxide 
poisoning  the  quantity  increased  to  1.227  p.  m.    Fat  and  fatty  acids  occur 


1  v.  Schroder,  Zeitschr.  f.  physiol.  Chem.,  14;  Baglioni,  Centralbl.  f.  Physiol.,  19. 

2  Zeitschr.  f.  physiol.  Chem.,  35,  which  also  gives  the  older  literature. 

3  Wien.  klin.  Wochenschr.,  1897,  and  Zeitschr.  f.  klin.  Med.,  35. 

4  Lud wig's  Festschrift,  1887. 

6  Journ.  of  biol.  Chem.,  13  and  14. 

8  Irisawa,  Zeitschr.  f.  physiol.  Chem.,  17,  which  also  gives  the  older  literature; 
Saito  and  Katsuyama,  ibid.,  32. 


BLOOD   IN   DIFFERENT  VASCULAR  REGIONS.  335 

perhaps  only  in  the  serum.     The  small  traces  of  bile  acids  occurring  in 
normal  blood,  according  to  Ckoftan,1  are  contained  in  the  leucocytes. 

The  calcium  occurs,  with  the  exception  perhaps  of  the  blood  cor- 
puscles of  the  ox,  only  in  the  plasma  and  the  same  applies  at  least  for 
the  principal  part  of  the  magnesium.  The  division  of  the  alkali  between 
the  blood-ccrpuscles  and  the  plasma  is  very  different,  namely,  the  blood- 
corpuscles  of  the  pig,  horse  and  rabbit  contain  no  sodium,  the  human 
corpuscles  are  richer  in  potassium  and  those  of  the  ox,  sheep,  goat,  dog 
and  cat  are  much  richer  in  sodium  than  potassium.  Chlorine  occurs  in 
greater  abundance  in  the  serum  of  all  animals  than  in  the  blood-corpuscles. 
The  iodine  only  occurs  in  serum,  while  iron  regularly,  almost  without  ex- 
ception occurs  in  the  form-elements,  especially  in  the  erythrocytes.  As  the 
nucleoproteins  contain  iron,  some  iron  occurs  in  the  leucocytes  and  traces 
of  iron  also  occur  in  the  serum.  This  quantity  is  very  small  under  normal 
conditions  while  in  disease  the  relationship  between  the  haemoglobin- 
iron  and  the  other  blood-iron  may,  it  seems,  changes  very  distinctly. 
Manganese  has  also  been  found  in  the  blood,  as  well  as  traces  of  lithium 
copper,  lead,  silver,  and  also  arsenic  in  menstrual  blood.  The  entire 
blood  contains  in  ordinary  cases  770-820  p.  m.  water  with  180-230 
p.  m.  solids,  among  these  173-220  p.  m.  are  organic  and  6-10  p.  m., 
inorganic.  The  organic  consist,  after  substracting  6-12  p.  m.  extractives, 
of  protein  and  haemoglobin.  The  quantity  of  the  latter  in  man  is  130- 
150  p.  m.  In  the  dog,  cat,  pig  and  horse  the  haemoglobin  content  is 
about  the  same;  in  ox,  bull,  sheep,  gcat  and  rabbit  blood  it  is  lower 
(Abderhalden). 

The  Composition  of  the  Blood  in  Different  Vascular  Regions  and  under 

Different  Conditions. 

Arterial  and  Venous  Blood.  The  most  striking  difference  between 
these  two  kinds  of  blood  is  the  variation  in  color  caused  by  their  con- 
taining different  amounts  of  gas  and  different  amounts  of  oxyhaemoglobin 
and  haemoglobin.  The  arterial  blood  is  light  red;  the  venous  blood  is 
dark  red,  dichroic,  greenish  by  transmitted  light  through  thin  layers. 
The  arterial  coagulates  more  quickly  than  the  venous  blood.  The  latter, 
on  account  of  the  transudation  which  takes  place  in  the  capillaries,  was 
formerly  said  to  be  somewhat  poorer  in  water  but  richer  in  blood-cor- 
puscles and  haemoglobin  than  the  arterial  blood;  but  this  is  denied  by 
modern  investigators.     According  to  Krtjger  2  and  his  pupils  the  quan- 


1  Pfliiger's  Arch.,  90. 

2  Zeitschr.  f.  Biologie,  26.     This  also  gives  the  literature  on  the  composition  of 
the  blood  in  different  vascular  regions. 


336  THE  BLOOD. 

tity  of  dry  residue  and  haemoglobin  in  blood  from  the  carotid  artery  and 
from  the  jugular  vein  (in  cats)  is  the  same.  Rohmann  and  Muhsam 
could  not  detect  any  difference  in  the  quantity  of  fat  in  arterial  and  venous 
blood.  The  serum  from  dog's  blood  has,  according  to  Wiener,1  a  rela- 
tively higher  globulin  content  relative  to  the  albumin  in  the  venous  blood 
as  compared  with  the  arterial  blood. 

Blo»d  from  the  Portal  Vein  and  the  Hepatic  Vein.  In  consequence  of 
the  small  quantities  of  bile  and  lymph  formed  relatively  to  the  large 
-quantity  of  blood  circulating  through  the  liver  in  a  given  time,  we  can 
hardly  expect  to  detect  by  chemical  analysis  a  positive  difference  in  the 
composition  between  the  blood  of  the  portal  and  hepatic  veins.  The 
•statements  in  regard  to  such  a  difference  are  in  fact  contradictory.  For 
example,  Drosdoff  found  more  haemoglobin  in  the  hepatic  than  in 
the  portal  vein,  while  Otto  found  less.  Kruger  finds  that  the  quantities 
cf  haemoglobin,  as  well  as  of  the  solids,  in  the  blood  from  the  vessels 
passing  to  and  from  the  liver  are  different,  but  a  constant  relation  can- 
not be  determined.  The  hepatic  vein,  according  to  Doyon  and  collab- 
orators,2 is  richer  in  fibrinogen  than  the  blood  from  the  portal  vein. 
The  disputed  question  as  to  the  varying  quantities  of  sugar  in  the  por- 
tal and  hepatic  veins  will  be  discussed  in  a  following  chapter  (see  Chap- 
ter VII,  on  the  formation  of  sugar  in  the  liver).  After  a  meal  rich  in 
carbohydrates,  the  blood  of  the  portal  vein  not  only  becomes  richer 
in  glucose,  but  may  also  contain  dextrin  and  other  carbohydrates  (v. 
Mering,  Otto3).  The  amount  of  urea  in  the  blood  from  the  hepatic 
vein  is  greater  than  in  other  blood  (Grehant  and  Quinquaud).  In 
portal  blood  Folin  and  Denis  found  about  the  same  amount  of  urea 
as  in  the  carotid  blood.  Like  Horodjnski,  Salaskin  and  Zaleski,4 
they  found  that  the  portal  blood  was  richer  in  ammonia  than  the  carotid 
Hood.  The  largest  amount  of  ammonia  was  always  found  in  the  blood 
from  the  mesentery  vessels  of  the  large  intestine. 

Blood  of  the  Splenic  Vein  is  decidedly  richer  in  leucocytes  than  the 
blood  from  the  splenic  artery.  The  red  blood-corpuscles  of  the  blood 
from  the  splenic  vein  are  smaller  than  the  ordinary,  are  less  flattened,  and 
show  a  greater  resistance  to  water.  The  blood  from  the  splenic  vein  is 
also  claimed  to  be  richer  in  water,  fibrin,  and  protein  than  the  ordinary 
venous  blood.     According  to  v.  Middendorff,  it  is  richer  in  haemoglobin 

1  Rohmann  and  Miihsam,  Pfliiger's  Arch.,  46;  Wiener,  Zeitschr.  f.  physiol.  Chem., 
$2. 

2  See  footnote  2,  page  253. 

1  Drosdoff,  Zeitschr.  f.  physiol.  Chem.,  1;  Otto,  Maly's  Jahresber,  17;  v.  Mering, 
Arch.  f.  (Anat.  u.)  Physiol.,  1877,  214. 

4  Gr6hant  et  Quinquaud,  1.  c.  footnote  3,  page  333;  Folin  and  Denis,  Journ.  of  biol. 
Chem.,  11  and  12;  Horodjnski,  Salaskin  and  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  35. 


BLOOD  OF  THE  TWO  SEXES.  337 

than  arterial  blood.  Kruger  1  and  his  pupils  found  that  the  blood 
from  the  vana  lienalis  is  generally  richer  in  haemoglobin  and  solids  than 
arterial  blood;  still  the  contrary  is  often  found.  The  blood  from  the 
splenic  vein  coagulates  slowly. 

The  Blood  from  the  Veins  of  the  Glands.  The  blood  circulates  with 
greater  rapidity  through  a  gland  during  activity  (secretion)  than  when 
at  rest,  and  the  outflowing  venous  blood  has  therefore  during  activity  a 
lighter  red  color  and  a  greater  amount  of  oxygen.  Because  of  the  secre- 
tion, the  venous  blood  also  becomes  somewhat  poorer  in  water  and  richer 
in  solids. 

The  blood  from  the  Muscular  Veins  shows  an  opposite  behavior,  for 
during  activity  it  is  darker  and  more  venous  in  its  properties  because 
of  the  increased  absorption  of  oxygen  by  the  muscles  and  still  greater 
production  of  carbon  dioxide  than  when  at  rest. 

Menstrual  Blood,  according  to  an  old  belief,  has  not  the  power  of 
coagulating.  This  statement,  is  nevertheless,  false,  and  the  apparent 
uncoagulability  depends  in  part  on  the  retarding  action  of  the  mucous 
membrane  of  the  uterus  upon  coagulation  (Cristea  and  Denk  2)  and  in 
part  on  a  contamination  with  vaginal  mucus,  which  disturbs  the  coagula- 
tion. Menstrual  blood,  according  to  Gautier  and  Bourcet,  contains 
arsenic  and  is  also  richer  in  iodine  than  other  blood  (see  Blood-serum, 
page  269). 

The  Blood  of  the  Two  Sexes.  Women's  blood  coagulates  somewhat 
more  quickly,  has  a  lower  specific  gravity,  a  greater  amount  of  water, 
and  a  smaller  quantity  of  solids  than  the  blood  of  man.  The  amount 
of  blood-corpuscles  and  haemoglobin  is  somewhat  smaller  in  woman's 
blood.  The  amount  of  haemoglobin  is  146  p.  m.  for  man's  blood  and  133 
p.  m.  for  woman's. 

During  pregnancy  Nasse  has  observed  a  decrease  in  the  specific  gravity, 
with  an  increase  in  the  amount  of  water,  until  the  end  of  the  eighth  month. 
From  then  the  specific  gravity  increases,  and  at  delivery  it  is  again 
normal.  The  amount  of  fibrin  is  somewhat  increased  (Becquerel  and 
Rodier,  Nasse).  The  number  of  blood-corpuscles  seems  to  decrease. 
In  regard  to  the  amount  of  haemoglobin  the  statements  are  somewhat 
contradictory.  Cohnstein  found  the  number  of  red  corpuscles  diminished 
in  the  blood  of  pregnant  sheep  as  compared  with  non-pregnant,  but 
the  red  corpuscles  were  larger  and  the  quantity  of  haemoglobin  in  the  blood 
was  greater  in  the  first  case.  Mollenberg  found  in  most  cases  an 
increase  in  the  amount  of  haemoglobin  in  pregnancy  in  the  last  months, 


1  v.  Middendorff,  Centralbl.  f.  Physiol.,  2,  753;  Kriiger,  1.  c. 
-  Cristea  and  Denk,  Maly's  Jahresb.,  40,  181. 


338  THE  BLOOD. 

and  according  to  Hermann  and  Naumann  l  an  increase  in  the  cholesterin 
ester  and  the  neutral  fats  occurs  in  the  blood  during  pregnancy. 

The  Blood  at  Different  Periods  of  Life.  Fetal  and  infant  blood  is 
richer  in  erythrocytes  and  haemoglobin  than  the  blood  of  the  mother. 
In  animals  this  is  true  at  least  for  the  haemoglobin  while  the  number 
of  erythrocytes  in  growing  or  adult  animals  may  be  greater  than  in  new- 
born animals.  The  highest  percentage  of  haemoglobin  in  the  blood  has 
been  observed  by  several  investigators,  such  as  Cohnstein  and  Zuntz, 
Otto,  Winternitz,  Abderhalden,  Schwinge,  and  others,  immediately 
or  very  soon  after  birth  or  at  least  within  the  first  few  days.  In  man 
two  or  three  days  after  birth  the  haemoglobin  reaches  a  maximum  (200- 
210  p.  m.)  which  is  greater  than  at  any  other  period  of  life.  This  is  the 
cause  of  the  great  abundance  of  solids  in  the  blood  of  new-born  infants, 
as  observed  by  several  investigators.  The  quantity  of  haemoglobin  and 
blood-corpuscles  sinks  gradually  from  this  first  maximum  to  a  minimum 
of  about  110  p.  m.  haemoglobin,  which  minimum  appears  in  human  beings 
between  the  fourth  and  eighth  years.  The  quantity  of  haemoglobin  then 
increases  again  until  about  the  twentieth  year,  when  a  second  maximum 
of  137-150  p.  m.  is  reached.  The  haemoglobin  remains  at  this  point 
only  to  about  the  forty-fifth  year,  and  then  gradually  and  slowly 
decreases  (Leichtenstern,  Otto  2) .  According  to  earlier  reports,  the 
blood  at  old  age  is  poorer  in  blood-corpuscles  and  protein  bodies,  but 
richer  in  water  and  salts. 

The  Influence  of  Food  on  the  Blood.  In  complete  starvation  no 
decrease  in  the  amount  of  solid  blood-constituents  is  found  to  take  place 
(Panum  and  others).  The  amount  of  haemoglobin  is  increased  a  little, 
at  least  in  the  early  period  (Subbotin,  Otto,  Hermann  and  Groll, 
Luciani  and  Bufalini),  and  also  the  number  of  red  blood-corpuscles 
increases  (Worm  Muller,  Buntzen3),  which  probably  depends  partly 
on  the  fact  that  the  blood-corpuscles  are  not  so  quickly  transformed  as 
the  serum  and  partly  on  a  greater  concentration  due  to  loss  of  water. 

1  Nasse,  Maly's  Jahresber.,  7;  Becquerel  and  Rodier,  Traitc  de  chim.  pathol., 
Paris,  1854;  Cohnstein,  Pfliiger'  Arch.,  34,  233;  Mollenberg,  Maly's  Jahresber.,  31, 
185.  See  also  Payer,  Arch.  f.  Gynak.,  71;  Herrmann  and  Naumann,  Bioch.  Zeitschr., 
43. 

2  Cohnstein  and  Zuntz,  Pfluger's  Arch.,  34;  Winternitz,  Zeitschr.  f.  physiol.  Chem., 
22;  Leichtenstern,  Untersuch.  liber,  den  Hamoglobingehalt  des  Blutes,  etc.,  Leipzig, 
1878;  Otto,  Maly's  Jahresber.,  15  and  17;  Abderhalden,  Zeitschr.  f.  physiol.  Chem., 
34;  Schwinge,  Pfluger's  Arch.,  73  (literature).  See  also  Fehrsen,  Journ.  of  Physiol., 
30. 

3  Panum,  Virchow's  Arch.,  29;  Subbotin,  Zeitschr.  f.  Biologie,  7;  Otto.  1.  c, 
Worm  Muller,  Transfusion  und  Plethora,  Christiania,  1875;  Buntzen,  see  Maly's 
Jahresber.,  9;  Hermann  and  droll,  Pfluger's  Arch.,  43;  Luciani  and  Bufalini,  Maly's 
Jahresber.,  12. 


INFLUENCE  OF  FOOD  ON  THE  BLOOD.  339 

In  rabbits  and  to  a  less  extent  in  dogs,  Popel  found  that  complete  absti- 
nence had  a  tendency  to  increase  the  specific  gravity  of  the  blood.  The 
amount  of  fat  in  the  blood  may  be  somewhat  increased  in  starvation 
because  the  fat  is  taken  up  from  the  fat  deposits  and  carried  to  the  various 
organs  by  the  blood  (N.  Schulz,  Daddi  '). 

After  a  rich  meal,  or  after  secretion  of  digestive  juices  or  absorption 
of  nutritive  liquids,  the  relative  number  of  blood-corpuscles  may  be 
increased  or  diminished  (Buntzen,  Leichtenstern).  The  number  of 
white  blood-corpuscles  may  be  considerably  increased  after  a  diet  rich 
in  proteins.  After  a  diet  rich  in  fat  the  plasma  becomes,  even  after  a 
short  time,  more  or  less  milky-white,  like  an  emulsion.  According  to 
Just,  in  rabbits,  on  the  contrary,  the  various  food-stuffs  such  as  carbohy- 
drate, fat  and  protein  or  peptone  has  no  influence  on  the  number  of  red 
and  white  corpuscles,  which  he  considers  as  a  proof  for  the  difference 
between  the  digestive  processes  in  carnivora  and  herbivora  (rabbits). 
The  composition  of  the  food  acts  essentially  on  the  amount  of  haemo- 
globin in  the  blood.  Subbotin  has  observed  in  dogs  after  a  one-sided 
feeding  with  food  rich  in  carbohydrates  that  the  amount  of  haemoglobin 
sank,  from  the  physiological  average  of  137.5  p.  m.  to  103.2-93.7  p.  m. 
Tsuboi  2  has  also  shown  in  experiments  on  rabbits  and  dogs  that  with 
an  insufficient  diet  of  bread  and  potatoes,  where  the  body  gave  up  pro- 
tein and  contained  relatively  considerable  carbohydrate,  the  amount 
of  haemoglobin  decreased  and  the  blood  became  richer  in  water.  Accord- 
ing to  Leichtenstern,  a  gradual  increase  in  the  amount  of  haemoglobin 
is  found  to  take  place  in  the  blood  of  human  beings  on  enriching  the  food, 
and  according  to  the  same  investigator  the  blood  of  lean  persons  is  gen- 
erally somewhat  richer  in  haemoglobin  than  blood  from  fat  ones  of  the 
same  age.  The  addition  of  iron  salts  to  the  food  greatly  influences 
the  number  of  blood-corpuscles  and  especially  the  amount  of  haemoglobin 
they  contain.  The  action  of  the  iron  salts  is  obscure.3  There  does  not 
seem  to  be  any  doubt  that  the  iron  contained  in  the  food  in  the  form 
of  organic  compounds  is  active,  but  also  iron  salts  and  therapeutic  iron. 
According  to  Bunge  and  his  pupils  the  iron  preparations  act  indirectly 
only.  They  may  combine  with  the  sulphureted  hydrogen  of  the  intes- 
tinal canal  and  thereby  prevent  the  iron  associated  in  the  food  as  assim- 


1  Popel,  Arch,  des  scienc.  biol.  de  St.  P€tersbourg,  4,  354;  Schulz,  Pfluger's  Arch., 
65;  Daddi,  Maly's  Jahresber.,  30. 

2  Just,  Centralbl.  f.  Physiol.,  23;  Subbotin,  1.  c;   Tsuboi,  Zeitschr.  f.  Biologie,  44. 

3  See  Bunge,  Zeitschr.  f.  physiol.  Chem.,  9;  Hausermann,  ibid.,  23,  where  the 
works  of  Woltering,  Gaule,  Hall,  Hochhaus,  and  Quincke  are  cited  (the  same  work 
contains  a  table  of  the  quantity  of  iron  in  various  foods);  Kunkel,  Pfluger's  Arch., 
61;  Macallum,  Journal  of  Physiol.,  16;  Abderhalden,  Zeitschr.  f.  Biologie,  39. 


340  THE  BLOOD. 

ilal  le  protein  compounds  from  being  eliminated  as  iron  sulphide  (Bunge), 
a  view  which  is  now  generally  discarded. 

An  increase  in  the  number  of  red  corpuscles,  takes  place  after  trans- 
fusion of  blood  of  the  same  species  of  animal.  According  to  the  observa- 
tions of  Panum  and  Worm  Muller,1  the  blood-liquid  is  quickly  eliminated 
and  transformed  in  this  case — the  water  being  eliminated  principally 
by  the  kidneys  and  the  protein  burned  into  urea,  etc. — while  the  blood- 
corpuscles  are  preserved  longer  and  cause  a  "  polycythcemia."  A  relative 
increase  in  the  number  of  red  corpuscles  is  found  after  abundant  transuda- 
tion from  the  blood,  as  in  cholera  and  heart-failure  with  considerable 
congestion.  An  increase  in  the  number  of  red  blood-corpuscles  has 
also  been  observed  under  the  influence  of  diminished  pressure  or  in  high 
altitudes.  Viault  first  called  attention  to  the  fact  that  the  number  of 
red  corpuscles  was  very  great  in  the  blood  of  man  and  animals  living 
in  high  regions.  According  to  him  the  llama  has  about  16  million  blood- 
corpuscles  per  cubic  millimeter.  By  observations  on  himself  and  others, 
as  well  as  on  animals,  Viault  found  the  first  effect  of  sojourning  in  high 
altitudes  was  a  very  considerable  increase  in  the  number  of  red  corpuscles, 
in  his  own  case  5-8  millions.  In  a  young  man  residing  for  four  weeks 
in  2900  meters  altitude,  Lacqueur  observed  an  increase  in  the  erythro- 
cytes as  well  as  the  haemoglobin  in  the  unit  volume  of  the  blood.  The 
maximum,  which  appeared  first  after  15  days,  was  15  per  cent  increase 
for  the  erythrocytes  and  16  per  cent  for  the  haemoglobin.  He  also 
found  that  dogs  from  whom  about  one-half  of  the  blood  was  drawn  required 
at  2900  meters  altitude,  on  an  average  of  16  days  to  replace  the  same, 
while  at  the  normal  level  this  requires  on  an  average  of  27  days  or  in 
round  numbers  an  increase  of  70  per  cent.  Both  observations  show 
a  re-formation  of  blood  under  the  influence  of  high  altitude.  Cohnheim 
and  Weber  2  found  in  23  men  engaged  on  the  Jungfrau  railroad,  who  had 
lived  for  a  long  time  in  high  altitudes  that  the  number  of  erythrocytes 
(5.2-6.3  million)  as  well  as  the  haemoglobin  (generally  87-94  as  com- 
pared to  80  per  cent  as  normal)  was  higher  than  under  normal  conditions, 
and  they  consider  this  as  a  proof  for  the  actual  formation  of  blood- 
corpuscles  in  high  altitudes.  A  similar  increase  of  the  red  blood-cor- 
puscles, as  also  an  increase  in  the  quantity  of  haemoglobin  under  the 
influence  of  diminished  pressure,  has  been  observed  by  many  other  inves- 
tigators, in  human  beings  as  well  as  in  animals.  Investigators  are  not 
united  as  to  how  this  increase  is  brought  about.  The  increase  in  the 
blood-corpuscles  is  not  absolute,  but  is  only  relative,  and  it  is  considered 
by  several  observers  that  there  is  neither  a  new  formation  nor  a  dimin- 

1  Panum,  Yin-how's  Arch.,  29;  Worm  Muller,  1.  c. 
I  :  iquim,  Deutscb.  Arch.  f.  klin.  Med.,  110;  Cohnheim  and  Weber,  ibid.,  110. 


VARIATIONS  IN  THE  NUMBER  OF  RED-CORPUSCLES.  341 

ished  destruction  of  the  blood-corpuscles.  A  relative  increase  may  be 
brought  about  in  different  ways.  For  example,  another  division  of  the 
blood-corpuscles  in  the  vascular  system  has  been  supposed,  whereby 
the  blood-corpuscles  accumulate  in  the  capillaries,  from  which  region 
the  blood  has  been  examined  most  often  (Zuntz).  It  is  also  claimed 
that  a  concentration  of  the  blood  takes  place  by  increased  evaporation 
(Grawitz),  and  finally  an  increase  in  the  blood-corpuscles  has  also  been 
explained  by  assuming  a  contraction  of  the  vascular  system  with  the 
pressing  out  of  plasma  (Bunge,  Abderhalden  1).  In  connection  with 
these  experiments,  it  must  be  remarked  that  several  trustworthy  observa- 
tions show  that  under  the  influence  of  diminished  blood-pressure  an 
actual  increase  in  the  red  blood-corpuscles  takes  place.  These  and 
especially  those  of  Zuntz  and  his  co-workers  have  shown  that  under 
these  conditions  an  increased  activity  occurs  in  the  red  bone-marrow. 
This  question  is  still  not  clear.  Cohnheim  and  collaborators 2  have 
observed  in  man  and  dogs,  that  no  essential  increase  in  the  blood-corpuscles 
and  haemoglobin  occurs  in  high  altitudes  after  12  days.  They  do  not 
dispute  the  action  of  a  continued  residence  in  high  altitudes,  and  they  also 
do  not  dispute  such  an  action  upon  rabbits  and  mice.  The}'  explain 
this  in  these  animals  by  a  concentration  of  the  blood  due  to  a  loss  of  water 
which  is  not  replaced.  In  man  and  dogs  on  the  contrary  the  loss  of 
water  brought  about  by  perspiration  is  immediately  replaced  and  the 
concentration  of  the  blood  prevented  and  the  increase  in  the  number 
of  blood-corpuscles  and  of  haemoglobin  is  not  observed. 

A  decrease  in  the  number  of  red  corpuscles  occurs  in  anaemia  from  differ- 
ent causes.  Every  excessive  hemorrhage  causes  an  acute  anaemia,  or,  more 
correctly,  oligaemia.  Even  during  the  hemorrhage,  the  remaining  blood 
becomes  by  diminished  secretion  and  excretion,  as  also  by  an  abundant 
absorption  of  parenchymous  fluid,  richer  in  water,  somewhat  poorer  in 
proteins,  and  strikingly  poorer  in  red  blood-corpuscles.  The  oligaemia 
soon  passes  into  an  hydraemia.  The  amount  of  protein  then  gradually 
increases  again;  but  the  re-formation  of  the  red  blood-corpuscles  is  slower, 
and  after  the  hydraemia  follows  also  an  oligocythaemia.  After  a  little 
time  the  number  of  blood-corpuscles  rises  to  normal.  Inagaki  3  has 
made  thorough  investigations  on  the  changes  which  the  number,  volume 
and  haemoglobin  content  of  the  erythrocytes  undergo  after  drawing  blood 
as  well  as  during  regeneration.     It  is  impossible  here  to  enter  more  in 

1  The  literature  on  this  subject  may  be  found  in  Abderhalden,  Zeitschr.  f.  Biologie, 
43;  van  Voornveld,  Pfliiger's  Arch.,  92. 

2  Hohenklima  und  Bergwanderungen,  by  N.  Zuntz,  A.  Loewy,  Franz  Muller,  and 
W.  Caspari,  Berlin,  1906;  Otto  Cohnheim,  G.  Kreglinger,  L.  Tobler,  O.  H.  Weber, 
Zeitschr.  f.  physiol.  Chem.,  78  and  Cohnheim,  Ergebn.  d.  Physiologie,  1912,  12. 

3  Zeitschr.  f.  Biol.,  49. 


342  THE  BLOOD. 

detail  as  to  the  results,  but  simply  to  state  that  they  substantiate  the 
previously  known  observation  that,  during  regeneration,  irregularties 
may  occur  in  the  relation  between  the  quantity  of  haemoglobin  and  the 
number  of  erythrocytes.  A  considerable  decrease  in  the  number  of  red 
corpuscles  also  occurs  in  chronic  anaemia  and  chlorosis;  still  in  such  cases 
an  essential  decrease  in  the  amount  of  haemoglobin  occurs  without  an 
essential  decrease  in  the  number  of  blood-corpuscles.  The  decrease  in  the 
amount  of  haemoglobin  is  more  characteristic  of  chlorosis  than  a  decrease 
in  the  number  of  red  corpuscles.  The  opinions  on  the  changes  in  the 
blood  in  anaemia  and  chlorosis  differ  very  considerably.1 

A  very  considerable  decrease  in  the  number  of  red  corpuscles  (300,000- 
400,000  in  1  c.mm.)  and  diminution  in  the  amount  of  haemoglobin 
(|  -jfo)  occurs  in  pernicious  anaemia  (Hayem,  Laache,  and  others2). 
On  the  contrary,  the  individual  red  corpuscles  are  larger  and  richer  in 
haemoglobin  than  they  ordinarily  are,  and  the  number  stands  in  an  inverse 
relation  to  the  amount  of  haemoglobin  (Hayem).  Besides  this  the  red 
corpuscles  often,  but  not  always,  show  in  pernicious  anaemia  remarkable 
and  extraordinary  irregularities  of  form  and  size,  which  has  been  termed 
poikilocytosis. 

The  number  of  leucocytes  may,  as  stated  above,  be  increased  under 
physiological  conditions  as  well  as  after  a  meal  rich  in  protein  (physiological 
leucocytosis).  Under  pathological  conditions  a  high  leucocytosis  may 
occur,  and  this  is  especially  found  in  leucaemia,  which  is  characterized 
by  a  very  great  abundance  of  leucocytes  in  the  blood.  The  number  of 
leucocytes  is  markedly  increased  in  this  disease,  and  indeed,  not  only 
absolutely,  but  also  in  relation  to  the  number  of  red  blood-corpuscles, 
which  are  diminished  to  a  considerable  extent  in  leucaemia.  Leucaemic 
Hood  has  a  lower  specific  gravity  than  the  ordinary  blood  (1035-1040), 
and  a  paler  color,  as  if  it  were  mixed  with  pus.  The  reaction  is  alkaline, 
but  after  death  it  is  frequently  acid,  probably  due  to  a  decomposition 
of  lecithin,  which  is  often  considerably  increased  in  leucaemia.  Volatile 
fatty  acids,  lactic  acid,  glycero-phosphoric  acid,  large  amounts  of  purine 
bases,  and  so-called  Charcot's  crystals  (see  Semen,  Chapter  XII)  have 
also  been  found  in  leucaemic  blood.  The  peptone  (proteose)  which  is 
found  in  the  leucaemic  blood  after  death,  and  which  does  not  exist  in 
the  fresh  blood,  is,  according  to  Erben,3  a  digestive  product  which  is 

1  Complete  analyses  of  chlorotic  blood  may  be  found  in  Erben,  Zeitschr.  f.  klin. 
Med.,  47. 

2  Laache,  Die  Anamie  (Christiania,  1883),  which  also  contains  the  older  liter- 
ature. A  complete  chemical  analysis  of  the  blood  has  been  made  by  Erben,  Zeitschr. 
f.  klin.  Med.,  40. 

Erben,  Zeitschr.  f.  Heilkunde,  24,  and  Hofmeister's  Beitrage,  5.     See  also  Schumm, 
ibid.,  4  and  5.     See  also  footnote  3,  page  342. 


QUANTITY  OF  BLOOD.  343 

produced  by  a  tryptic  enzyme  which  originates  from  the  leucocytes 
as  well  as  by  traces  of  a  peptic  enzyme.  A  chemical  analysis  of  leucrcmic 
blood  has  been  made  by  Erben.1 

A  great  number  of  investigations  have  been  made  on  the  chemical 
composition  of  blood  in  disease.  But  as  we  have  only  a  few  analyses 
of  the  blood  of  healthy  individuals,  and  as  the  possible  variations  under 
physiological  conditions  are  little  known,  it  is  difficult  to  draw  any  pos- 
itive conclusions  from  the  analyses  of  pathological  blood.  Unfortunately, 
on  account  of  the  large  number  of  contradictory  deductions  concern- 
ing the  composition  of  the  blood  of  diseased  human  beings,  it  is  impossible 
to  give  a  brief  summary  of  the  results,  still  the  changes  in  the  blood  in 
disease  must  be  of  the  greatest  importance. 

The  quantity  of  blood  is  indeed  somewhat  variable  in  different  species 
of  animals  and  in  different  conditions  of  the  b©dy;  in  general  we  consider 
the  entire  quantity  of  blood  in  adults  as  about  tV-tV  (;f  the  weight  of  the 
body,  and  in  new-born  infants  about  tV-  Haldane  and  Lorrain  Smith,2 
who  have  determined  the  quantity  of  blood  by  a  new  method,  find  in 
fourteen  persons  that  it  varies  between  -fg-  and  -£$  of  the  weight  of  the  body. 
According  to  the  same  method  Oerum3  has  determined  the  quantity 
of  blood  in  men  as  about  iV  and  in  woman  2V  of  the  weight  of  the  body. 
Fat  individuals  are  relatively  poorer  in  blood  than  lean  ones.  During 
inanition  the  quantity  of  blood  decreases  less  quickly  than  the  weight 
of  the  body  (Panum4),  and  it  may  therefore  be  also  proportionally  greater 
in  starving  individuals  than  in  well-fed  ones. 

By  careful  bleeding,  the  quantity  of  blood  may  be  considerably  dimin- 
ished without  any  dangerous  symptoms.  A  loss  of  blood  amounting 
to  one-fourth  of  the  normal  quantity  has  as  a  sequence  no  lasting  sink- 
ing of  the  blood-pressure  in  the  arteries,  because  the  smaller  arteries 
accommodate  themselves  to  the  small  quantities  of  blood  by  contract- 
ing (Worm  Muller5).  A  loss  of  blood  amounting  to  one-third  of 
the  quantity  reduces  the  blood-pressure  considerably,  and  a  loss  of 
one-half  of  the  blood  in  adults  is  dangerous  to  life.  The  more  rapid  the 
bleeding  the  more  dangerous  it  is.  New-born  infants  are  very  sensitive 
to  loss  of  blood,  and  likewise  fat,  old,  and  weak  persons  cannot  stand 
much  loss  of  blood.  Women  can  stand  loss  of  blood  better  than  men. 
The  quantity  of  blood  may  be  considerably  increased  by  the  injection 
of  blood  from  the  same  species  of  animal   (Panum,   Landois,   Worm 


1  Zeitschr.  f.  klin.  Med.,  66  (1908). 

2  Journ.  of  Physiol.,  25. 

3  Deutsch.  Arch.  f.  klin.  Med.,  93  (1908). 

4  Yirchow's  Arch.,  29. 

5  Transfusion  und  Plethora,  Christiania,  1875. 


344  THE  BLOOD. 

Muller,  Ponfick).  According  to  Worm  Muller  the  normal  quantity 
of  blood  may  indeed  be  increased  as  much  as  83  per  cent  without  pro- 
ducing any  abnormal  conditions  or  lasting  high  blood-pressure.  An 
increase  of  150  per  cent  in  the  quantity  of  blood  may,  with  a  considerable 
variation  in  the  blood-pressure,  be  directly  dangerous  to  life  (Worm 
Muller).  If  the  quantity  of  blood  of  an  animal  is  increased  by  trans- 
fusion with  blood  of  the  same  kind  of  animal,  an  abundant  formation 
of  lymph  takes  place.  The  water  in  excess  is  eliminated  by  the  urine; 
and  as  the  protein  of  the  blood-serum  is  quickly  decomposed,  while  the 
red  blood-corpuscles  are  destroyed  much  more  slowly  (Tschirjew,  Fors- 
ter,  Panum,  Worm  Muller1),  a  polycythemia  is  gradually  produced. 
The  quantity  of  blood  in  the  different  organs  depends  essentially  on 
their  activity.  During  work  the  exchange  of  material  in  an  organ  is 
more  pronounced  than  during  rest,  and  the  increased  metabolism  is 
connected  with  a  more  abundant  flow  of  blood.  Although  the  total 
quantity  of  blood  in  the  body  remains  constant,  the  distribution  of  the 
blood  in  the  various  organs  may  be  different  at  different  times.  As  a 
rule  the  quantity  of  blood  in  an  organ  is  an  approximate  measure  of  the 
more  or  less  active  metabolism  going  on  in  it,  and  from  this  point  of 
view  the  distribution  of  the  blood  in  the  different  organs  is  of  interest. 
According  to  Ranke,2  to  whom  we  are  especially  indebted  for  our  knowl- 
edge of  the  relation  of  the  activity  of  the  organs  to  the  quantity  of  blood 
contained  therein,  of  the  total  quantity  of  blood  (in  the  rabbit)  about 
one-fourth  comes  to  the  muscles  in  rest,  one-fourth  to  the  heart  and  the 
large  blood-vessels,  one-fourth  to  the  liver,  and  one-fourth  to  the  other 
organs. 

1  Panum,  Xord.  med.  Ark.,  7;  Virchow's  Arch.,  63;  Landois,  Centralbl.  f.  d. 
med.  Wissensch.,  1875,  and  Die  Transfusion  des  Blutes,  Leipzig,  1875;  Worm  Muller, 
Transfusion  und  Plethora;  Ponfick,  Virchow's  Arch.,  62;  Tschirjew,  Arbeiten  aus 
der  physiol.  Anstalt  zu  Leipzig,  1874,  292;  Forster,  Zeitschr.  f.  Biologie,  11;  Panum, 
Virchow's  Arch.,  29. 

2  Die  Blutvertheilung  und  der  Thatigkeitswechsel  des  Organe,  Leipzig,  1871. 


CHAPTER  VI. 
CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

I.     CHYLE  AND   LYMPH. 

The  lymph  is  at  least  in  part  the  mediator  in  the  exchange  of  con- 
stituents between  the  blood  and  the  tissues.  The  bodies  necessary  for 
the  nutrition  of  the  tissues  pass  from  the  blood  into  the  lymph,  and  the 
tissues  deliver  water,  salts,  and  products  of  metabolism  to  the  lymph. 
The  lymph,  therefore,  originates  partly  from  the  blood  and  partly  from 
the  tissues.  From  a  purely  theoretical  standpoint  one  can,  according 
to  Heidehain,  differentiate  between  blood-lymph  and  tissue-lymph 
according  to  origin.  It  is  impossible  at  the  present  time  to  separate 
completely  that  which  comes  from  the  one  or  the  other  source. 

The  lymph  formed  in  the  different  organs  and  tissues  has  a  different 
composition,  and  as  the  lymph  is  not  obtained  directly  but  only  from  the 
large  lymph  vessels,  hence  the  lymph  that  we  use  for  investiga- 
tions is  generally  a  mixture,  whose  composition  may  vary  under  certain 
conditions.  The  most  easily  obtained  and  best  studied  is  the  lymph 
from  the  thoracic  duct.  In  starving  individuals  this  lymph,  which  is 
called  starvation  lymph,  does  not  essentially  differ  from  other  lymphs. 
After  fatty  food  the  lymph,  which  is  called  digestion  lymph  or  chyle, 
differs  from  other  lymphs  by  its  great  richness  in  very  finely  divided  fat, 
which  gives  it  a  milky  appearance,  and  which  has  led  to  the  old  name 
"  lacteal  fluid." 

Chemically  the  lymph  is  the  same  as  plasma,  and  contains,  at  least 
to  a  great  extent,  the  same  bodies.  The  observation  of  Asher  and  Bar- 
bera,1  that  the  lymph  contains  poisonous  metabolic  products,  does 
not  contradict  such  an  assumption,  as  no  doubt  these  products  are  trans- 
ferred to  the  blood  with  the  lymph.  Although  the  blood  does  not  show 
the  same  poisonous  action  as  the  lymph,  still  this  can  be  explained  by  the 
great  dilution  these  bodies  undergo  in  the  blood,  and  the  difference 
between  blood-plasma  and  lymph  is  no  doubt  of  a  quantitative  nature. 
This  difference  consists  chiefly  in  that  the  lymph  is  poorer  in  proteins. 

1  Zeitschr.  f .  Biologie  36. 

345 


346  CHYLE,   LYMPH,  TRANSUDATES  AND  EXUDATES. 

Lymph,  like  the  plasma,  contains  seralbumin,  serglobulins,  fibrinogen, 
and  fibrin  ferment.  The  two  last-mentioned  bodies  occur  only  in  very 
small  amounts;  therefore  the  lymph  coagulates  slowly  (but  spontaneously) 
and  yields  but  little  fibrin.  Like  other  liquids  poor  in  fibrin  ferment, 
lymph  does  not  at  once  coagulate  completely,  but  repeated  coagula- 
tions take  place. 

The  extractive  bodies  seem  to  be  the  same  as  in  plasma.  Sugar  (or 
at  least  a  reducing  substance)  is  found  in  about  the  same  quantity  as  in 
the  blood-serum,  namely,  about  1  p.  m.  The  glycogen  detected  by  Dastre  * 
in  the  lymph  occurs  only  in  the  leucocytes.  According  to  Rohmann  and 
Bial,  lymph  contains  a  diastatic  enzyme  similar  to  that  in  blood-plasma, 
and  Lepine2  found  that  the  chyle  of  a  dog  during  digestion  has  great  gly- 
colytic activity.  Lipases  may  also  occur  in  lymph.  The  amount  of  urea 
has  been  determined  by  Wurtz3  as  0.12-0.28  p.  m.  The  mineral  bodies 
appear  to  be  the  same  as  in  plasma. 

As  form-elements,  leucocytes  and  in  certain  cases  red  blood-corpuscles 
are  common  to  both  chyle  and  lymph.  Chyle  in  fasting  animals  has  the 
appearance  of  lymph.  After  fatty  food  it  is,  on  the  contrary,  milky, 
due  partly  to  small  fat-globules,  as  in  milk,  and  partly,  indeed,  mostly 
to  finely  divided  fat.  The  nature  of  the  fat  occurring  in  chyle  depends 
upon  the  kind  of  fat  in  the  food.  By  far  the  greater  part  consists  of 
neutral  fat,  and  even  after  feeding  with  large  quantities  of  free  fatty 
acids,  Munk4  found  that  the  chyle  contained  chiefly  neutral  fat  with 
only  small  amounts  of  fatty  acids  or  soaps. 

The  gases  of  the  entirely  normal  human  lymph  have  not  thus  far  been 
investigated.  The  gases  from  dog-lymph  contain,  according  to  Hammar- 
sten,  only  traces  of  oxygen,  and  consist  of  37.4-53.1  per  cent  CO2  and 
1.6  per  cent  N,  calculated  at  0°  C,  and  760  mm.  mercury.  The  chief  mass 
of  the  carbon  dioxide  of  the  lymph  seems  to  be  in  firm  chemical  com- 
bination. Comparative  analyses  of  blood  and  lymph  have  shown  that  the 
lymph  contains  more  carbon  dioxide  than  arterial,  but  less  than  venous 
blood.  The  tension  of  the  carbon  dioxide  of  lymph  is,  according  to 
Pfluger  and  Strassburg,5  smaller  than  in  venous,  but  greater  than  in 
arterial,  blood. 

The  quantitative  composition  of  the  chyle  must  evidently  be  very 
variable.     The  specific  gravity  varies  between  1.007   and  1.043.     As  an 

1  Compt.  rend,  de  soc.  biol.,  47,  and  Compt.  Rend.,  120;  Arch,  de  Physiol.  (5),  7. 

2  Rohmann  and  Bial,  Pfluger's  Arch.,  52,  53,  and  55;  Lepine,  Compt.  Rend.,  110. 

3  Compt,  Rend.,  49. 

*  Virchow's  Arch.,  80  and  123.  In  regard  to  the  analysis  of  the  fat  of  chyle,  see 
Erben,  Zeitschr.  f.  physiol.  Chem.,  30. 

5  Hammarsten,  Die  Gase  der  Hundelymphe,  Arbeiten  aus  d.  physiol.  Anstalt  zu 
Leipzig,  1871 ;  Strassburg,  Pfluger's  Archiv,  6. 


CHYLE  AND  LYMPH.  347 

example  of  the  composition  of  human  chyle  two  analyses  will  be  given. 
The  first  is  by  Owen-Rees,  of  the  chyle  of  an  executed  person,  and  the 
second  by  Hoppe-Seyler,1  of  the  chyle  in  a  case  of  rupture  of  the  thoracic 
duct.  In  the  latter  case  the  fibrin  had  previously  separated.  The  results 
are  in  parts  per  1000. 

No.  1.  No.  2. 

Water 904.8  940.72  water 

Solids 95.2  59.28  solids 

Fibrin Traces  

Albumin 70.8  36.67  albumin 

Fat 9.2  7.23  fat 


Remaining  organic  bodies.  . .     10.8 
Salts 4.4 


2 .  35  soaps 
0.83  lecithin 

1 .  32  cholesterin 

3 .  63  alcohol  extractives 
0 .  58  water  extractives 

J  6.80  soluble  salts 
i  0 .  35  insoluble  salts 


The  quantity  of  fat  is  very  variable  and  may  be  considerably  increased 
by  partaking  of  food  rich  in  fats.  I.  Munk  and  A.  Rosenstein  2  have 
investigated  the  lymph  or  chyle  obtained  from  a  lymph  fistula  at  the 
end  of  the  upper  third  of  the  leg  of  a  girl  eighteen  years  old  and  weigh- 
ing 60  kg.,  and  the  highest  quantity  of  fat  in  the  chylous  lymph  was  47 
p.  m.  after  partaking  of  fat.  In  the  starvation  lymph  from  the  same 
patient  they  found  only  0.6-2.6  p.  m.  fat.  The  quantity  of  soaps  was 
always  small,  and  on  partaking  of  41  grams  of  fat  the  quantity  of  soaps  was 
only  about  2V  of  the  neutral  fats.  Schumm  3  found  in  the  creamy 
contents  of  a  chylous  cyst  of  the  mesentery,  357.8  p.  m.  fat  and  compara- 
tively large  amounts  of  calcium  soaps. 

A  great  many  analyses  of  chyle  from  animals  have  been  made,  and 
they  chiefly  show  the  fact  that  the  chyle  is  a  liquid  with  a  very  changeable 
composition  which  stands  closely  related  to  blood-plasma,  but  with  the 
principal  difference  that  it  contains  more  fat  and  less  solids.  The  reader 
is  referred  to  special  works  for  these  analyses,  as,  for  example,  to  v.  Gorup- 
Besanez's  "  Lehrbuch  der  physiologischen  Chemie,"  4th  edition. 

The  composition  of  the  lymph  is  also  very  changeable,  and  its  specific 
gravity  shows  about  the  same  variation  as  the  chyle.  In  the  following 
analyses,  1  and  2,  made  by  Gubler  and  Quevenne,  are  the  results 
obtained  from  lymph  of  the  upper  part  of  the  thigh  of  a  woman  aged 
thirty-nine;    and  3,  made  by  v.  Scherer,  is  an  analysis  of  lymph  from 


'Owen-Rees,  cited  from  Hoppe-Seyler's  Physiol.  Chem.,  595;  Hoppe-Seyler, 
ibid.,  597.  See  also  Carlier,  Brit.  Med.  Journ.,  1902,  175,  and  T.  Sollmann,  Amer. 
Journ.  of  Physiol.,  17. 

•  Yirchow's  Arch.,  123. 

3  Zeitschr.  f.  physiol.  Chem.,  49. 


348  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

the  sac-like  dilated  lymphatic  vessels  of  the  spermatic  cord.  No.  4 
was  made  by  C.  Schmidt  1  the  data  being  obtained  from  lymph  from  the 
neck  of  a  colt.    The  results  are  expressed  in  parts  per  1000. 

12  3  4 

Water 939.9  934.8  957.6  955.4 

Solids 60.1  65.2  42.4  44.6 

Fibrin 0.5  0.6'  0.4  2.2 

Albumin 42.7  42.8  34.71       

Fat,  cholesterin,  lecithin 3.8  9.2         Y  35.0 

Extractive  bodies 5.7  4.4         J       

Salts 7.3  8.2  7.2  7.5 

The  salts  found  by  C.  Schmidt  in  the  lymph  of  the  horse  have  the 
following  composition,  calculated  in  parts  per  1000  parts  of  the  lymph: 

Sodium  chloride 5 .  67 

Soda 1.27 

Potash 0. 16 

Sulphuric  acid 0 .  09 

Phosphoric  acid  united  with  alkalies 0 .  02 

Earthy  phosphates 0 .  26 

In  the  cases  investigated  by  Munk  and  Rosenstein  the  quantity  of 
solids  in  the  fasting  condition  varied  between  35.7  and  57.2  p.  m.  This 
variation  was  essentially  dependent  upon  the  extent  of  secretion,  so  that 
the  low  amount  coincides  with  a  more  active  secretion,  and  the  reverse 
in  the  other  case.  The  chief  portion  of  the  solids  consisted  of  proteins, 
and  the  relation  between  globulin  and  albumin  was  as  1 :2.4  to  4.  The 
mineral  bodies  in  1000  parts  lymph  (chylous)  were:  NaCl  5.83;  Na2CC>3 
2.17;  K2HPO40.28;  Ca3(P04)2  0.28;  Mg3(P04)2  0.09;  and  Fe(PO4)0.025. 
The  quantity  of  titratable  alkali  in  the  lymph  is  much  smaller  than  in 
the  blood.  Carlson,  Greer  and  Luckhardt2  have  recently  made  com- 
parative estimations  of  NaCl  in  blood-serum  and  lymph  of  the  same 
individual  (horse  and  dog)  and  find  that  the  lymph  is  regularly  richer  in 
chlorides,  a  condition  which,  according  to  them,  is  difficult  to  reconcile 
with  the  view  of  the  filtration  and  transudation  processes  in  the  forma- 
tion of  lymph. 

In  this  connection  it  must  be  recalled  that  according  to  many  inves- 
tigators the  lymph  has  a  somewhat  higher  osmotic  pressure  and  therefore 
a  somewhat  greater  molecular  concentration  than  the  serum.  Carlson, 
Greer  and  Becht3  found,  nevertheless,  that  the  osmotic  pressure  of 
neck-lymph  in  the  dog  is  often  lower  than  the  serum. 

1  Gubler  and  Quevenne,  cited  from  Hoppe-Seyler's  Physiol.  Chem.,  591;  v.  Scherer, 
ibid.,  591;  C.  Schmidt,  ibid.,  592.  See  also  Zaribnicky,  Zeitschr.  f.  physiol.  Chem., 
78. 

2  Amer.  Journ.  of  Physiol.,  22  (1908). 
'  Ibid.,  19  (1907). 


CHYLE  AND  LYMPH.  340 

Under  special  conditions  the  lymph  may  be  so  rich  in  finely  divided  fat  that 
it  appears  like  chyle.  Such  lymph  has  been  investigated  by  Hensen  in  a  case  of 
lymph  fistula  in  a  ten-year-old  boy,  and  by  Lang  l  in  a  case  of  lymph  fistula  in 
the  upper  part  of  the  left  thigh  of  a  girl  of  seventeen.  The  lymph  investigated 
by  Hensen  varied  in  the  quantity  of  fat,  as  an  average  of  nineteen  analyses, 
between  2.8  and  36.9  p.  m.;  while  that  investigated  by  Lang  contained  24.85 
p.  m.  of  fat. 

The  quantity  of  lymph  secreted  must  naturally  change  considerably 
under  various  conditions,  and  there  are  no  means  of  measuring  it.  The 
size  of  the  flow  of  lymph  is,  as  Heidenhain  suggests,  no  measure  of  the 
abundance  of  supply  of  nutritive  material  to  the  organs,  and  the  lymph- 
tubes  act  according  to  him  as  "  drain-tubes,"  removing  the  excess  of 
fluid  from  the  lymph  fissures  as  soon  as  the  pressure  therein  rises  to  a 
certain  height.  Attempts  have  been  made  to  determine  the  quantity 
of  lymph  flowing  in  24  hours  through  the  thoracic  duct  of  animals.  Ac- 
cording to  Heidenhain  the  quantity  averages  640  cc.  for  a  dog  weighing 
10  kilos. 

Detei-minations  of  the  quantity  of  lymph  in  man  have  also  been 
attempted.  Noel-Paton  2  obtained  1  cc.  of  lymph  per  minute  from  the 
severed  thoracic  duct  of  a  patient  weighing  60  kilos.  The  quantity  in 
the  24  hours  cannot  be  calculated  from  this  amount.  In  the  case  of 
Munk  and  Kosenstein,  1134-1372  grams  of  chyle  were  collected  within 
12-13  hours  after  partaking  of  food.  In  the  fasting  condition  or  after 
starving  for  18  hours  they  found  50  to  70  grams  per  hour,  sometimes  120 
grams  and  above,  especially  in  the  first  few  hours  after  powerful  muscular 
exercise. 

Several  circumstances  have  a  marked  influence  on  the  extent  of  lymph 
secretion.  During  starvation  less  lymph  is  secreted  than  after  partak- 
ing of  food.  Nasse  3  has  observed  that  the  formation  of  lymph  in  dogs 
is  increased  36  per  cent  more  after  feeding  with  meat  than  after  feeding 
with  potatoes,  and  about  54  per  cent  more  than  after  24  hours'  depriva- 
tion of  food.  In  this  connection  mention  must  be  made  of  the  important 
observations  of  Asher  and  Barbera4  that  with  pure  protein  diet  the 
lymph  current  is  increased  in  the  thoracic  cavity,  and  also  that  the  increase 
in  the  lymph  secretion  runs  parallel  with  the  elimination  of  nitrogen  in 
the  urine,  i.e.,  with  the  absorption  of  the  protein  from  the  digestive  tract. 

An  increase  in  the  total  quantity  of  blood,  as  by  transfusion  of  blood, 
also  especially  in  preventing  the  flow  of  blood  by  means  of  ligatures, 


1  Hensen,  Pfliiger's  Arch.,  110;  Lang,  see  Maly's  Jahresber.,  4. 

2  Journ.  of  Physiol.,  11. 

3  Cited  from  Hoppe-Seyler,  Physiol.  Chem.,  593. 

4  The  works  of  Asher  and  collaborators,  Barbera,  Gies,  and  Busch,  upon  lymph 
fonration  may  be  found  in  Zeitschr.  f.  Biologie,  36,  37,  40. 


350  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES, 

causes  an  increase  in  the  quantity  of  lymph.  According  to  Heidenhain, 
on  the  contrary,  a  very  considerable  change  in  the  pressure  in  the  aorta 
causes  only  a  little  change  in  the  abundance  of  the  lymph  flow.  The 
quantity  of  lymph  may  be  raised  by  powerfully  active  and  passive  move- 
ments of  the  limbs  (Lesser).  Under  the  influence  of  curare,  an  increase 
of  the  lymph  secretion  is  observed  (Paschutin,  Lesser1),  and  the  quan- 
tity of  solids  in  the  lymph  is  also  increased. 

The  bodies  inciting  lymph  flow,  the  so-called  lymphagogues,  are  of 
especially  great  interest,  and  they  may,  according  to  Heidenhain,2  be 
divided  into  two  different  chief  groups.  The  lymphagogues  of  the  first 
series — extracts  of  crab-muscles,  blood-leech,  anodons,  liver  and  intestine 
of  dogs,  as  well  as  peptone  and  egg  albumin,  strawberry  extracts,  meta- 
bolic products  of  bacteria  and  others — cause  a  greatly  increased  secre- 
tion of  lymph  without  raising  the  blood-pressure,  and  in  this  way  the 
blood-plasma  becomes  poorer  in  proteins  and  the  lymph  richer  than 
before.  For  the  formation  of  this  lymph,  which  Heidenhain  designates 
blood-lymph,  we  must  admit  with  him  that  a  special  secretory  activity 
of  the  capillary-wall  endothelium  exists.  The  lymphagogues  of  the  second 
series,  such  as  sugar,  urea,  sodium  chloride,  and  other  salts,  also  cause 
an  abundant  lymph  formation.  The  blood,  as  well  as  the  lymph,  thereby 
becomes  richer  in  water.  This  increased  amount  of  water  depends, 
according  to  Heidenhain,  upon  an  increased  delivery  of  water  by  the 
tissue-elements,  and  this  lymph  is  chiefly  tissue-lymph,  in  his  opinion. 
Diffusion  is  no  doubt  of  great  importance  in  the  formation  of  this  lymph, 
but  the  secretory  activity  of  the  endothelium  is  also  of  importance, 
at  least  for  certain  bodies,  such  as  sugar. 

In  the  past,  the  formation  of  lymph  was  explained  in  a  purely  phj'sical 
way  by  the  united  action  of  filtration  from  the  blood  and  the  osmosis 
between  the  blood  and  tissue-fluid.  Later  Heidenhain  and  also  Ham- 
burger ascribed  a  special  activity  to  the  capillary  endothelium,  assum- 
ing that  they  take  part  in  the  formation  of  lymph  in  a  secretory  manner. 
The  above-mentioned  observations  on  the  greater  NaCl  content  in  the 
lymph  as  compared  to  the  plasma  as  well  as  the  regularly  found  higher 
osmotic  pressure  of  the  lymph  speak  for  such  a  view. 

According  to  Asher  and  his  collaborators  (Barbera,  Gies  and 
Busch)  the  lymph  is  a  product  of  the  work  of  the  organs.  Its  amount 
is  dependent  upon  an  increased  or  diminished  activity  of  the  organs, 


1  Lesser,  Arbeiten  aue  der  physiol.  Anstalt  zu  Leipzig,  Jahrgang,  6;  Paschutin, 
ibid.,  7. 

2  Heidenhain,  Pfliiger's  Arch.,  49;  Hamburger,  Zeitschr.  f.  Biologie,  27  and  30. 
See  especially  Ziegler's  Beitr.  zur  Path.  u.  zur  allg.  Pathol.,  14,  443;  also  Arch.  f. 
(Anat.  u.)  Physiol.,  1895  and  1896. 


CHYLE  AND   LYMPH.  351 

and  the  lymph  is  therefore  a  measure  of  the  work  in  these.  The  close 
relation  between  lymph  formation  and  the  work  of  organs  has  also  been 
showrn  for  several  of  them,  especially  for  the  liver.  Starling  has 
shown  that  after  the  introduction  of  lymphagogues  of  the  first  series, 
chiefly  liver  lymph  is  secreted,  which  he  claims  is  a  proof  against  Heiden- 
hain's  view,  and  he  explains  the  increased  permeability  of  the  vessel 
wall  by  the  fact  that  these  bodies  have  an  irritating,  poisonous  action. 
On  the  contrary,  Ashek  explains  this  increased  lymph  flow  by  the  state- 
ment that  the  substance  in  question — as  well  as  those  influences  which 
incite  the  activity  of  the  liver — produces  an  increased  formation  of  lymph 
in  these  organs.  This  view  is  supported  by  experiments  upon  the  action 
of  lymphagogues  on  blood  coagulation  and  liver  activity  (Delezenne 
and  others),  for,  according  to  Gley,  these  bodies  have  at  the  same  time 
a  lymphagogue  action  and  an  action  upon  the  secretion  of  the  glands. 
We  have  no  direct  evidence  of  the  action  of  the  lymphagogues  of  the 
first  series  upon  the  organs,  but  we  know  from  Kusmine's  work  that 
peptone,  leech  extract,  and  the  extractives  of  the  crab-muscles  act  directly 
upon  the  liver-cells  and  bring  about  morphological  changes.  The  con- 
nection betwreen  organ  activity  and  lymph  formation  has  also  been 
shown  upon  muscles  and  glands  by  others  besides  the  above-mentioned 
investigators  (Hamburger,  Bainbridge  1). 

The  extent  of  organ  work  essentially  influences  the  quantity  and 
properties  of  the  lymph.  Still  from  this  we  cannot  draw  any  posi- 
tive conclusions  as  to  whether  the  lymph  formation  is  brought  about 
by  physico-chemical  processes  alone  or  whether  in  this  process  a  specific, 
not  closely  definable  secretory  force  is  at  work  at  the  same  time.  In 
regard  to  this  much-disputed  question,  attention  must  be  called  in  the 
first  place  to  the  fact  that  the  important  works  of  Heidenhain,  Ham- 
burger, Lazarus-Barlow,  and  others,  as  well  as  the  investigations  of 
Asher  and  Gies  and  of  Mendel  and  Hooker  2  upon  the  lengthy  post- 
mortem lymph  flow,  have  shown  that  the  older  filtration  hypothesis  is 
untenable. 

That  osmotic  processes  play  an  important  role  in  the  lymph  formation 
is  generally  admitted  and  that  the  work  of  the  glands  and  tissue  cells 
must  cause  a  difference  in  the  osmotic  pressure  on  both  sides  of  the  capillary 
wralls,  has  been  shown  by  the  researches  of  many  investigators  (Koranyi, 
Starling,  Roth,  Asher  and  others).  That  this  is  so  follows  from 
several  circumstances,  and  especially  from  the  fact  that,  in  disassimila- 


1  In  regard  to  the  works  cited,  as  well  as  the  literature  upon  lymph  formation,  see 
Ellinger,  "Die  Bildung  der  Lymphe,"  Ergebnisse  der  Physiol.,  I,  Abt.  1,  1902,  and 
Asher,  Biochem.  Centralbl.,  4. 

2  Amer.  Journ.  of  Physiol.,  7.     See  also  footnote  1. 


352  CHYLE,  LYMPH,   TKANSUDATES  AND  EXUDATES. 

tion  in  the  cells,  bodies  of  high  molecular  weight  are  split  into  a  number 
of  smaller  molecules,  which  latter,  either  directly,  if  they  leave  the  cells 
and  pass  into  the  tissue-fluid,  or  indirectly,  when  they  remain  in  the  cells, 
produce  an  increase  in  the  osmotic  tension  within  the  cells,  and  in  this 
way  cause  a  taking  up  of  water  from  the  fluid,  and  must  therefore  increase 
the  osmotic  pressure  of  the  tissue-fluids.  As  the  cells  can  by  synthesis 
build  up  highly  complex  constituents  from  simple  molecules,  and  as  the 
chief  products  of  catabolism  are  carbon  dioxide  and  water,  it  is  difficult 
to  explain  these  intricate  conditions.  Still,  irrespective  of  whatever 
view,  a  change  in  one  or  the  other  direction  in  the  osmotic  pressure 
upon  both  sides  of  the  capillary  wall  must  be  produced  thereby.  Whether 
this  and  other  physico-chemical  processes  are  alone  sufficient  to  explain 
the  lymph  formation  (Cohnstein,  Ellinger)  remains  an  open  and 
disputed  question.1 

H.  TRANSUDATES  AND  EXUDATES. 

The  serous  membranes  are  normally  kept  moistened  by  liquids  whose 
quantity  is  sufficient  only  in  a  few  instances,  as  in  the  pericardial  cavity 
and  the  subarachnoidal  space,  for  a  complete  chemical  analysis  to  be 
made  of  them.  Under  diseased  conditions  an  abundant  transudation 
may  take  place  from  the  blood  into  the  serous  cavities,  into  the  sub- 
cutaneous tissues,  or  under  the  epidermis;  and  in  this  way  pathological 
transudates  are  formed.  Such  true  transudates,  which  are  similar  to  lymph, 
are  generally  poor  in  form-elements  and  leucocytes,  and  yield  only  very 
little  or  almost  no  fibrin,  while  the  inflammatory  transudates,  the  so-called 
exudates,  are  generally  rich  in  leucocytes  and  yield  proportionally  more 
fibrin.  As  a  rule,  the  richer  a  transudate  is  in  leucocytes  the  closer  it 
stands  to  pus,  while  a  diminished  quantity  of  leucocytes  renders  it  more 
nearly  like  a  real  transudate  or  lymph. 

It  is  ordinarily  accepted  that  filtration  is  of  the  greatest  importance 
in  the  formation  of  transudates  and  exudates.  The  facts  coincide  with 
this  view  that  all  these  fluids  contain  the  salts  and  extractive  bodies 
occurring  in  the  blood-plasma  in  about  the  same  quantity  as  the  blood- 
plasma,  while  the  amount  of  proteins  is  habitually  smaller.  While  the 
different  fluids  belonging  to  this  group  have  about  the  same  quantities 
of  salts  and  extractive  bodies,  they  differ  from  one  another  chiefly  in 
containing  differing  quantities  of  protein  and  form-elements,  as  well  as 
varying  quantities  of  transformation  and  decomposition  products  of 
these    latter — changed    blood-eoloring    matters,    cholesterin,    etc.     The 

xOn  this  question  see  Ellinger,  "Die  Bildung  der  Lymphe,"  Ergebnisse  der  Phys- 
iologic, I,  Abt.  1,  355,  and  Asher,  Biochem.  Centralbl.,  4,  pp.  1  and  45. 


TRANSUDATES  AND  EXUDATES.  353 

correspondence  in  the  amount  of  salts  and  extractive  bodies  present  in 
the  blood  and  in  transudates  supplies  just  as  little  proof  for  a  filtration 
as  it  does  for  the  formation  of  lymph;  but  still  it  cannot  be  doubted  for 
other  reasons  that  filtration  is  often  of  great  importance  in  the  forma- 
tion of  a  transudate.  To  what  extent  filtration  is  active  in  the  perfectly 
normal  vascular  wall  cannot  be  answered. 

The  altered  permeability  of  the  capillary  walls  in  disease  is  a  second 
important  factor  in  the  formation  of  transudates.  The  circumstance 
that  the  greatest  quantity  of  protein  occurs  in  transudates  in  inflammatory 
processes,  to  which  is  also  due  the  abundant  quantity  of  form-elements 
in  such  transudates,  has  been  explained  by  this  hypothesis.  The  greater 
quantity  of  protein  in  the  transudates  in  formative  irritation  is  in  great 
part  explained  by  the  large  amount  of  destroyed  form-elements.  The 
interesting  observation  made  by  Paijkull,1  that  in  those  cases  in  which 
an  inflammatory  irritation  has  taken  place  the  fluid  contains  nucleoal- 
bumin  (or  nucleoprotein?),  while  this  substance  does  not  occur  in 
transudates  in  the  absence  of  inflammatory  processes,  can  be  explained 
by  the  presence  of  form-elements.  Still,  such  a  phosphorized  protein 
substance  does  not  occur  in  all  inflammatory  exudates. 

As  the  secretory  importance  of  the  capillary  endothelium  has  been 
made  probable  by  the  investigations  of  Heidenhain,  it  is  a  priori  to  be 
expected  that  an  abnormally  increased  secretory  activity  of  the  endothe- 
lium is  a  cause  of  transudates.  Those  observations  which  substantiate 
such  an  assumption  can  also  be  explained  just  as  well  by  assuming  a 
changed  permeability  of  the  capillary  walls. 

The  varying  quantities  of  protein  observed  by  C.  Schmidt2  in  the 
tissue-fluids  in  different  vascular  regions  can  perhaps  be  explained  by  the 
different  condition  of  the  capillary  endothelium.  For  example,  the 
amount  of  protein  in  the  pericardial,  pleural,  and  peritoneal  fluids 
is  considerably  greater  than  in  those  fluids  which  are  found  in  the  sub- 
arachnoidal space,  in  the  subcutaneous  tissues,  or  in  the  aqueous 
humor,  which  are  poor  in  protein.  The  condition  of  the  blood  also 
greatly  affects  the  transudates,  for  in  hydrsemia  the  amount  of  protein  in 
the  transudate  is  very  small.  With  the  increase  in  the  age  of  a  transudate, 
of  a  hydrocele  fluid  for  instance,  the  quantity  of  protein  is  increased, 
probably  by  resorption  of  water,  and  indeed  exceptional  cases  may  occur 
in  which  the  amount  of  protein,  without  any  previous  hemorrhage,  is 
even  greater  than  in  the  blood-serum. 

The  proteins  of  transudates  are  chiefly  seralbumin,  serglobulin,  and 
a   little   fibrinogen.     Proteoses   and   peptones    do   not   occur,    excepting 

1  See  Maly's  Jahresber.,  22. 

2  Cited  from  Hoppe-Seyler,  Physiol.  Chera.,  607. 


354  CHYLE,   LYMPH,    TRANSUDATES   AND   EXUDATES. 

perhaps  in  the  cerebrospinal  fluid,  and  in  those  cases  where  an  autolysis 
has  taken  place  in  the  liquid.1  The  non-inflammatory  transudates  do 
not  as  a  rule  undergo  spontaneous  coagulation  or  do  so  only  very  slow- 
ly. On  the  addition  of  blood  or  blood-serum  they  coagulate.  Inflam- 
matory exudates  coagulate  spontaneously,  and  Paijkull  has  shown 
that  these  often  contain  nucleoprotein  (or  nucleoalbumin) .  In  inflam- 
matory exudates  a  protein  substance  has  been  habitually  observed  which 
is  precipitated  by  acetic  acid,  but  which  does  not  occur  in  transudates, 
or  only  in  very  small  quantities.  This  substance,  which  has  been  observed 
and  studied  by  Moritz,  Staehelin,  Umber,  and  Rivalta,  is  claimed 
by  the  first  three  observers  to  be  free  from  phosphorus,  while  Rivalta 
considers  it  to  be  a  phosphorized  pseudoglobulin.  Umber  calls  it  sero- 
samucin, although  it  yields  only  very  little  reducing  carbohydrate. 
According  to  Joachim  2  it  is  only  a  part  of  the  globulin,  a  view  which  can- 
not be  correct  for  all  cases,  v.  Holst  3  has  so  far  substantiated  Umber's 
observation  in  that  he  has  isolated  a  mucin  substance  from  an  ascitic 
fluid  in  carcinoma  of  the  stomach  and  the  peritoneum,  which  seemed  to 
be  identical  with  Umber's  serosamucin,  as  well  as  with  the  synovial 
mucin.  There  does  not  seem  to  be  any  doubt  that  in  transudates  and 
exudates,  different  protein  substances  may  occur  under  different  cir- 
cumstances, although  the  globulins  form  besides  seralbumin  the  principal 
mass  of  the  protein  bodies.  Mucoid  substances,  which  were  first 
observed  by  Hammarsten  in  certain  cases  of  ascites  without  complica- 
tions with  ovarial  tumors,  and  which  are  cleavage  products  of  a  more 
complicated  substance,  seem  according  to  Paijkull4  to  be  regular 
constituents  of  transudates  and  are  closely  related  to  the  above-men- 
tioned serosamucin.  The  occurrence  of  the  above-mentioned  substances 
precipitable  by  acetic  acid,  the  globulins  (Rivalta)  and  the  nucleo- 
proteins,  in  puncture  fluids,  has  been  recognized  as  of  very  great  impor- 
tance in  the  differential  diagnosis  between  transudates  and  exudates. 
There  are  numerous  investigations  on  the  relation  between  glob- 
ulin and  seralbumin,  and  Joachim  has  determined  the  relation  between 
euglobulin  and  the  total  globulin.  No  conclusive  results  can  be  drawn 
from  these  determinations.  The  relation  between  globulin  and  seral- 
bumin  varies   very   much   in   different   cases,    but,    as   Hoffmann   and 

1  Umber,  Munch,  med.  Wochenschr.,  1902,  and  Berlin,  klin.  Wochenschr.,  1903. 
In  regard  to  the  autolysis  in  transudates,  see  also  Galdi,  Biochem.  Centralbl.,  3; 
Eppinger,  Zeitsehr.  f.  Heilkunde,  25,  and  Zak,  Wien.  klin.  Wochenschr.,  1905. 

-  Paijkull,  1.  c;  Moritz,  Munch,  med.  Wochenschr.,  1903;  Staehelin,  ibid.,  1902, 
Qmber,  Zeitsehr.  f.  klin.  Med.,  48;  Rivalta,  Biochem.  Centralbl.,  2  and  5;  Joachim; 
Pfltlger'e  Arch.,  93. 

3  Zeitsehr.  f.  physiol.  Chem.,  43. 

4  Hammarsten,  ibid.,  15;  Paijkull,  1.  c. 


TRANSUDATES  AND   EXUDATES.  355 

Pigeand  l  have  shown,  the  variation  is  in  each  case  the  same  as  in  t he 
blood-serum  of  the  individual. 

The  specific  gravity  runs  almost  parallel  with  the  quantity  of  protein. 
The  varying  specific  gravity  has  been  suggested  as  a  means  of  differentia- 
tion between  transudates  and  exudates  by  Reuss,2  as  the  first  often  show 
a  specific  gravity  below  1015-1010,  while  the  others  have  a  specific  gravity 
of  1018  or  above.     This  rule  holds  good  in  many,  but  not  in  all  cases. 

The  gases  of  the  transudates  consist  of  carbon  dioxide  besides  small 
amounts  of  nitrogen  and  traces  of  oxygen.  The  tension  of  the  carbon 
dioxide  is  greater  in  the  transudates  than  in  the  blood.  When  mixed 
with  pus,  the  amount  of  carbon  dioxide  is  decreased. 

The  extractives  are,  as  above  stated,  the  same  as  in  the  blood-plasma. 
Urea  seems  to  occur  in  very  variable  amounts.  Sugar  also  occurs  in 
transudates,  but  it  is  not  known  to  what  extent  the  reducing  power  is 
due  to  other  bodies,  as  in  blood-serum.  A  reducing,  non-fermentable 
substance  has  been  found  by  Pickardt  in  transudates.  The  sugar  is 
generally  glucose,  but  fructose  seems  to  have  been  found 3  in  several 
cases.  Sarcolactic  acid  has  been  found  by  C.  Kulz  in  the  pericardial 
fluid  from  oxen.  Succinic  acid  has  been  found  in  a  few  cases  in  hydrocele 
fluids,  while  in  other  cases  it  is  entirely  absent.  Leucine  and  tyrosine 
have  been  found  in  transudates  from  diseased  livers  and  pus-like  trans- 
udates which  have  undergone  decomposition,  and  after  autolysis.  Among 
other  extractives  found  in  transudates  must  be  mentioned  allantoin 
(Moscatelli4),  uric  acid,  purine  bases,  creatine,  inosite,  and  pyrocate- 
chin  (?). 

The  division  of  the  nitrogenous  substances  in  human  transudates 
and  exudates  has  so  far  been  little  studied.  Otori  found  that  no 
essential  difference  exists  between  serous  exudates  and  transudates  in 
regard  to  the  quantity  of  urea  and  amino-acids.  The  amount  of  total 
nitrogen  and  proteins  runs  parallel  with  the  specific  gravity,  and  the 
same  is  generally  true  for  the  absolute  values  for  ammonia  nitrogen  and 
purine  nitrogen.  According  to  the  investigations  of  Czernecki,5  in 
pathological  puncture  fluids,  also  oxyproteic  acids  (see  Chapter  XIV 
on  the  urine)  occur  and  which  represent  13.3 — 25.9  per  cent  of  the  total 
nitrogen  of  the  protein  free  filtrate.     The  question  as  to  the  amount  of 

1  Joachim,  1.  c;  Hoffmann,  Arch.  f.  exp.  Path.  u.  Pharm.,  16;  Pigeand,  see  Maly's 
Jahresber.,  16. 

2  Reuss,  Deutsch.  Arch.  f.  klin.  Med.,  28.     See  also  Otto,  Zeitschr.  f.  Heilkunde,  17. 
8  Pickardt,  Berl.  klin.  Wochenschr.,  1897.     See  also  Rotmann,  Munch,  med.  Woch- 

enschr.,  1898;  Neuberg  and  Strauss,  Zeitschr.  f.  physiol.  Chem.,  36;  Sittig,  Bioch. 
Zeitschr.  21. 

4C.  Kulz,  Zeitschr.  f.  Biologie,  32;  Moscatelli,  Zeitschr.  f.  physiol.  Chem.  13. 

6  Otori,  Zeitschr.  f.  Heilk.  25;  Czernecki,  Maly's  Jahresb.,  39. 


356  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

urea  nitrogen  and  amino-acid  nitrogen  in  such  fluids  must,  under  these 
circumstances,  require  further  study. 

The  investigations  upon  the  molecular  concentration  have  shown 
that  no  essential  and  constant  difference  exists  between  exudates  and 
transudates.  The  osmotic  concentration  and  the  concentration  of  the 
electrolytes  are  as  a  rule  the  same  as  in  blood-serum,  although  some- 
times rather  divergent  results  have  been  found.  The  concentration  of 
the  electrolytes  shows,  according  to  Bodon,1  like  the  blood-serum,  much 
less  variation  than  the  total  concentration.  The  alkalinity  determined 
by  titration  is  about  the  same  in  transudates  and  exudates,  and  is  equal 
to  that  of  the  blood-serum.  The  determination  of  the  HO  ion  concen- 
tration has  shown  that  the  transudates  and  exudates  in  this  regard  are 
about  as  neutral  as  the  blood-serum  (Bodon). 

As  above  stated,  irrespective  of  the  varying  number  of  form-elements 
contained  in  the  different  transudates,  the  quantity  of  protein  is  the  most 
characteristic  chemical  distinction  in  the  composition  of  the  various 
transudates;  therefore  a  quantitative  analysis  is  of  importance  only 
in  so  far  as  it  considers  the  quantity  of  protein.  On  this  account,  in  the 
following,  relative  to  the  quantitative  composition,  stress  will  be  put  on 
the  quantity  of  protein. 

Pericardial  Fluid.  The  quantity  of  this  fluid  is,  even  under  physio- 
logical conditions,  so  large  that  a  sufficient  quantity  for  chemical  inves- 
tigation has  been  obtained  (from  persons  who  had  been  executed).  This 
fluid  is  lemon-yellow  in  color,  somewhat  sticky,  and  yields  more  fibrin 
than  other  transudates.  The  amount  of  solids,  according  to  the  analyses 
performed  by  v.  Gorup-Besanez,  Wachsmuth,  and  Hoppe-Seyler,2 
is  37.5-44.9  p.  m.,  and  the  amount  of  protein  is'  22.8-24.7  p.  m.  The 
analysis  made  by  Hammarsten  of  a  fresh  pericardial  fluid  from  a  young 
man  who  had  been  executed  yielded  the  following  results,  calculated  in 
1000  parts  by  weight. 

Water 960.85 

Solids 39.15 

f  Fibrin 0.31 

Proteins 28 .  60    Globulin ....     5 .95 

(Albumin 22.34 

Soluble  salts 8.60     NaCl 7.28 

Insoluble  salts 0.15 

Extractive  bodies 2 .  00 

Friend  3  found  almost  the  same  composition  for  a  pericardial  fluid 
from  a  horse,  with  the  exception  that  this  liquid  was  relatively  richer 

1  Pflviger's  Arch.,  104,  where  literature  on  this  subject  may  be  found. 

2  v.  Gorup-Besanez,  Lehrbuch  d.  physiol  Chem.,  4.  Aufl.,  401;  Wachsmuth,  Vir- 
chow's  Arch.,  7;  Hoppe-Seyler,  Physiol.  Chem.,  605. 

•Halliburton,  Text-book  of  Chem.  Physiol.,  etc.,  London,  1891. 


PLEURAL  FLUID.  357 

in  globulin.  The  ordinary  statement  that  pericardial  fluids  are  richer 
in  fibrinogen  than  other  transudates  is  hardly  based  on  sufficient  proof. 
In  a  case  of  chylopericardium,  which  was  probably  due  to  the  rupture 
of  a  chylous  vessel,  or  caused  by  a  capillary  exudation  of  chyle  because 
of  stoppage,  Hasebroek  l  found  in  1000  parts  of  the  fluid  103.61  parts 
solids,  73.79  parts  proteins,  10.77  parts  fat,  3.34  parts  cholesterin,  1.77 
parts  lecithin,  and  9.34  parts  salts. 

The  pleural  fluid  occurs  under  physiological  conditions  in  such  small 
quantities  that  a  chemical  analysis  of  it  cannot  be  made.  Under  patho- 
logical conditions  this  fluid  may  show  very  variable  properties.  In 
certain  cases  it  is  nearly  serous,  in  others  again  sero-fibrinous,  and  in  others 
similar  to  pus.  There  is  a  corresponding  variation  in  the  specific  gravity 
and  the  properties  in  general.  If  a  pus-like  exudate  is  kept  enclosed  for 
a  long  time  in  the  pleural  cavity,  a  more  or  less  complete  maceration 
and  solution  of  the  pus-corpuscles  is  found  to  take  place.  The  ejected 
yellowish-brown  or  greenish  fluid  may  then  be  as  rich  in  solids  as  the 
blood-serum;  and  an  abundant  flocculent  precipitate  of  a  nucleoalbumin 
or  nuceloprotein  (the  pyrin  of  early  writers)  may  be  obtained  on  the 
addition  of  acetic  acid.  This  precipitate  is  soluble  with  difficulty  in 
an  excess  of  acetic  acid. 

Numerous  analyses,  by  many  investigators,2  of  the  quantitative 
composition  of  pleural  fluids  under  pathological  conditions  have  been 
published.  From  these  analyses  we  learn  that  in  hydrothorax  the 
specific  gravity  is  lower  and  the  quantity  of  protein  less  than  in  pleuritis. 
In  the  first  case  the  specific  gravity  is  generally  less  than  1.015,  and  the 
quantity  of  protein  10-30  p.  m.  In  acute  pleuritis  the  specific  gravity 
is  generally  higher  than  1.020,  and  the  quantity  of  protein  30-65  p.  m. 
The  quantity  of  fibrinogen,  which  in  hydrothorax  is  about  0.1  p.  m., 
may  amount  to  more  than  1  p.  m.  in  pleuritis.  In  pleurisy  with  an 
abundant  accumulation  of  pus,  the  specific  gravity  may  rise  even  to  1.030 
according  to  the  observations  of  Hammarsten.  The  quantity  of  solids 
is  often  60-70  p.  m.,  and  may  be  even  more  than  90-100  p.  m.  (Ham- 
marsten). Mucoid  substances  have  also  been  detected  in  pleural  fluids 
by  Paijkull.  Cases  of  chylous  pleurisy  are  also  known;  in  such  a 
case  M£hu  3  found  17.93  p.  m.  fat  and  cholesterin  in  the  fluid. 

The  quantity  of  peritoneal  fluid  is  very  small  under  physiological 
conditions.     The  investigations  refer  only  to  the  fluid   under  diseased 


1  Zeitschr.  f.  physiol.  Chem.,  12. 

2  See  the  works  of  M6hu,  Runeberg,  F.  Hoffmann,  Reuss,  all  of  which  are  cited  in 
Bernheim's  paper  in  Virchow's  Arch.,  131,  274.  See  also  Paijkull,  1.  c,  and  Halli- 
burton's Text-book,  346;  Joachim,  1.  c. 

1  Arch.  gen.  de  meYl.,  1886,  2,  cited  from  Maly's  Jahresber.,  16. 


358  CHYLE,    LYMPH,  TRANSUDATES  AND  EXUDATES. 

conditions  (ascitic  fluid).  The  color,  transparency,  and  consistency  of 
these  may  vary  greatly. 

In  cachectic  conditions  or  a  hydrsemic  condition  of  the  blood  the  fluid 
has  little  color,  is  milky,  opalescent,  watery,  does  not  coagulate  spon- 
taneously, has  a  very  low  specific  gravity,  1.006-1.010-1.015,  and  is 
almost  free  from  form-elements.  The  ascitic  fluid  in  portal  stagnation, 
or  in  general  venous  congestion,  has  a  low  specific  gravity  and  contains  or- 
dinarily less  than  20  p.  m.  protein,  although  in  certain  cases  the  quantity 
of  protein  may  rise  to  35  p.  m.  In  carcinomatous  peritonitis  it  may  have  a 
cloudy,  dirty-gray  appearance,  due  to  its  richness  in  form-elements  of 
various  kinds.  The  specific  gravity  is  then  higher,  the  quantity  of  solids 
greater,  and  it  often  coagulates  spontaneously.  In  inflammatory  proc- 
esses it  is  straw-  or  lemon-yellow  in  color,  somewhat  cloudy  or  reddish, 
due  to  leucocytes  and  red  blood-corpuscles,  and  from  great  richness  in 
leucocytes  it  may  appear  more  like  pus.  It  coagulates  spontaneously 
and  may  be  relatively  richer  in  solids.  It  contains  regularly  30  p.  m. 
or  more  protein  (although  exceptions  with  less  protein  occur),  and  may 
have  a  specific  gravity  of  1.030  or  above.  On  account  of  the  rupture 
of  a  chylous  vessel,  the  ascitic  fluid  may  be  rich  in  very  finely  emulsified 
fat  (chylous  ascites).  In  such  cases  3.86-10.30  p.  m.  fat  has  been 
found  in  the  ascitic  fluid  (Guinochet,  Hay  1),  and  even  17-43  p.  m. 
has  been  found  by  Minkowski. 

As  first  shown  by  Gross,  an  ascitic  fluid  may  have  a  chylous  appearance 
without  the  presence  of  fat,  i.e.,  pseudochylous.  The  cause  of  the  chylous 
properties  of  a  transudate  is  not  known,  although  numerous  investigators, 
such  as  Gross,  Bernert,  Mosse,  and  Strauss,  have  studied  the  sub- 
ject; several  observations,  however,  seem  to  show  that  it  is  connected 
with  the  amount  of  lecithin  contained  therein.  In  a  case  investigated 
by  H.  Wolff2  the  oleic-acid  ester  of  cholesterin  was  combined  either 
chemically  or  molecularly  with  the  euglobulin. 

By  admixture  of  ascitic  fluid  with  that  from  an  ovarian  cyst  the 
former  may  sometimes  contain  pseudomucin  (see  Chapter  XII).  There 
are  also  cases  in  which  the  ascitic  fluid  contains  mucoids  which  may  be 
precipitated  by  alcohol  after  removal  of  the  proteins  by  coagulation  at 
boiling  temperature.  Such  mucoids,  which  yield  a  reducing  substance 
on  boiling  with  acids,  have  been  found  by  Hammarsten  in  tuberculous 
peritonitis  and  in  cirrhosis  hepatis  syphilitica  in  men.  According  to  the 
investigations  of  Paijkull,  these  substances  seem  to  occur  often  and 
perhaps  habitually  in  the  ascitic  fluids. 

1  Guinochet,  see  Strauss,  Arch,  de  Physiol.,  18.     See  Maly's  Jahresber.,  16,  475. 

2  Gross,  Arch.  f.  exp.  Path.  u.  Pharm.,  44;  Bernert,  ibid.,  49;  Mosse,  Leyden's 
Festschrift,  1901;  Strauss,  cited  in  Biochem.  Centralbl.,  1,  437;  Wolff,  Hofmeister's 
Beitrage,  5. 


HYDROCELE  AND  SPERMATOCELE  FLUIDS.  359 

As  the  quantity  of  protein  in  ascitic  Quids  ie  dependent  upon  the  same 
factors  as  in  other  transudates  and  exudates,  it  is  sufficient  to  give  the 
following  example  of  the  composition,  taken  from  Bernheim's  l  treatise. 
The  results  arc  expressed  in  1000  parts  of  the  fluid: 

Max.  Min.  Mean. 

Cirrhosis  of  the  liver 34.5  5.6  9.69—21.06 

Bright's  disease 16.11  10.10  5.6—10.36 

Tuberculous  and  idiopathic  peritonitis.  .  .     55.8  18.72  30.7  — 37.95 

Carcinomatous  peritonitis 54.20  27.00  35.1—58.96 

Joachim  found  the  highest  relative  globulin  amounts  and  lowest  albumin 
percentages  in  cirrhosis;  in  carcinoma,  on  the  contrary,  the  lowest  globulin  and 
the  highest  albumin.  The  values  in  cardiac  stagnation  stand  between  the  cirrhosis 
and  carcinoma  percentages. 

Urea  has  also  been  found  in  ascitic  fluids,  sometimes  only  as  traces,  some- 
times in  larger  quantities  (4  p.  m.  in  albuminuria),  also  uric  acid,  allantoin  in 
cirrhosis  of  the  liver  (Moscatelli),  xanthine,  creatine,  cholesterin,  sugar,  diastatic 
and  proteolytic  enzymes,  and  according  to  Hamburger  2  also  a  lipase. 

Hydrocele  and  Spermatocele  Fluids.  These  fluids  differ  essentially 
from  each  other  in  various  ways.  The  hydrocele  fluids  are  generally 
colored  light  or  dark  yellow,  sometimes  brownish  with  a  shade  of  green. 
They  have  a  relatively  higher  specific  gravity,  1.016-1.026,  with  a  variable 
but  generally  higher  amount  of  solids,  an  average  of  60  p.  m.  They 
sometimes  coagulate  spontaneously,  sometimes  only  after  the  addition  of 
fibrin  ferment  or  blood.  They  contain  leucocytes  as  chief  form-elements. 
Sometimes  they  contain  smaller  or  larger  amounts  of  cholesterin  crystals. 

The  spermatocele  fluids,  on  the  contrary,  are  as  a  rule  colorless, 
thin,  and  cloudy  like  water  mixed  with  milk.  They  sometimes  have  an 
acid  reaction.  They  have  a  lower  specific  gravity,  1.006-1.010,  a  lower 
amount  of  solids — an  average  of  about  13  p.  m. — and  do  not  coagulate 
either  spontaneously  or  after  the  addition  of  blood.  They  are,  as  a  rule, 
poor  in  protein  and  contain  spermatozoa,  cell-detritus,  and  fat-globules  as 
form  constituents.  To  show  the  unequal  composition  of  these  two  kinds 
of  fluids  we  will  give  the  average  results  (calculated  in  parts  per  1000 
parts  of  the  fluid)  of  seventeen  analyses  of  hydrocele  fluids  and  four 
of  spermatocele  fluids  made  by  Hammarsten.3 

Hydrocele.       Spermatocele. 

Water 938.85  986.83 

Solids 61.15  12.17 

Fibrin 0 .  59             

Globulin 13.25  0.59 

Seralbumin. . 35.94  1 .82 

Ether  extractive  bodies 4 .  02 

Soluble  salts 8.60  \  10.76 

Insoluble  salts 0 .  66 

1 1.  c.  As  it  was  impossible  to  derive  mean  figures  from  those  given  by  Bernheim, 
the  author  has  given  the  maximum  and  minimum  of  the  averages  given  by  him. 
'Arch.  f.  (Anat.  u.)  Physiol.,  1900,  433. 
*  Upsala  Lakaref.  Forh.,  14,  and  Mary's  Jahresber.,  8,  347. 


360  CHYLE,   LYMPH,  TRANSUDATES  AND  EXUDATES. 

In  the  hydrocele  fluid  traces  of  urea  and  a  reducing  substance  have  been 
found,  and  in  a  few  cases  also  succinic  acid  and  inosite.  A  hydrocele  fluid  may, 
according  to  Devillard,1  sometimes  contain  paralbumin  or  metalbumin  (?). 
Cases  of  chylous  hydrocele  are  also  known. 

Cerebrospinal  Fluid.  The  cerebrospinal  fluid  is  thin,  water-clear, 
of  low  specific  gravity,  1.007-1.008.  The  spina  bifida  fluid  is  very  poor 
in  solids,  8-10  p.  m.  with  only  0.19-1.6  p.  m.  protein.  The  fluid  of 
chronic  hydrocephalus  is  somewhat  richer  in  solids  (13-19  p.  m.)  and 
proteins.  The  amount  of  protein  in  the  cerebrospinal  fluid  seems  to  be 
rather  variable  under  diseased  conditions  and  Frenkel-Heiden  2  found 
0.875-3  p.  m.  protein  in  the  lumbar  fluid  in  progressive  paralysis  and 
0.7-2.8  p.  m.  protein  in  tuberculous  meningitis.  In  the  perfectly  fresh 
fluid  from  healthy  calves  Nawratzki  found  an  average  of  0.22  p.  m. 
protein. 

According  to  Halliburton  the  protein  of  the  cerebrospinal  fluid 
is  a  mixture  of  globulin  and  proteose;  occasionally  some  peptone  occurs, 
and  more  rarely,  in  special  cases,  seralbumin  appears.  The  conclusions 
of  Halliburton  on  the  occurrence  of  proteose  do  not  coincide  with  the 
observations  of  other  investigators  (Panzer,  Salkowski  3) .  In  general 
paralysis,  Halliburton  and  Mott  obtained  a  nucleoprotein  in  the 
cerebrospinal  fluid.  Choline  occurs  in  several  diseases,  as  in  general 
paralysis,  brain-tumors,  tabes  dorsalis,  and  epilepsy  (Halliburton 
and  Mott,  Donath,  Rosenheim).  According  to  Kaufmann4  we 
are  not  here  dealing  with  choline  but  with  another  base.  Glucose,  or  at 
least  a  fermentable  sugar,  occurs  habitually  in  the  cerebrospinal  fluid, 
while  the  claims  of  Halliburton  as  to  the  occurrence  of  a  substance 
similar  to  pyrocatechin  could  not  be  substantiated  in  calves  and  men 
by  Nawratzki,5  and  hence  this  substance  does  not  exist  in  all  cerebro- 
spinal fluids.  Urea  occurs  in  cerebrospinal  fluids,  but  not  always.  In 
the  cases  investigated  by  Frenkel-Heiden  indeed  all  the  rest-nitrogen 
occurred  as  urea  and  the  urea-nitrogen  varied  in  different  pathological 
cases  between  0.196-1.12  p.  m.  Lactic  Acid  has  been  found  by  Lehnt- 
dorff  and  Baumgarten6  in  many  pathological  cases.  The  quantity 
of  NaCl  is  regularly  much  greater  than  the  KC1,  6-7  p.  m.  NaCl  against 


1  Bull.  Soc.  chim.,  42,  617. 

2  Bioch.  Zeitschr.,  2. 

'Halliburton's  Text-book;  Panzer,  Wein.  klin.  Wochenschr.,  1899;  Salkowski, 
Jaffe"  Festschrift,  265. 

*  Halliburton  and  Mott,  Phil.  Transact.  Roy.  Soc.  London,  Series  B,  191;  Donath, 
Zeitschr.  f.  physiol  Chem.,  39  and  42;  see  also  Mansfield,  ibid.,  42;  Rosenheim,  Journ. 
of  Physiol.,  35;  Kaufmann,  Zeitschr.  f.  physiol.  Chem.,  66. 

6  Zeitschr.  f.  physiol.  Chem.,  23.     See  also  Rossi,  ibid.,  39  (literature). 

"Zeitschr.  f.  exp.  Path.  u.  Therap.,  4  (literature). 


AQUEOUS  HUMOR  AND  BLISTER-FLUID.  361 

about  0.4  p.  m.  KC1,  and  the  variable  relation  between  potassium  and 
sodium  is  probably  due,  according  to  Salkowski,1  to  the  absence  or  pres- 
ence  of  fever  during  the  formation  of  the  exudate;  the  amount  of  potas- 
sium is  high  in  the  acute  cases  and  low  in  the  chronic  ones.  According 
to  Landau  and  Halpern  2  a  certain  antagonism  seems  to  exist  between 
nitrogen  and  sodium  chloride,  as  the  highest  results  of  the  first  correspond 
to  the  lowest  results  of  the  other.  According  to  Cavazzani,3  who  has 
especially  studied  the  cerebrospinal  fluids,  the  alkalinity  of  these  fluids 
is  considerably  less  than  that  of  the  blood  and  independent  of  this  last 
fluid.  For  this  and  several  other  reasons  Cavazzani  draws  the  con- 
clusion that  the  cerebrospinal  fluid  is  formed  by  a  true  secretory  process. 

A  large  number  of  investigations  on  the  cerebrospinal  fluid  have  been  made 
on  the  fluid  obtained  from  cadavers  and  in  consideration  of  this  it  must  be  remarked 
that  this  fluid  quickly  changes  after  death  and  that  the  results  obtained  therefore 
are  not  comparable  with  the  fluid  during  life. 

Aqueous  Humor.  This  fluid  is  clear,  alkaline  toward  litmus,  and  has 
a  specific  gravity  of  1.003-1.009.  The  amount  of  solids  is  on  an  average 
13  p.  m.,  and  the  amount  of  proteins  only  0.8-1.2  p.  m.  The  protein 
consists  of  seralbumin  and  globulin  and  very  little  fibrinogen  and  mucin. 
According  to  Gruenhagen  it  contains  paralactic  acid,  another  dextro- 
gyrate substance,  and  a  reducing  body  which  is  unlike  sugar  or  dextrin. 
Pautz  4  found  urea  and  sugar  in  the  aqueous  humor  of  oxen. 

Blister-fluid.  The  content  of  blisters  caused  by  burns,  and  of  vesi- 
catory blisters  and  the  blisters  of  the  'pemphigus  chronicus,  is  generally  a 
fluid  rich  in  solids  and  proteins  (40-65  p.  m.).  This  is  especially  true 
of  the  contents  of  vesicatory  blisters.  In  a  burn-blister  K.  Morner  5 
found  50.31  p.  m.  proteins,  among  which  were  13.59  p.  m.  globulin  and 
0.11  p.  m.  fibrin.  The  fluid  contains  a  substance  which  reduces  copper 
oxide,  but  no  pyrocatechin.  The  fluid  of  the  pemphigus  is  alkaline  in 
reaction.  A  wound  secretion  collected  by  Lieblein  6  under  aseptic 
conditions  was  alkaline  in  reaction,  and  contained  less  protein  than  the 
blood-serum.  It  formed  a  slight  fibrin  clot,  and  contained  proteoses 
only  at  first  or  at  the  beginning  of  the  abscess  formation.  As  the  wound 
healed,  the  relation  between  the  globulin  and  albumin  changed,  and  on 


1  See  Salkowski,  1.  c.    New  quantitative  analyses  of  cerebrospinal  and  hydrocephalus 
fluids  may  be  found  in  the  cited  works  of  Nawratzki,  Panzer,  and  Salkowski. 

2  Bioch.  Zeitschr.,  9. 

sSee  Maly's  Jahresber.,  22,  346,  and  Centralbl.  f.  Physiol.,  15,  216. 
*  Gruenhagen,  Pfluger's  Arch.,  43;  Pautz.  Zeitschr.  f.  Biologie,  31. 
8  Skand.  Arch.  f.  Physiol.,  5. 
8  Habilitationsschrift  Prag.  1902,  printed  by  H.  Laupp,  Tubingen. 


3132  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

the  third  day  of  the  healing  the  quantity  of  albumin  was  at  least  nine- 
tenths  of  the  total  protein. 

The  fluid  of  subcutaneous  oedema.  This  is,  as  a  rule,  very  poor  in 
solids,  purely  serous,  does  not  contain  fibrinogen,  and  has  a  specific 
gravity  of  1.005-1.013.  The  quantity  of  proteins  is  in  most  cases  lower 
than  10  p.  m. — according  to  Hoffmann  1-8  p.  m. — and  in  serious 
affections  of  the  kidneys,  generally  with  amyloid  degeneration,  less  than 
1  p.  m.  has  been  shown  (Hoffmann  *).  The  cedematous  fluid  also  habit- 
ually contains  urea,  1-2  p.  m.,  and  sugar. 

The  fluid  of  the  echinococcus  cyst  is  related  to  the  transudates,  and  is 
poor  in  proteins.  It  is  thin  and  colorless,  and  has  a  specific  gravity  of  1.005- 
1.015.  The  quantity  of  solids  is  14-20  p.  m.  The  chemical  constituents  are 
sugar  (2.5  p.  m.),  inosite,  traces  of  urea,  creatine,  succinic  acid,  and  salts  (8.3-9.7 
p.  m.).  Proteins  are  found  only  in  traces,  and  then  only  after  an  inflammatory 
irritation.     In  the  last-mentioned  case  7  p.  m.  proteins  have  been  found  in  the  fluid. 

The  Synovial  Fluid  and  Fluid  in  Synovial  Cavities  around  Joints, 
etc.  The  synovia  is  hardly  a  transudate,  but  it  is  often  discussed  in  an 
appendix  to  the  transudates. 

The  synovia  is  an  alkaline,  sticky,  fibrous,  yellowish  fluid  which 
is  cloudy,  from  the  presence  of  cell-nuclei  and  the  remains  of  destroyed 
cells,  but  is  also  sometimes  clear.  Besides  proteins  and  salts,  it  also 
contains  a  mucin  substance,  synoviamucin  (v.  Holst2).  In  pathological 
synovia,  Hammarsten  found  a  mucin-like  substance  which  is  not  mucin. 
It  behaves  like  a  nucleoalbumin  or  a  nucleoprotein,  and  gives  no  reducing 
substance  on  boiling  with  acids.  Salkowski  3  also  found  a  mucin-like 
substance  in  a  pathological  synovial  fluid,  which  was  neither  mucin  nor 
nucleoalbumin.     He  called  the  substance  synovin. 

The  composition  of  synovia  is  not  constant,  but  is  different  in  rest 
and  in  motion.  In  the  last-mentioned  case  the  quantity  of  fluid  is  less, 
but  the  amount  of  the  mucin-like  body,  of  proteins,  and  of  the  extractive 
bodies  is  greater,  while  the  quantity  of  salts  is  diminished.  This  may 
be  seen  from  the  following  analyses  by  Frerichs.4  The  figures  repre- 
sent parts  per  1000. 

I.  Synovia  from  II.   Synovia  from 

__  a  Stall-fed  Ox.  a  Field-fed  Ox. 

^ater 969.9  948.5 

Solids 30.1  51.5 

Mucin-like  body 2.4  5.6 

Albumin  and  extractives 15.7  35.1 

Fat 0.6  -0.7 

Salts 11.3  9.9 


1  Deutsch.  Arch.  f.  klin.  Med.,  44. 

2  Zeitschr.  f.  physiol.  Chem.,  43. 

'  Hammarsten,  Maly's  Jahresber.,  12;  Salkowski,  Virchow's  Arch.,  131. 
4  Wagner's  Handwdrterbuch,  3,  Abt.  1,  463. 


PUS.  363 

The  synovia  of  new-born  babes  corresponds  to  that  of  resting  animal-. 
The  fluid  of  the  bursae  mucosae,  as  also  the  fluid  in  the  synovial  cavities 
around  joints,  etc.,  is  similar  to  synovia  from  a  qualitative  standpoint. 


III.  PUS. 

Pus  is  a  yellowish-gray  or  yellowish-green,  creamy  mass  of  a  faint 
odor  and  an  unsavory,  sweetish  taste.  It  consists  of  a  fluid,  the  pus- 
serum,  in  which  solid  particles,  the  pus-cells,  swim.  The  number  of  these 
cells  varies  so  considerably  that  the  pus  may  at  one  time  be  thin  and  at 
another  time  so  thick  that  it  scarcely  contains  a  drop  of  serum.  The 
specific  gravity,  therefore,  may  also  greatly  vary,  namely,  between 
1.020  and  1.040,  but  ordinarily  it  is  1.031-1.033.  The  reaction  of  fresh 
pus  is  generally  alkaline,  but  it  may  become  neutral  or  acid  from  a  decom- 
position in  which  fatty  acids,  glycerophosphoric  acid,  and  also  lactic 
acid  are  formed. 

In  the  chemical  investigation  of  pus,  the  pus-serum  and  the  pus- 
corpuscles  must  be  studied  separately. 

Pus-serum.  Pus  does  not  coagulate  spontaneously  nor  after  the 
addition  of  defibrinated  blood.  The  fluid  in  which  the  pus-corpuscles 
are  suspended  is  not  to  be  compared  with  the  blood-plasma,  but  rather 
with  the  serum.  The  pus-serum  is  pale  yellow,  yellowish-green,  or  brownish- 
yellow,  and  has  an  alkaline  reaction  toward  litmus.  It  contains,  for  the 
most  part,  the  same  constituents  as  the  blood-serum;  but  sometimes 
besides  these — when,  for  instance,  the  pus  has  remained  in  the  body 
for  a  long  time — it  contains  a  nucleoalbumin  or  a  nucleoprotein  which 
is  precipitated  by  acetic  acid  and  is  soluble  with  great  difficulty  in  an 
excess  of  the  acid  (pyin  of  the  earlier  authors).  This  nucleoalbumin 
seems  to  be  formed  from  the  hyaline  substance  cf  the  pus-cells  by  macera- 
tion. The  pus-serum  contains,  moreover,  at  least  in  many  cases,  no 
fibrin  ferment.  According  to  the  analyses  of  Hoppe-Seyler  l  the  pus- 
serum  contains  in  1000  parts: 

i.  ii. 

Water 913.70  905.65 

Solids 86 .  30  94  35 

Proteins 63.23  77  21 

Lecithin 1 .  50                0 .  56 

Fat 0.26                 0.29 

Cholesterin 0 .  53                0 .  87 

Alcohol  extractives 1 .  52                0 .  73 

Water  extractives 11 .  53                6 .  92 

Inorganic  salts 7 .  73                7 .  77 


1  Med.-Chem.  Untersuch.,  490. 


364  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

The  ash  of  pus-serum  has  the  following  i  composition,  calculated  to 

1000  parts  of  the  serum: 

i.  ii. 

NaCl 5.22  5.39 

Na2SO< 0.40  0.31 

Na2HP04 0.98  0.46 

Na2C03 0.49  1.13 

Ca3(P04)2 0.49  0.31 

Mg3(P04)2 0.19  0.12 

P04  (in  excess) 0.05 

The  pus-corpuscles  are  generally  thought  to  consist  chiefly  of  emi- 
grated white  blood-corpuscles,  and  their  chemical  properties  have  there- 
fore been  given  in  discussing  these.  The  molecular  granules,  fat- 
globules,  and  red  blood-corpuscles  are  considered  rather  as  casual  form- 
elements. 

The  pus-cells  may  be  separated  from  the  serum  by  centrifugal  force, 
or  by  decantation  directly  or  after  dilution  with  a  solution  of  sodium 
sulphate  in  water  (1  vol.  saturated  sodium-sulphate  solution  and  9  vols, 
water)  and  then  washed  by  this  same  solution  in  the  same  manner  as 
the  blood-corpuscles. 

The  chief  constituents  of  the  pus-corpuscles  are  proteins,  of  which 
the  largest  portion  seems  to  be  a  nucleoprotein  which  is  insoluble  in 
water  and  which  expands  into  a  tough,  slimy  mass  when  treated  with  a 
10-per  cent  common-salt  solution.  This  protein  substance,  which  is 
soluble  in  alkali  but  is  quickly  changed  thereby,  is  called  Rovida's  hyaline 
substance,  and  the  property  of  the  pus  of  being  converted  into  a  slime- 
like mass  by  a  solution  of  common  salt  depends  on  this  substance.  Besides 
this  substance,  to  which  the  nucleoprotein  of  the  pus-cells  investigated 
by  Strada  1  seems  to  stand  in  close  relation,  we  also  have  a  globulin 
which  coagulates  at  48-49°  C,  as  well  as  serglobulin  (?),  seralbumint 
a  substance  similar  to  coagulated  protein  (Miescher),  and  lastly  peptone 
or  proteose  (Hofmeister2).  It  is  very  remarkable  that  no  nucleo- 
histone  or  histone  has  been  detected  in  the  pus-cells,  although  histone 
occurs  in  the  cells  of  the  lymph  glands. 

There  are  also  found  in  the  protoplasm  of  the  pus-cells,  besides  the 
proteins,  lecithin,  cholesterin,  glucolhionic  acid,3  purine  bodies,  fat,  and  soaps. 
Hoppe-Seyler  has  found  cerebrin,  a  decomposition  product  of  a  pro- 
tagon-like  substance,  in  pus  (see  Chapter  XI).  Kossel  and  Freytag4 
have   isolated   from   pus   two   substances,   pyosin  and  pyogenin,   which 

1  Bioch.  Zeitschr.,  16. 

2  Miescher  in  Hoppe-Seyler's  Med.-Chem.  Untersuch.,  441;  Ch.  Pone.  Maly's  Jahresb., 
39;  Hofmeister,  Zeitschr.  f.  physiol.  Chem.,  4. 

3  Mandel  and  Levene,  Bioch.  Zeitschr.,  4. 
♦Zeitschr.  f.  physiol.  Chem.,  17,  452. 


PUS.  365 

belong  to  the  cerebrin  group  (see  Chapter  XI).  Hoppe-Seyler  x 
claims  that  glycogen  appears  only  in  the  living,  contractile  white  blood- 
cells  and  not  in  the  dead  pus-corpuscles.  Several  other  investigators 
have,  nevertheless,  found  glycogen  in  pus.  The  cell-nucleus  contains 
nuclein  and  nucleoproteins. 

In  regard  to  the  occurrence  of  enzymes  in  the  pus-cells  it  must  be 
remarked  that  neither  thrombin  nor  prothrombin  is  found  therein, 
although  these  bodies  are  generally  considered  as  being  derived  from 
the  leucocytes,  and  also  obtainable  from  the  thymus  leucocytes.  The 
occurrence  in  the  pus-cells,  besides  catalases  and  oxidases,  of  a  proteolytic 
enzyme,  is  of  great  interest.  It  is  not  only  important  for  the  intracellular 
digestion  and  for  the  amount  of  proteoses  in  the  pus-cell,  but  also  for 
the  solution  of  the  fibrin  clot  and  pneumonic  infiltrations  (Fr.  Muller, 
O.  Simon  2).  A  lipase,  which  splits  neutral  fats,  also  occurs,  according  to 
Fiessinger  and  Marie,  in  pus. 

The  mineral  constituents  of  the  pus-corpuscles  are  potassium,  sodium, 
calcium,  magnesium,  and  iron.  A  part  of  the  alkalies  exists  as  chlorides, 
and  the  remainder,  as  well  as  the  chief  part  of  the  other  bases,  exists 
as  phosphates. 

The  quantitative  composition  of  the  pus-cells  from  the  analyses  of 
Hoppe-Seyler  is  as  follows,  in  parts  per  1000  of  the  dried  substance: 

i.  ii. 

Proteins 137 .  62  ] 

Nuclein 342.57^685.85        673.69 

Insoluble  bodies 205 .  66  J 

Lecithin \    .  .Q  „Q  75.64 

Fat /    14d8d  75.00     • 

Cholesterin 74.00  72.83 

Cerebrin 51 .99 


Extractive  bodies 44 .  33 


102.84 


MINERAL  SUBSTANCES  IN  1000  PARTS  OF  THE  DRIED  SUBSTANCE. 

NaCl 4.34 

Ca3(P04)2 2.05 

Mg3(P04)2 1 .  13 

FePO-4 1.06 

P04 9.I6 

Na 0.68 

K Traces  (?) 

Miescher  obtained  other  results  for  the  alkali  compounds,  namely,  potas- 
sium phosphate  12,  sodium  phosphate  6.1,  earthy  phosphate  and  iron  phos- 
phate 4.2,  sodium  chloride  1.4,  and  phosphoric  acid  combined  with  organic  sub- 
stances 3.14-2.03  p.  m. 

In  pus  from  congested  abscesses  which  has  stagnated  for  some  time 
there  occur  peptone  (proteose),  leucine  and  tyrosine,  free  fatty  acids  and 

1  Hoppe-Seyler,  Physiol.  Chem.,  790. 

2Fr.  Muller,  Verhandl.  Nat.  Gesellsch.  zu.  Basel,  1901;  O.  Simon,  Deutsch.  Arch, 
f.  klin.  Med.,  70. 


366  CHYLE,   LYMPH,  TRANSUDATES  AND  EXUDATES. 

volatile  fatty  acids,  such  as  formic  acid,  butyric  acid  and  valeric  acid. 
There  are  also  found  urea,  glucose  (in  diabetes),  bile-pigments,  and  bile- 
acids  (in  catarrhal  icterus). 

As  more  specific  but  not  constant  constituents  of  the  pus  must  be 
mentioned  the  following:  pyin,  which  seems  to  be  a  nucleoprotein  pre- 
cipitable  by  acetic  acid,  and  also  pyinic  acid  and  chlorrhodinic  acid,  which 
have  been  so  little  studied  that  they  cannot  be  more  fully  treated  here. 

In  many  cases  a  blue,  more  rarely  a  green,  color,  has  been  observed 
in  the  pus.  This  depends  on  the  presence  of  micro-organisms  (Bacillus 
pyocyaneus).  From  such  pus  Fordos  and  Lucke  x  have  isolated  a  crys- 
talline blue  pigment,  pyocyanin,  and  a  yellow  pigment,  pyoxanthose, 
which  is  produced  from  the  first  by  oxidation. 

Appendix. 
Lymphatic  Glands,  Spleen  and  Endocrinic  Glands. 

The  Lymphatic  Glands.  The  cells  of  the  lymphatic  glands  are 
found  to  contain  the  protein  substances  generally  occurring  in  cells 
(Chapter  V).  According  to  Bang2  they  also  contain  histone  nucleates 
(nucleohistone),  but  in  smaller  amounts  and  of  a  different  variety  from 
the  better-studied  nucleohistone  from  the  thymus  gland.  Proteoses 
occur  as  products  of  autolysis.  By  a  lengthy  autolysis  of  lymph  glands 
Reh3  found  ammonia,  tyrosine,  leucine  (somewhat  scanty),  thymine, 
and  uracil  among  the  cleavage  products.  Besides  the  other  ordinary 
tissue  constituents,  such  as  collagen,  reticulin,  elastin,  and  nuclein,  there 
occur  in  the  lymphatic  glands  also  cholesterin,  fat,  glycogen,  sarcolactic 
acid,  purine  bases,  and  leucine.  In  the  inguinal  glands  of  an  old  woman 
Oidtmann  found  714.32  p.  m.  water,  284.5  p.  m.  organic  and  1.16  p.  m. 
inorganic  substances.  In  the  cells  of  the  mesenteric  lymphatic  glands 
of  oxen,  Bang4  found  804.1  p.  m.  water,  195.9  p.  m.  solids,  137.9  total 
proteins,  6.9  p.  m.  histone  nucleate,  10.6  p.  m.  nucleoprotein,  47.6  p.  m. 
bodies  soluble  in  alcohol,  and  10.5  p.  m.  mineral  constituents. 

The  Thymus.  The  cells  of  this  gland  are  very  rich  in  nuclein  bodies 
and  relatively  poor  in  the  ordinary  proteins,  but  their  nature  has  not  been 
closely  studied.  The  chief  interest  is  attached  to  the  nuclein  substances. 
Kossel  and  Lilienfeld  first  prepared  from  the  watery  extract  of  the 
gland,  by  precipitating  with  acetic  acid  and  then  further  purifying,  a 


1  Fordos,  Compt.  Rend.,  51  and  56;  Lucke,  Arch.  f.  klin.  Chirurg.,  3;  Boland,  Cen- 
tralbl.  f.  Bakt.  u.  Parasit.,  I.,  25. 

2  Studio-  over  Nucleoproteider,  Krietiania,  1902,  and  Hofmeister'e  Beitrage,  4. 

3  Hofmeister'a  Beitrage,  3. 
M.  c. 


THYMUS.  367 

protein  substance  which  has  been  generally  called  nucleohislone.  By  the 
action  of  dilute  hydrochloric  acid  upon  nucleohistone  it  splits,  according 
to  these  investigators,  into  histone  and  leuconuclein.  The  leuconuclein 
is  a  true  nuclein;  hence  it  is  a  nucleic-acid  compound  with  protein  which 
is  relatively  poor  in  protein  and  rich  in  phosphorus.  The  more  recent 
investigations  of  Bang,  Malengreau,  Huiskamp  and  Gouban  *  upon 
nucleohistone  all  show  that  this  nucleoprotein  is  not  a  unit  substance,  but 
a  mixture  of  at  least  two  bodies.  The  views  of  the  investigators  men- 
tioned differ  quite  essentially  from  one  another  as  to  the  nature  of  these 
bodies,  but  this  is  partly  due  to  the  different  methods  used  by  them  and 
partly  to  the  ready  changeability  of  the  substances  in  question. 

Besides  the  real  nucleohistone,  B-nucleoalbumin  of  Malengreau, 
Lilienfeld's  histone  contains  a  second  nucleoprotein  which  Bang  and 
Huiskamp  call  simple  nucleoprotein,  while  Malengreau  designates 
it  A-nucleoalbumin.  This  protein,  which  contains  only  about  1  per  cent 
phosphorus  and  which  is  possibly  identical  with  the  nucleoprotein  found 
by  Lilienfeld  in  the  thymus,  yields  a  nuclein,  but  no  free  nucleic  acid, 
on  cleavage.  As  a  second  cleavage  product  it  yields,  according  to  Mal- 
engreau, the  A-histone,  which  can  be  readily  precipitated  by  magnesium 
and  ammonium  sulphates  from  the  ordinary  B-histone  of  the  thymus 
gland.  The  occurrence  of  A-histone  in  the  gland  has  been  verified  by 
Bang,  and  according  to  Bang  and  Huiskamp  the  A-histone  is  not  derived 
from  the  nucleoprotein,  as  these  investigators  claim  that  it  yields  no  his- 
tone. According  to  Bang  the  nucleoprotein  yields  only  an  albuminate, 
besides  the  nuclein,  as  cleavage  products.  According  to  Gouban  we 
have  been  dealing  with  three  substances,  namely  a  nucleoprotein  which 
does  not  yield  any  histone,  and  two  nucleohistones,  which  correspond 
to  the  nucleoalbumins  A  and  B  of  Malengreau  and  form  the  mixture 
of  lime-nucleohistone  of  Huiskamp.  They  occur  in  this  last  mentioned 
mixture  in  a  somewhat  modified  form  due  to  the  method  of  preparation. 

The  true  nucleohistone,  which  is  much  richer  in  phosphorus  (the- 
calcium  salt  containing,  according  to  Bang,  on  an  average  5.23  per  cent 
P),  yields  ordinary  histone  (or  2  histones)  as  one  cleavage  product  and 
free  nucleic  acid  as  the  other.  According  to  Bang,  whose  statements 
on  this  point  have  been  substantiated  by  Malengreau,  it  splits  on  saturat- 
ing with  NaCl  into  nucleic  acid  and  histone  without  yielding  any  other 
protein.  On  this  account  Bang  does  not  consider  this  body  as  nucleo- 
histone in  the  ordinary  sense,  i.e.,  not  as  a  nucleoprotein,  but  as  a  histone 


1  Lilienfeld,  Zeitschr.  f.  physiol.  Chem.,  18;  Kossel,  ibid.,  30  and  31;  Bang.,  ibid. 
30  and  31.  See  also  Arch.  f.  Math,  og  Naturvidenskab,  25,  Kristiania,  1902,  and 
Hofmeister's  Beitrage,  1  and  4;  Malengreau,  La  Cellule,  17  and  19;  Huiskamp,  Zeit- 
schr. f.  physiol.  Chem.,  32,  34  and  39;  Gouban,  Bioch.  Centralbl.,  9,  803. 


36S  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

nucleate.  We  cannot  say  whether  this  applies  to  the  two  nucleohistones 
(if  there  are  two).  The  nucleohistone  or  mixture  of  nucleohistones 
behave  like  an  acid,  whose  salts,  especially  the  calcium  salt,  have  been 
closely  studied  by  Huiskamp.  On  the  electrolysis  of  a  solution  of  alkali 
nucleohistone  in  water  Huiskamp  also  found  that  the  nucleohistone 
collected  in  traces  at  the  anode,  and  that  the  sodium  compound  is  there- 
fore ionized  in  the  solution.  The  nucleic  acid-calcium  histone-com- 
pound  has  been  prepared,  it  seems,  in  a  pure  state  by  Bang,  and  he  found 
the  following  average  composition:  C  43.69;  H  5.60;  N  16.87;  S  0.47; 
P  5.23;  Ca  1.71  per  cent. 

The  nucleohistone  prepared  by  Huiskamp' s  method  of  precipitating  with 
CaCb  is,  according  to  him,  a  mixture  of  two  nucleohistones,  of  which  one,  the 
a-nucleohistone,  contains  4.5  per  cent  phosphorus,  and  the  other,  |3-nucleohistone 
contains,  on  the  contrary,  only  in  round  numbers  3  per  cent  phosphorus.1  As 
the  two  nucleohistones  are  poorer  in  phosphorus  than  the  nucleic  acid-histone 
compound  analyzed  by  Bang,  and  as  Huiskamp  on  cleavage  of  his  preparation 
did  not,  like  Bang  and  Malengreau,  obtain  pure  nucleic  acid,  it  is  still  a  ques- 
tion whether  Huiskamp  was  working  with  sufficiently  pure  substances. 

In  regard  to  the  methods  used  by  the  above  investigators  in  the 
isolation  of  the  bodies  in  question  we  must  refer  to  the  original  publications. 

In  connection  with  the  so-called  nucleohistone,  attention  must  be  called  to 
tissue  fibrinogen  and  cell  fibrinogen,  which  are  compound  proteins,  and  are  claimed 
by  certain  investigators  to  stand  in  close  relation  to  the  coagulation  of  the  blood. 
These  may  be  in  part  nucleoproteins  and  in  part  also  nucleohistones.  To  this  same 
group  belong  also  the  important  cell  constituents  described  by  Alex.  Schmidt  2 
and  called  cytoglobin  and  preglobulin.  The  cytoglobin,  which  is  soluble  in  water, 
may  be  considered  as  the  alkali  compound  of  preglobulin.  The  residue  of  the 
cells  left  after  complete  extraction  with  alcohol,  water,  and  salt  solution  has 
been  called  cytin  by  Alex.  Schmidt. 

Besides  the  above-mentioned  and  the  ordinary  bodies  belonging  to 
the  connective-tissue  group,  small  quantities  of  fat,  leucine,  succinic 
acid,  lactic  acid,  sugar,  and  traces  of  iodothyrin  are  present.  According 
to  Gautier  3  arsenic  also  occurs  in  very  small  amounts,  and  no  doubt 
here  as  well  as  in  other  organs  it  is  related  to  the  nuclein  substances.  The 
richness  in  nuclein  bodies  explains  the  occurrence  of  large  quantities 
of  purine  bases,  chiefly  adenine,  whose  quantity,  according  to  Kossel 
and  Sciiindler,4  is  1.79  p.  m.  in  the  fresh  organ  and  19.19  p.  m.  in  the 
dry  substance,  and  guanine.  The  bodies  thymine  and  (uracil?)  obtained, 
besides  lysine  and  ammonia,  by  Kutscher,  as  products  of  autodiges- 
tion  of  the  gland,  probably  have  a  similar  origin.     Among  the  enzymes, 

1  Zeitschr.  f.  phy.siol.  Chem.,  39. 

2  See  footnote  1,  p.  307. 
8Compt.  Rend.,  129. 

*  Zeitschr.  f.  physiol.  Chem.,  13;  Kutacher,  Zeitachr.  f.  phyaiol.  Chem.,  34. 


THYMUS  AND   SPLEEN.  369 

besides  argimue,  guanase,  adenase,  and  proteolytic  enzyme  we  must 
especially  mention  the  enzyme  studied  by  Jones,1  which  acts  like  a  nu- 
clease, splitting  off  phosphoric  acid  and  purine  bases,  from  the  nucleo- 
proteins.  This  enzyme,  contrary  to  trypsin,  acts  best  in  acid  liquids,  and 
is  readily  destroyed  by  alkalies  at  body  temperature.  The  quantitative 
composition  of  the  lymphocytes  from  the  thymus  of  a  calf  is,  according  to 
Lilienfeld's  analysis,  as  follows.  The  results  are  given  in  1000  parts 
of  the  dried  substance: 

Proteids 17.7 

Leuconuclein 687 . 9 

Histone 86 . 7 

Lecithin 75 . 1 

Fat 40.2 

Cholesterin 44 . 0 

Glycogen 8.0 

The  dried  substance  of  the  leucocytes  amounted  to  an  average  of 
114.9  p.  m.  Potassium  and  phosphoric  acid  are  prominent  mineral 
constituents.  Lilienfeld  found  KH2PO4  among  the  bodies  soluble  in 
alcohol. 

Attention  must  be  called  to  the  analyses  of  Bang,2  which  show  that 
the  thymus  contains  about  the  same  quantity  of  nucleoprotein,  but  about 
five  times  as  much  histone  nucleate  as  the  lymphatic  glands — calculated 
in  both  cases  upon  the  same  amount  of  dry  substance.  Oidtman  3  found 
807.06  p.  m.  water,  192.74  p.  m.  organic  and  0.2  p.  m.  inorganic  sub- 
stances in  the  gland  of  a  child  two  weeks  old. 

In  regard  to  the  functions  of  the  thymus  it  seems  to  be  the  general 
view  that  this  gland  takes  part  in  the  recruiting  of  the  blood  lympho- 
cytes and  correspondingly  belong  to  the  lymphoid  organs.  On  the  other 
hand  also  certain  other  observations  indicate  that  it  may  belong  to  the 
endocrinic  organs.  It  is  generally  admitted  that  the  extirpation  of  the 
thymus  leads  to  a  reduction  and  change  in  the  formation  of  bone.  A 
certain  relation  also  exists  with  the  organs  of  generation  and  perhaps 
a  reciprocal  action  also  exists  between  it  and  other  organs  with  internal 
secretion. 

The  Spleen.  The  pulp  of  the  spleen  cannot  be  freed  from  blood. 
The  mass  which  is  separated  from  the  spleen  capsule  and  the  structural 
tissue  by  pressure,  and  which  ordinarily  serves  as  material  for  chemical 
investigations  is,  therefore  a  mixture  of  blood  and  spleen  constituents. 
For  this  reason  the  proteins  of  the  spleen  are  little  known.  The  nucleo- 
protein isolated  by  Levene  and  Mandel  4  is  to  be  considered  as  a  true 

1  Zeitschr.  f.  physiol.  Chem.,  41. 
2 1.  c,  Arch.  f.  Math.,  etc. 

3  Cited  from  v.  Gorup-Besanez,  Lehrb.  d.  physiol.  Chem.,  4.  Aufl.,  p.  732. 

4  Bioch.  Zeitschr.,  5. 


370  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

spleen  constituent,  and  this  nucleoprotein  yields  25  per  cent  glutamic 
acid  on  hydrolysis.  Histone  has  not  been  directly  detected  in  the  spleen; 
but  its  presence  is  to  be  admitted  because  Krasnosselsky  x  was  able 
to  isolate  a  histone-peptone  as  sulphate  from  the  spleen.  The  ferruginous 
albuminate  has  been  considered  as  a  spleen  constituent  for  a  long  time, 
and  especially  also  a  protein  substance  which  does  not  coagulate  on  boil- 
ing and  which  is  precipitated  by  acetic  acid  and  yields  an  ash  contain- 
ing much  phosphoric  acid  and  iron  oxide.  This  substance  is  probably 
identical  with  the  nucleoproteins  which  later  investigators  such  as  Sato 
and  Capezzuoli  2  have  prepared  from  the  spleen.  These  nucleoproteins, 
which  are  modified  products,  contain  iron  in  variable  amounts  and  more 
or  less  firmly  combined. 

The  pulp  of  the  spleen,  when  fresh,  has  an  alkaline  reaction,  but 
quickly  turns  acid,  due  partly  to  the  formation  of  free  paralactic  acid 
and  partly  perhaps  to  glycerophosphoric  acid.  Besides  these  two  acids 
there  are  found  in  the  spleen  also  volatile  fatty  acids,  as  formic,  acetic, 
and  butyric  acids,  as  well  as  succinic  acid,  neutral  fats,  cholesterin,  traces 
of  leucine,  inosite  (in  ox-spleen),  scyllite,  a  body  related  to  inosite  (in  the 
spleen  of  Plagiostoma) ,  glycogen  (in  dog-spleen),  uric  acid,  purine  bases, 
and  jecorin.  Levene  found  a  glucothionic  acid  in  the  spleen,  i.e.,  an 
acid  which  is  related  to  chondroitin-sulphuric  acid  but  not  identical 
therewith,  and  which  gives  a  beautiful  violet  coloration  with  orcin  and 
hydrochloric  acid.  The  question  whether  this  glucothionic  acid  originates 
from  the  above-mentioned  nucleoprotein  or  from  the  mucoid  substance 
has  not  been  decided  (Levene  and  Mandel).  In  regard  to  the  question 
whether  this  acid  is  a  unit  body  or  not  we  refer  to  the  work  of  Mandel 
and  Neuberg  and  Levene  and  Jacobs.3 

In  the  human  and  ox-spleen  Burow  4  has  found  three  phosphatides 
which  all  contain  iron  in  organic  combination.  Among  these  one  is  a 
saturated  diaminomonophosphatide  and  the  other  two  are  unsaturated 
phosphatides. 

Many  enzymes  are  found  in  the  spleen  also,  and  certain  of  these 
are  of  special  interest.  To  these  belong  the  uric-acid-forming  enzyme, 
the  xanthine  oxidase  (Burian),  which  occurs  in  the  spleen  of  many 
animals,  but  not  in  man,  and  which  transforms  the  oxypurines, 
hypoxanthine,  and  xanthine  into  uric  acid;   also  the  deamidizing  enzymes 


1  Zeitschr.  f.  physiol.  Chem.,  49. 

2  Sato,  Bioch.  Zeitschr.,  22;  Capezzuoli;  Zeitschr.  f.  physiol.  Chem.,  60. 

8  Levene,  Zeitschr.  f.  physiol.  Chem.,  37;  Levene  and  Mandel,  ibid.,  45  and  47; 
Mandel  and  Neuberg,  Bioch.  Zeitschr.,  13;  Levene,  ibid.,  16;  Neuberg,  ibid.,  16;  Levene 
and  Jacobs,  Joum.  of  experim.  Medic,  10. 

*  Bioch.  Zeitschr.,  25. 


SPLEEN.  371 

guanase  and  adenase  (Levene,  Schittenhelm,  Jones  and  Partridge, 
Jones  and  Winternitz),  by  the  first  of  which  the  guanine  is  transformed 
into  xanthine,  and  by  the  latter  the  adenine  into  hypoxanthine.  The 
guanase  also  occurs  in  the  spleen  of  the  ox  and  horse,  but  not  (Jones), 
or  only  in  small  amounts  (Schittenhelm),  in  the  pig-spleen.1  The 
spleen  also  contains  two  enzymes,  lienases,  as  shown  by  Hedin  (and 
Rowland),  one  of  which,  the  a-lienase,  acts  chiefly  in  alkaline  solution, 
while  the  other,  0-lienase,  is  active  only  in  acid  reaction.  These  enzymes, 
which  without  doubt  stand  in  close  relation  to  the  leucocytes,  not  only 
act  autolytically  upon  the  proteins  of  the  spleen,  but  they  also  dissolve 
fibrin  and  coagulated  blood-serum.  The  spleen  also  contains  nucleases 
and  besides,  as  Tanaka  2  has  found  for  the  pig-spleen,  diastase,  invertin, 
lipase,  urease,  trypsin  and  an  erepsin  like  enzyme. 

Among  the  constituents  of  the  spleen  the  deposit  rich  in  iron,  which 
consists  of  ferruginous  granules  or  conglomerate  masses  of  them,  and 
which  is  derived  from  a  transformation  of  the  red  blood-corpuscles,  is  of 
special  interest.  It  was  closely  studied  by  Nasse.  This  deposit  does 
not  occur  to  the  same  extent  in  the  spleen  of  all  animals.  It  is  found 
especially  abundant  in  the  spleen  of  the  horse.  Nasse  3  on  analyzing 
the  grains  (from  the  spleen  of  a  horse)  obtained  840-630  p.  m.  organic 
and  160-370  p.  m.  inorganic  substances.  These  last  consisted  of  566- 
726  p.  m.  Fe203,  205-388  p.  m.  P205,  and  57  p.  m.  earths.  The  organic 
substances  consisted  chiefly  of  proteins  (660-800  p.  m.),  nuclein  (52  p.  m. 
maximum),  a  yellow  coloring-matter,  extractive  bodies,  fat,  cholesterin, 
and  lecithin. 

In  regard  to  the  mineral  constituents,  it  is  to  be  observed  that  the 
amount  of  iron  in  new-born  and  young  animals  is  small  (Lapicque, 
Kruger,  and  Pernou),  in  adults  more  appreciable,  and  in  old  animals 
sometimes  very  considerable.  Nasse  found  nearly  50  p.  m.  iron  in  the 
dried  pulp  of  the  spleen  of  an  old  horse.  Guillemonat  and  Lapicque  4 
have  determined  the  iron  in  man.  They  find  no  regular  increase  with 
growth,  but  in  most  cases  0.17-0.39  p.  m.  (after  subtracting  the  blood- 
iron)  calculated  on  the  fresh  substance.  A  remarkably  high  amount  of 
iron  is  not  dependent  upon  old  age,  but  is  a  residue  from  chronic  diseases. 
Magnus-Levy  found  0.72  p.  m.  iron  in  the  fresh  human  spleen. 


1  See  Chapter  XIV  for  the  literature. 

2  Hedin  and  Rowland,  Zeitschr.  f.  physiol.  Chem.,  32,  and  Hedin,  Journ.  of  Physiol., 
30,  and  Hammarsten's  Festschr.,  1906;  Tanaka,  Bioch.  Zeitschr,  37. 

3  Maly's  Jahresber.,  19,  p.  315. 

4  Lapicque,  ibid.,  20;  Lapicque  and  Guillemonat,  Cornpt,  rend,  de  soc.  biol.,  48, 
and  Arch  de  Physiol.  (5)  8;  Kruger  and  Pernou,  Zeitschr.  f.  Biologie,  2";  Nasse,  cited 
from  Hoppe-Seyler,  Physiol.  Chem.,  720. 


372  CHYLE,   LYMPH,  TRANSUDATES  AND  EXUDATES. 

On  the  analysis  of  the  human  spleen  Magnus-Levy  found  784.7 
parts  water,  215.3  parts  solids,  27.7  parts  fat  and  27.9  parts  nitrogen  in 
1000  parts  of  the  fresh  organ.  In  the  dog  spleen,  Corper  1  found  750 
to  770  p.  m.  water,  and  120-150  p.  m.  ether  soluble  substances,  of  which 
one-fourth  consisted  of  cholesterin  and  three-fourths  of  lecithin.  As 
purine  bases  he  found  1.1  p.  m.  guanine,  0.6  p.  m.  adenine,  0.15  p.  m. 
hypoxanthine  and  0.04  p.  m.  xanthine. 

In  regard  to  the  pathological  processes  going  on  in  the  spleen  we  must 
specially  recall  the  abundant  re-formation  of  leucocytes  in  leucaemia  and 
the  appearance  of  amyloid  substance  (see  page  172) . 

The  physiological  functions  of  the  spleen  are  little  known,  with  the 
exception  of  its  importance  in  the  formation  of  leucocytes.  Some 
consider  the  spleen  as  an  organ  for  the  dissolution  of  the  red  blood- 
corpuscles,  and  the  occurrence  of  the  above-mentioned  deposit  rich  in 
iron  seems  to  confirm  this  view,  but  this  iron  could  in  part  have  another 
origin.  Asher  and  his  collaborators  Grossenbacher,  Zimmermann  and 
H.  Vogel  have  found  that  the  spleen  is  an  organ  for  the  iron  metabolism, 
as  they  found  in  a  splenectomized  dog  that  the  iron  elimination  was  much 
greater  than  in  a  dog  with  its  spleen.  R.  Bayer2  has  made  a  similar 
observation  on  a  splenectomized  human  being,  and  the  spleen  it  seems 
has  the  purpose  of  retaining  for  the  organism  the  iron  set  free  in  the 
metabolism  and  also  in  starvation  metabolism. 

The  spleen  has  also  been  claimed  to  play  a  certain  part  in  digestion 
especially  in  pancreatic  digestion.  This  organ  is  said  by  Schiff,  Herzen, 
and  others  to  be  of  importance  in  the  production  of  trypsin  in  the  pan- 
creas. The  investigations  of  Herzen  seem  to  confirm  this  relation,  but 
the  recent  work  of  Prym  3  has  made  the  assumption  doubtful. 

Splenectomized  dogs  require  according  to  Richet  4  for  their  mainte- 
nance more  food,  about  one-third  more,  than  normal  dogs.  The  spleen 
makes  a  complete  utilization  of  the  food  possible  or  diminishes  its  con- 
sumption. 

An  increase  in  the  quantity  of  uric  acid  eliminated  in  splenic  leucaemia 
has  been  observed  by  many  investigators  (see  Chapter  XIV),  while  the 
reverse  has  been  observed  under  the  influence  of  quinine  in  large  doses, 
which  produces  an  enlargement  of  the  spleen.  These  facts  give  a  rather 
positive  proof  that  there  is  a  close  relation  between  the  spleen  and  the 


1  Magnus-Levy,  Bioch.  Zeitschr.,  24;  H.  J.  Corper,  Journ.  of  biol.  Chem.,  11. 

2  Asher  and  Grossenbacher,  Centralbl.  f.  Physiol..  22,  375,  and  Bioch.  Zeitschr,  17  ; 
Zimmermann,  Bioch.  Zeitschr.,  17;  R.  Bayer,  Bioch.  Centralbl.,  9,  815. 

:;  Schiff,  cited  by  Herzen,  Pfliiger's  Arch.,  30,   295,  308,  and  84,  and  Maly's  Jahr- 
esber.,  18;  Prym,  Pfliiger's  Arch.,  104  and  107;  see  also  Chapter  VIII. 
4  Journ.  de  Physiol,  et  de  Pathol,  gen.,  14  and  15. 


THYROID   GLAND.  373 

formation  of  uric  acid.  This  relation  has  been  studied  by  Horbac- 
zewski.  He  has  shown  that  when  the  spleen-pulp  and  blood  of  calves 
arc  allowed  to  act  on  each  other,  under  certain  conditions  and  certain  tem- 
perature, in  the  presence  of  air,  large  quantities  of  uric  acid  are  formed, 
and  he  has  also  shown  that  the  uric  acid  originates  from  the  nucleins  of 
the  spleen.1  This  behavior  is  explained  by  the  above-mentioned  inves- 
tigations of  Burian,  Schittenhelm,  Jones,  and  others  on  the  enzymotic 
formation  of  uric  acid,  and  the  deamidization  of  the  purine  bodies,  and  a 
relation  between  the  spleen  and  uric-acid  formation  is  indisputable. 
Still  we  cannot  say  that  the  spleen  shows  a  special  relation  to  the  uric-acid 
formation  as  compared  with  other  organs  (see  Chapter  XIV). 

The  spleen  has  the  same  property  as  the  liver  of  retaining  foreign 
bodies,  metals  and  metalloids. 

The  Thyroid  Gland.  The  nature  of  the  different  protein  substances 
occurring  in  the  thyroid  gland  has  not  been  sufficiently  studied,  but  at 
present,  through  the  researches  of  Oswald,  there  are  known  at  least  two 
bodies  which  are  constituents  of  the  so-called  secretion  of  the  glands, 
the  colloids.  One  of  these,  iodothyreoglobulin,  behaves  like  a  globulin,  while 
the  other  is  a  nucleoprotein  (see  also  Gotjrlay2).  The  iodine  present 
in  the  gland  occurs  chiefly  in  the  first  body,  while  the  arsenic,  which  has 
been  shown  to  be  a  normal  constituent  by  Gautier  and  Bertrand,* 
seems  to  be  related  to  the  nuclein  substances. 

According  to  Oswald  the  iodothyreoglobulin  occurs  only  in  those 
glands  which  contain  colloid,  while  the  colloid-free  glands,  the  parenchyma- 
tous goitre,  and  the  glands  of  the  new-born  contain  thyreoglobulin  free 
from  iodine.  The  thyreoglobulin  first  becomes  iodized  into  iodothyreo- 
globulin on  passing  from  the  follicle-cells.  Besides  these  mentioned 
bodies  leucine,  xanthine,  hypoxanthine,  choline,  iodothyrine,  lactic  and 
succinic  acids  occur  in  the  thyreoidea.  Like  certain  other  organs,  sub- 
stances also  occur  in  the  thyroid  which  act  upon  the  blood  pressure  and 
indeed  partly  as  vasodilator  and  partly  depressing  but  whose  chemical 
nature  has  not  been  positively  established.  Among  the  enzymes  we 
•find  lipases  and  catalases  which,  according  to  Juschtschenko,4  are 
related  to  the  corresponding  enzymes  of  the  blood.  Magnus-Levy  5 
found  757  parts  water,  243  parts  solids,  43.8  parts  fat,  26.8  parts  nitrogen, 
and  0.058  parts  iron  in  1000  parts  of  the  human  thyroid  gland. 

1  Monatshefte  f.  Chem.,  10,  and  Wein.  Sitzungsber.  Math.  Nat.  Klasse,  100,  Abt.  3. 

2  Gourlay,  Journ.  of  Physiol.,  16;  Oswald,  Zeitschr.  f.  physiol.  Chem.,  32,  and 
Biochem.  Centralbl.,  1,  249. 

3  Gautier,  Compt.  Rend.,  129.  See  also  ibid.,  130,  131,  134,  135;  Bertrand,  ibid., 
134,  135. 

4  Juschtschenko,  Bioch.  Zeitschr.,  25  and  Arch,  scienc.  biol.  de  St.  Petersbourg,  15. 

5  Bioch.  Zeitschr.,  24. 


374     CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

In  "  strumacystica  "  Hoppe-Seyler  found  hardly  any  protein  in  the  smaller 
glandular  vessels,  but  an  excess  of  mucin,  while  in  the  larger  he  found  a  great 
deal  of  proteinl  70-80  p.  m.1  Cholesterin  is  regularly  found  in  such  cysts,  some- 
times in  such  large  quantities  that  the  entire  contents  form  a  thick  mass  of  cho- 
lesterin plates.  Crystals  of  calcium  oxalate  also  occur  frequently.  The  contents 
of  the  struma  cysts  are  sometimes  of  a  brown  color,  due  to  decomposed  coloring- 
matter,  methcemoglobin  (and  haematin?).  Bile-coloring  matters  have  also  been 
found  in  such  cysts.  (In  regard  to  the  paralbumins  and  colloids  which  have  been 
found  in  struma  cysts  and  colloid  degeneration,  see  Chapter  XII.) 

Those  substances  which  bear  a  close  relation  to  the  functions  of  the 
gland  seem  to  be  of  special  interest. 

The  complete  extirpation,  as  also  the  pathological  destruction,  of  the 
thyroid  gland  causes  great  disturbances,  ending  finally  in  death.  In 
dogs,  after  the  total  extirpation,  a  disturbance  of  the  nervous  and  muscular 
systems  occurs,  such  as  trembling  and  convulsions,  ^and  death  generally 
supervenes  shortly  after,  most  often  during  such  an  attack.  The  researches 
of  Gley,  Vassale  and  Generalt  2  upon  various  animals  have  shown 
that  for  the  success  cf  the  operation  it  is  of  the  greatest  importance 
whether  the  parathyroids,  discovered  by  Sandstrov,3  are  removed  at 
the  same  time  or  not.  In  herbivora  (rabbits)  because  of  the  anatomical 
relations,  the  parathyroids  are  seldom  extirpated  in  the  operation  of 
the  removal  of  the  thyroid,  the  tetany  does  not  regularly  occur  and 
the  disturbance  in  metabolism  is  most  striking.  If  these  glands  are 
not  extirpated  in  dogs,  the  tetany  also  does  not  appear,  and  the  dis- 
turbances in  metabolism  occur.  In  human  beings,  after  the  removal  of 
the  gland  by  operation,  different  disturbances  appear,  such  as  nervous 
symptoms,  diminished  intelligence,  dryness  of  the  skin,  falling  out  of 
the  hair,  and,  on  the  whole,  those  symptoms  which  are  included  under 
the  name  cachexia  thyreopriva,  death  coming  gradually.  Among  these 
symptoms  must  be  mentioned  the  peculiar  slimy  infiltration  and  exuber- 
ance of  the  connective  tissue  called  myxedema. 

All  these  conditions  indicate  that  the  thyroids  belong  to  those  glands 
with  internal  secretion,  so  called  endocrinic  glands.  The  most  con- 
vincing proof  of  this  is  the  fact  that  the  ordinary  symptoms  do  not  occur 
if  a  small  piece  of  the  gland  is  allowed  to  remain  in  the  body,  or  even 
when  a  piece  of  the  gland  is  transplanted  in  any  part  of  the  body.  The 
observations  of  Asher  and  Flack4  that  the  irritation  of  the  nerves  of  the 
thyroid  causes  an  internal  secretion  from  the  thyroid  gland  into  the 
blood,  is  of  great  interest  in  this  connection.     A  further  proof  of  practical 

^Physiol.  Chem.,  p.  721. 

J  Gley,  Compt.  rend.  soc.  biol.,  1891,  and  Arch,  de  Physiol  (5),  4;  Vassale  and 
Generali,  Arch.  Ital.  d.  Biol.,  25  and  2fi. 
•Upaala  L&karef.  Fdrh.,   15  (1880). 
*  Asher  and  Flack,  Zeitscbr.  f.  Biol.,  55. 


THYROID  GLAND.  375 

importance  is  that  the  injurious  results  from  removal  of  the  thyroids 
can  be  counteracted  by  the  introduction  of  artificial  extracts  of  the 
thyroid  gland  into  the  body  or  by  feeding  with  thyroid  glands. 

Of  the  disturbances  in  metabolism  which  occur  on  the  extirpation 
or  reduction  of  the  thyroid  function  (athyreoidismus  or  hypothyreoid- 
ismus)  we  must  especially  mention  the  reduction  in  the  protein  catabolism 
which  in  a  starving  dog  without  thyroids  may  fall  to  about  one-half  of 
the  starvation  protein  metabolism  in  a  normal  dog  of  the  same  size 
(Falta  and  collaborators  J).  The  reverse  is  observed  when  large  quan- 
tities of  the  thyroid  gland  substance  is  fed,  namely,  a  strong  increase  in 
the  protein  metabolism,  besides  certain  other  symptoms.  Basedow's 
disease  is  also  considered  as  a  form  of  hyperthyreoidismus  which,  by  an 
increased  activity  of  the  glands,  brings  about  an  overproduction  of  the 
specific  secretion.  There  does  not  seem  to  be  any  doubt  that  the  thyroid 
glands  stand  in  close  relation  to  other  endocrinic  glands  although  for  the 
present  we  are  unable  to  survey  this  very  complicated  condition.  One 
side  of  this  reciprocal  action  with  other  organs,  which  is  of  special  impor- 
tance, is  the  relation  of  the  thyroids  to  glycosuria,  which  will  be  discussed 
in  a  following  chapter. 

The  glands  with  internal  secretion,  the  so-called  endocrinic  glands, 
to  which  the  adrenals  belong,  which  will  be  discussed  below,  and  the 
hypophysis,  are  of  especially  great  interest  because  of  the  reciprocal 
action  which  they  exert  among  each  other  and  with  other  organs.  A 
chemical  correlation  exists  between  different  organs,  of  a  kind,  that 
bodies  which  are  formed  in  one  organ  can  awaken  or  regulate  the  func- 
tions of  another  organ  or  other  organs.  These  chemically  active  sub- 
stances, which  awaken  or  regulate  the  activity  of  other  organs  have 
been  given  the  group  name  hormone  (6pfLaw  =  I  awaken  or  excite)  by  Star- 
ling and  to  this  group  belong  the  specifically  active  constituents  of  the 
endocrinic  glands. 

It  is  impossible  for  the  present  to  state  anything  about  the  kind  of 
bodies  having  a  specific  action  in  the  thyroid  gland  or  anything  about  the 
importance  of  the  bases  found  by  certain  investigators,  such  as  S.  Frank- 
el,  Drechsel,  and  Kocher,2  as  these  bodies  have  not  been  characterized 
sufficiently.  It  seems  proved  that  the  specifically  active  substance  is,  as 
first  shown  by  Notkin3  and  Oswald,4  a  protein  substance:  Notkin's 
thyreoproteid, Oswald's  thyreoglobulin .    This  does  not  conflict  with  the  views 


1  Eppinger,  Falta  and  Rudinger,  Zeitschr.  f.  klin.  Med.,  66. 

5  Frankel,  Wein.  med.  Blatter,  1895  and  1896;  Drechsel  and  Kocher,  CentralbL 
f.  Physiol.,  9,  705. 

3  Wien.  med.  Wochenschr.,  1895,  and  Virehow's  Arch.,  144,  Suppl.,  224. 
*  Zeitschr.  f.  physioi.  Ohem.,  32,  and  Bioch.  CentralbL,  1,  249. 


376  CHYLE,  LYMPH,  TRANSUDATES  AND  EXUDATES. 

of  Baumann  and  Roos  that  the  active  substance  is  iodothyrin,  as  thk 
can  be  produced  as  a  cleavage  product  from  the  iodothyreoglobulin.  In 
fact  Oswald  has  found  in  the  tryptic  digestion  of  iodothyreoglobulin 
that  a  substance  'similar  to  iodothyrin  is  produced;  important  inves- 
tigations x  nevertheless  make  it  probable  that  the  thyreoglobulin  is 
the  active  substance  and  not  the  iodothyrin.  There  are  several  reasons 
why  the  action  of  the  thyroid  gland  substance  is  not  due  to  one  substance, 
but  to  several. 

Iodothyrin  is  considered  by  Baumann,  who  first  showed  that  the  thyroid  con- 
tained iodine  and  who  with  Roos  2  proved  the  importance  of  this  substance  for  the 
physiological  activity  of  the  gland,  as  the  only  active  substance.  By  boiling  the 
finely  divided  gland  with  dilute  sulphuric  acid  Baumann  obtained  iodothyrin 
as  an  amorphous,  brown  mass,  nearly  insoluble  in  water  but  readily  soluble  in 
alkali  and  precipitated  again  by  the  addition  of  acid.  The  iodothyrin,  which 
is  not  a  unit  bod}',  has  a  variable  content  of  iodine  and  is  not  a  protein  substance. 
According  to  v.  Furth  and  Schwarz  it  is  probably  a  melanoid-like  transforma- 
tion product  of  the  iodized  protein  of  the  gland  produced  by  the  action  of  the 
acid. 

Thyreoglobulin  or  iodothyreoglobulin  was  obtained  by  Oswald  from 
the  watery  extract  of  the  gland  by  half  saturating  with  ammonium  sul- 
phate. It  has  the  properties  of  the  globulins  and  with  the  exception  of 
the  iodine  content  it  has  about  the  same  composition  as  the  proteins. 
The  amount  of  iodine  varies:  0.46  per  cent  in  pigs,  0.86  per  cent  in  oxen, 
and  0.34  per  cent  in  man.  In  the  iodothyreoglobulin  of  the  ox,  Nuren- 
berg3  found  0.59-0.86  per  cent  iodine  and  1.83-2.0  per  cent  sulphur. 
In  young  animals,  whose  glands  contain  no  iodine,  the  thyreoglobulin 
is  iodine-free.  Thyreoglobulin  on  taking  up  iodine  is  converted  into 
iodothyreoglobulin.  By  introducing  iodine  salts  the  iodine  content  of  the 
iodothyreoglobulin  can  be  raised  in  living  animals  and  thus  the  physiological 
activity  increased  (Oswald).  The  amount  of  iodine  in  the  gland  is 
markedly  dependent  upon  the  food. 

Jolin  has  examined  a  large  number  of  thyroid  glands  from  healthy   and 
diseased  persons  (in  Sweden),  for  their  iodine  content.     In  28  children,  ages 

1  See  Oswald.  Arch.  f.  exp.  Path.  u.  Pharm.,  60;  Pick  and  Pineles,  Zeitschr.  f. 
exp.  Path.  u.  Therap.,  7. 

2  In  regard  to  this  subject,  see  Baumann  and  Roos,  Zeitschr.  f.  physiol.  Chem.,  21 
and  22;  also  Baumann,  Miinch.  med.  Wochenschr.,  1896;  Baumann  and  Goldmann, 
ibid.;  Roos.,  ibid.;  v.  Furth  and  Schwarz,  Pfliiger's  Arch.,  124.  An  extensive  review 
of  the  literature  on  the  action  of  iodothyrin  and  the  thyroid  preparations  can  be  found 
in  Roos,  Zeitschr.  f.  physiol.  Chem.,  22,  18.  In  regard  to  their  action  in  protein  catabo- 
lism  and  in  metabolism,  see  F.  Voit,  Zeitschr.  f.  Biologie,  35;  Schondorff,  Pliiger's  Arch., 
67,  and  Andersson  and  Bergman,  Skand.  Arch.  f.  Physiol.,  8;  Magnus-Levy,  Zeitschr. 
f.  klin.  Med.,  32.  In  regard  to  the  function  of  the  thyroid  gland  see  also  Sw.  Vincent^ 
Innere  Sckretion  etc.     Ergebnisse  d.  Physiol.,  11,  218-302. 

3  Bioch.  Zeitschr.,  16. 


ADRENAL  BODIES.  377 

varying  between  1  and  10  years,  he  found  an  average  of  0.28  p.  m.  iodine  in  the 
glands.  In  108  normal  glands  above  10  years  old  or  adults  the  iodine  content 
varied  with  an  average  of  1.56  p.  in.  iodine.  In  glands  from  persons  after  using 
iodine  preparations  (o4  rases)  the  iodine  content  was  2.56  p.m.  The  amount 
of  silicic  acid  in  normal  thyroid  glands  was  found  by  11.  Schulz  '  to  be  on  an 
average  0.084  |».  m.,  calculated  on  the  dry  substance.  In  goitres  from  Grbifswald 
and  ZURICH  he  found  0.17")  and  0.434  p.  m.,  respectively.  There  does  not  seem 
to  be  any  connection  between  the  silicic  acid  content  of  the  drinking  water  and 
the  occurrence  of  goitre. 

YYe  cannot  enter  into  a  discussion  as  to  the  various  hypotheses  and 
theories  in  regard  to  the  mode  of  action  of  the  constituents  of  the  thyroids. 
In  the  tetany  appearing  after  parathyroidectomy  many  investigators 
find  an  increased  elimination  of  calcium,  nitrogen  and  ammonia  and  the 
hypothesis  has  been  suggested  that  the  tetany  depends  upon  an  increased 
irritability  of  the  nervous  system  due  to  lack  of  calcium.  The  fact  as 
found  by  several  experimenters  that  a  diminished  calcium  content  of  the 
organs  in  question  does  not  occur,  speaks  against  this  theory.  On  the 
contrary,  it  seems  to  be  generally  admitted  that  lime  salts  reduce  or 
prevent  the  tetany  and,  according  to  Frouin,2  this  depends  upon  the  lime 
combining  with  the  carbonic  acid  produced,  which  is  the  cause  of  the 
tetany.  The  tetany  is  produced  at  least  from  a  poison  which  is  formed 
only  on  the  removal  of  the  parathyroids  or  if  it  is  regularly  produced 
it  is  made  harmless  by  these  organs. 

G.  Mansfield  and  Fr.  Muller3  have  made  investigations  in  regard 
to  the  action  of  the  thyroids  upon  protein  metabolism  which  indicate 
that  lack  of  oxygen  acts  as  an  excitant  upon  the  thyroids  and  that  the 
increased  protein  catabolism,  which  occurs  to  a  mean  degree  with  lack 
of  oxygen,  depends  upon  a  hyperfunction  of  the  thyroid  glands  brought 
on  by  this  condition.  With  greater  lack  of  oxygen  besides  this  a  general 
damage  to  the  protoplasm  of  the  bod}'  cells  may  occur.4 

The  Adrenal  Bodies.  Besides  proteins,  substances  of  the  connect- 
ive tissue,  and  salts,  there  occur  in  the  suprarenal  capsule  inosite,  purine 
bases,  especially  xanthine  (Oker-Blom),  phosphatides  and  glycerophos- 
phoric  acid,  which  is  probabty  a  decomposition  product  of  the  latter. 
The  earlier  accounts  of  the  occurrence  of  benzoic  acid,  hippuric  acid, 
and  bile-acids  are,  on  the  contrary,  doubtful,  and  are  not  substantiated 
by    recent    investigations     (Stadelmann5).     The  medullary  substance 


1  John,  Hammarsten's  Festschr.,  1906;  H.  Schulz,  Bioch.  Zeitschr.,  46. 

2  Compt.  Rend.,  148. 

3  Pfluger's  Arch.,  143. 

4  A  very  complete  discussion  of  the  physiology  of  the  thyroid  gland  and  the  pertinent 
literature  may  be  found  in  Sw.  Vincent,  Ergebnisse  der  Physiologie,  11,  218-302. 

5  Oker-Blom,  Zeitschr.  f.  physiol.  Chein.,  28;  Stadelmann,  ibid.,   18,  which  also 
contains  the  literature  on  this  subject. 


378  CHYLE,   LYMPH,  TRANSUDATES  AND  EXUDATES. 

contains  the  so-called  chromaffine  tissue,  i.e.,  cells,  whose  substance  is 
colored  brown  by  chromic  acid  or  chromates. 

Earlier  investigators,  like  Vulpian  and  Arnold,  have  found,  in  the 
medulla,  a  chromogen  which  has  been  considered  as  connected  with  the 
abnormal  pigmentation  of  the  skin  in  Addison's  disease.  This  chromogen, 
which  is  transformed  by  air,  light,  alkalies,  iodine,  and  other  bodies  into 
a  red  pigment,  seems,  on  the  contrary,  to  be  related  to  the  substance 
adrenalin,  of  the  gland  which  produces  an  increase  in  the  blood-pressure. 
Choline  has  been  shown  to  have  a  reverse  effect  upon  this  blood-pressure 
raising  action,  and  Lohmann  has  shown  that  it  is  formed  in  the  cortical 
substance  of  the  adrenals.  In  the  cortical  this  last-mentioned  exper- 
imenter x  has  found  besides  neurin,  another  not  known  base.  That 
the  watery  extract  of  the  adrenals  has  a  blood-pressure  raising  action 
was  shown  by  Oliver  and  Schafer,  Cybulski  and  Szymonowicz.2 
The  substance  which  is  here  active  was  formerly  called  sphygmogenin 
and  has  also  other  actions  besides  bringing  about  a  marked  increase 
in  blood-pressure  by  the  strong  contraction  of  the  muscles  of  the  periphery 
vessels;  for  instance,  it  can  bring  about  glycosuria  and  mydriasis,  espe- 
cially in  the  frog's  eye,  has  been  chemically  investigated  by  numerous 
experimenters.3  v.  Furth  calls  it  suprarenin,  Abel  epinephrin.  and 
Takamine  adrenalin.  This  last  name  seems  to  be  the  most  generally 
accepted  one. 

Adrenalin  (suprarenin  epinephrin)  (methylaminoethanolpyrocatechin) 

CH 

(HO)C     C.CH(OH).CH2.NHCH3 

C9H13N03,=  I      || 

(HO)C     CH 

V 

CH 

The  constitution  of  adrenalin  has  been  essentially  proved  by  Friedmann,4 
and  he  has  shown  the  correctness  of  the  above  formula,  which  was  given  by 
Pauly.  The  synthesis  of  adrenalin,  which  was  first  performed  by  Stolz,5  is 
also  in  accordance  with  this  formula.  By  the  action  of  methylamine  upon 
chloracetopyrocatechin  we  obtain  methylaminoaceto-pyrocatechin: 

C6H3(OH)2.COCH,Cl +XH.CH3  =  C6H3(OH)2.COCH,.NHCH3.HCl, 

which  yields  adrenalin  on  reduction. 

'Centralbl.  f.  Physiol.,  21,  and  Pfliiger's  Arch.,  118  and  Zeitschr.  f.  Biol.,  56. 

2  Oliver  and  Schafer,  Proceed,  of  Physiol.  Soc,  London,  1895.  Further  literature 
on  the  function  of  the  adrenals  may  be  found  in  Sw.  Vincent,  Innere  Sekretion,  etc. 
Ergebnisse  d.  Physiol.,  9,  505-585. 

3  The  literature  on  this  subject  may  be  found  in  Abderhalden's  Bioch.  Handlexikon 
Bd.  5,  s.  454-495. 

4  Hofmeister's  Beitrage,  8. 

s  Ber.  d.  d.  chem.  Gesellsch.,  37. 


ADRENALIN.  379 

The  synthetically  prepared  adrenalin  is  optically  inactive  r/-/-adrcnalin, 
while  that  from  the  adrenals  is  optically  active  /-adrenalin.  FLACHEB  has 
divided  the  racemic  adrenalin  into  the  two  optically  active  components, 
and  the  identity  of  the  so-obtained  synthetical  adrenalin  with  the  natural 
has  been  shown  by  Abderhalden  and  Fr.  Muller.1  These  last  inves- 
tigators also  found  that  the  /-adrenalin  had  at  least  15  times  as  strong 
an  action  upon  the  blood-pressure  as  the  (/-adrenalin,  and  later  Abder- 
halden with  Thies  and  Slavu  found  that  the  /-adrenalin  had  also  in 
other  respects  a  much  stronger  action  than  d-adrenalin. 

Adrenalin  crystallizes  in  masses  of  needles  or  rhombic  leaves.  It  is 
soluble  in  water,  and  can  be  precipitated  from  its  solution  by  ammonia 
as  a  crystalline  substance.  Its  aqueous  solution  containing  hydrochloric 
acid  is  levorotatory :  (c*)D=  —50.72°  (Abderhalden  and  Guggenheim  2). 
On  heating  adrenalin  it  turns  yellowish-brown  at  about  205°  and  decom- 
poses at  about  218°  C.  Its  solution  turns  emerald  green  with  ferric  chlor- 
ide in  acid  solution  and  carmine  red  in  alkaline  solution.  Adrenalin 
reduces  Fehling's  solution    and  ammoniacal  silver  solution. 

Among  the  reactions  for  adrenalin  in  solution  we  must  especially 
mention  the  red  coloration  which  is  obtained  on  the  addition  of  an  oxidizing 
medium  such  as  iodine  or  bi-iodate  and  dilute  phosphoric  acid  and  warm- 
ing (Frankel  and  Allers),  or  of  mercuric  chloride  in  the  presence  of  a 
catalyst  such  as  the  lime  salts  in  tap-water  (Comesatti).  These  reactions 
are  extremely  delicate,  1 :  1000000-2000000.  A  still  more  delicate  reaction 
(1:5000000)  is  the  one  suggested  by  Ewins,3  namely  a  characteristic 
red  coloration  is  obtained  on  adding  a  0.1  per  cent  solution  of  potassium 
persulphate  and  wrarming  gently  in  a  boiling  water-bath. 

As  above  stated,  it  has  been  considered  for  some  time  that  the  color 
of  the  skin  in  Addison's  disease  was  connected  with  the  adrenals  or  their 
chromogen.  We  know  nothing  positive  in  regard  to  this  relation, 
but  it  is  nevertheless  of  interest  that  pigments,  and  finally  melanins  or 
at  least  dark-brown  substances,  can  be  produced  from  adrenalin  by  the 
action  of  enzymes.  Neuberg  has  brought  about  such  melanin  forma- 
tion by  the  extract  from  the  metastases  of  a  melanoma  of  the  adrenals 
and  also  with  the  extract  of  the  ink-sac  of  the  sepia,  and  Abderhalden 
and  Guggenheim  4  with  tyrosinase.     This  would  indicate  a  close  relation 

1  Flacher,  Zeitschr.  f.  physiol.  Chem.,  58;  Abderhalden  and  Franz  Muller,  ibid. 
58;  with  Thies,  ibid.,  59;  with  Slavu,  ibid.,  59;  with  Kautsch  and  Muller,  ibid.,  61  and 
62;  see  also  Frohlich,  Centralbl.  f.  physiol.,  23  and  Waterman,  Zeitschr.  f.  physiol. 
Chem.,  63. 

2  Zeitschr.  f.  physiol.  Chem.,  57. 

*  Frankel  and  Allers,  Bioch.  Zeitschr.,  18;  Comesatti,  Munch,  med.  Wochenschr. 
1908  and  Physiol.  Centralbl.,  23;  Ewins,  Journ.  of  Physiol.,  40. 

4  Neuberg,  Bioch.  Zeitschr.,  8;  Abderhalden  and  Guggenheim,  Zeitschr.  f.  physiol. 
Chem.,  57. 


380  CHYLE,   LYMPH,  TRANSUDATES  AND  EXUDATES. 

between  adrenalin  and  tyrosine,  which  also  gives  melanin  with  the  sepia 
enzyme,  and  indeed  tyrosine  has  been  considered  as  the  probable  mother- 
substance  of  adrenalin  (Halle).  The  investigations  of  Ewin's  and 
Laidaw  x  to  prove  this  last-mentioned  possibility  have  not  given  any 
support  thereto. 

Besides  the  action  of  producing  a  rise  in  the  blood-pressure,  adrenalin 
is  also  of  special  interest  because,  as  first  shown  by  Blum,2  it  also  has  a 
glycosuric  action.  We  will  discuss  the  question  of  adrenalin  glycosuria 
and  the  relation  which  seems  to  exist  between  the  internal  secretions 
of  the  thyroids,  the  adrenals  and  the  pancreas,  when  we  treat  of  the 
formation  of  sugar  and  pancreas  diabetes.  We  cannot  here  enter  into 
the  question  of  the  reciprocal  action  between  the  adrenals  and  the  other 
organs. 

The  hypophysis  or  pituitary  gland  has  been  little  studied  from  a  chemical 
standpoint.  An  extract  of  the  gland  shows,  by  its  action,  a  certain  similarity  to  an 
extract  of  the  adrenals  in  that  it  causes  a  rise  in  blood  pressure  and  by  causing  a 
dilation  of  the  pupils  of  the  frog's  eye.  Still  no  adrenalin  could  be  detected  in  the 
gland.     Also  no  iodine  occurs  in  the  glands  (Wells,  Denis  3) . 

The  gland  consists  essentially  of  two  parts,  one  an  outside  formation  of  vascular- 
glandular  epithelium  and  a  lower  nervous  part  the  infundibular  part.  The  out- 
side part  seems  to  have  a  relation  to  the  growth  of  the  tissues  and  skeleton  and 
acromegalie  and  gigantism  are  claimed  by  many  investigators  to  be  related  to  this 
part.  The  infundibular  part,  on  the  contrary,  contains  the  specific  bodies  which 
raises  the  blood -pressure  and  stimulates  the  smooth  muscles  of  the  uterus  and 
upon  the  kidney  secretion.  The  relation  of  the  hypophysis  to  other  endocrinic  glands 
is  still  very  much  disputed. 

1  Halle,  Hofmeister's  Beitrage  8;  Ewins  and  Laidaw,  Journ.  of  Physiol.,  40. 

2  Deutsch.  Arch.  f.  klin.  Med.,  91  and  Pfliiger's  Arch.,  90. 

3  H.  G.  Wells,  Journ.  of  biol.  Chem.,  7;  W.  Denis,  ibid.,  9. 


CHAPTER  VII. 
THE  LIVER. 

The  liver,  which  is  the  largest  gland  of  the  body,  stands  in  close 
relation  to  the  glands  mentioned  in  Chapter  VI.  The  importance 
of  this  organ  for  the  assimilation  of  the  food-stuffs  and  for  the  phys- 
iological composition  of  the  blood  is  evident  from  the  fact  that  the 
blood  coming  from  the  digestive  tract,  laden  with  absorbed  bodies,  must 
circulate  through  the  liver  before  it  is  driven  by  the  heart  through  the 
different  organs  and  tissues.  An  assimilation  of  food-stuffs  in  the  liver 
.has  been  positively  shown  in  the  first  place  for  carbohydrates  in  that  the 
liver  constructs  a  polysaccharide  glycogen  from  hexoses,  which  according  to 
the  needs  is  then  again  retransformed  into  glucose.  The  liver  is  a  storage 
organ  for  fats  and  takes  up  food  fat  as  well  as  fat  from  depots  (in 
starvation) and  as  it  seems,  at  least  in  part,  prepares  them  so  that  they  can 
be  further  used  in  the  animal  body. 

We  are  not  clear  as  to  what  extent  an  assimilation  of  products  of  pro- 
tein digestion  takes  place  in  the  liver.  The  subject  will  be  discussed  in  detail 
under  absorption  in  Chapter  VIII.  It  is  claimed  that  the  liver  can  serve 
as  a  storage  organ  for  proteins,1  and  it  is  at  least  certain  that  it  retains 
alien  protein  which  is  brought  to  it  by  the  blood.2  The  retention 
of  alien  protein  stands  probably  in  close  relationship  to  the  ability  of 
the  liver  to  take  up  and  retain  foreign  substances  as  a  group  from  the  blood. 
This  is  not  only  true  for  different  metals  but  also,  as  shown  by  several 
investigators.3  alkaloids  which  perhaps  are  also  partly  decomposed  in  the 
liver.  Toxins  are  also  withheld  by  the  liver  and  hence  this  organ  has 
a  protective  action  against  poisons. 

The  formation  of  glycogen  from  glucose  is  one  of  the  numerous  syn- 
theses occurring  in  the  liver  and  this  is  no  doubt  the  one  which  takes 
place  to  the  greatest  extent.     Other  syntheses  in  the  liver  are,  for  example, 


1  See  Seitz,  Pfliiger's  Arch.,  Ill  and  Asher  and  Boehm,  Zeitschr.  f.  Biol.  51. 

2  See  Reach,  Bioch.  Zeitschr.,  16  and  Pacchioni  and  Carlini,  Maly's  Jahresb.,  39. 

3  Roger,  Action  du  foie  sur  les  poisons  (Paris,  1887),  which  quotes  the  works  of 
Schiff,  Heger  and  others;  also  W.  N.  Woronzow,  Maly's  Jahresb.,  40  and  Z.  Vamossy, 
Orid.,  40. 

381 


382  THE   LIVER. 

the  formation  of  urea  or  uric  acid  (in  birds)  from  ammonium  salts,  the 
formation  of  etheral  sulphuric  acids  and  conjugated  glucuronic  acids 
from  the  phenols  produced  in  intestinal  putrefaction  and  the  recently 
shown  syntheses  of  amino-acids  in  the  liver.  On  the  other  hand  a 
deamidation  of  amino-acids  and  purine  bodies,  hydrolyses,  oxidations, 
reductions  and  fermentative  processes  of  various  kinds  occur  in  the  liver. 
Because  of  these  diverse  processes,  the  results  of  which  we  must  espe- 
cially mention  the  formation  of  bile  as  well  as  the  fact  that  the  liver  is 
introduced  between  the  intestine  and  the  general  circulation,  makes  the 
liver  a  central  organ  for  metabolism. 

Among  the  numerous  chemical  processes  which  take  place  in  the  liver 
there  are  especially  two  which  give  special  interest  to  this  organ,  namely, 
the  formation  of  glycogen  or  the  carbohydrate  metabolism  in  the  liver, 
and  the  formation  of  bile.  For  this  reason  only  these  two  processes  will 
be  discussed  in  this  chapter  while  the  others  will  be  discussed  in  other 
chapters  and  in  other  connection.  Before  we  begin  to  discuss  these  two 
processes  a  short  review  of  the  constituents  and  the  chemical  com- 
position of  the  liver  seems  to  be  appropriate. 

The  reaction  of  the  liver-cells  is  alkaline  toward  litmus  during  life, 
but  becomes  acid  after  death,  due  to  a  formation  of  lactic  acid,  chiefly 
fermentation  lactic  acid  and  other  organic  acids  (Morishima,  Magnus- 
Levy  J).  A  coagulation  of  the  protoplasmic  proteins  in  the  cells  probably 
takes  place.  A  positive  difference  between  the  proteins  of  the  dead 
and  the  living,  non-coagulated  protoplasm  has  not  been  observed. 

The  proteins  of  the  liver  were  first  carefully  investigated  by  Pl6sz. 
He  found  in  the  watery  extract  of  the  liver  an  albuminous  substance 
which  coagulates  at  45°  C.  (globulin,  Halliburton),  also  a  globulin 
which  coagulates  at  75°  C,  a  nucleoalbumin  which  coagulates  at  70°  C, 
and  lastly  a  protein  body  which  is  closely  related  to  the  coagulated  albumi?is 
and  which  is  insoluble  in  dilute  acids  or  alkalies  at  the  ordinary  tem- 
perature, but  dissolves  on  the  application  of  heat,  being  converted  into 
an  albuminate.  Halliburton  2  found  two  globulins  in  the  liver-cells, 
one  of  which  coagulates  at  68-70°  C,  and  the  other  at  45-50°  C.  He 
also  found,  besides  traces  of  albumin,  a  nucleoprotein  which  possessed 
1.45  per  cent  phosphorus  and  a  coagulation-point  of  60°  C.  Pohl  has 
obtained  an  '"  organ  plasma "  by  extracting  the  finely  divided  liver 
which  had  previously  been  entirely  freed  from  blood  by  washing  with 
8  p.  m.  NaCl  solution,  in  which  he  was  able  to  detect  a  globulin  having 
a  low  coagulation  temperature.      The  very  variable  phosphorus,  content 


1  Morishima,  Arch.  f.  exp.  Path.  u.  Pharm.,  43;    Magnus-Levy,  Hofmeister's  Bei- 
trage,  2. 

2  P16sz,  Pfliiger's  Arch.,  7;  Halliburton,  Journ.  of  Physiol.,  13,  Suppl.  1892. 


PROTEINS   OF  THE    LIVER.  383 

(0.28-1.3  per  cent)  of  this  globulin  as  well  as  the  insolubility  of  the  pre- 
cipitates produced  by  little  acid,  in  an  excess  of  acid,  and  in  neutral  salt- 
seem  to  indicate  that  we  here  have  a  mixture  which  consists  chiefly  of 
nucleoproteins  and  not  of  globulins.  The  almost  complete  digestibility 
with  pepsin-hydrochloric  acid  does  not  controvert  this  assumption, 
because,  as  is  known,  nucleoproteins  may  on  digestion  yield  no  residue 
(see  Chapter  II).  Nor  can  we  be  positive  concerning  the  nature  of 
the  liver-globulin  found  by  Dastre,1  having  a  coagulation  temperature 
of  56° C.  The  proteins  extractable  from  the  liver  without  modification 
must  be  thoroughly  investigated. 

Besides  the  above-mentioned  proteins,  which  are  very  soluble,  the 
liver-cells  contain  large  quantities  of  difficultly  soluble  protein  bodies 
(see  Pl6sz).  The  liver  also  contains,  as  first  shown  by  St.  Zaleski 
and  later  substantiated  by  several  other  investigators,  ferruginous 
proteins  of  different  kinds.2.  The  chief  portion  of  the  protein-  substances 
in  the  liver  seems  in  fact  to  consist  of  ferruginous  nucleoproteins.  On 
boiling  the  liver  with  water,  such  a  nucleoprotein  or  perhaps  several 
are  split,  and  a  solution  is  obtained  containing  a  nucleic-acid-rich  nucleo- 
protein or  a  mixture  of  these  which  are  precipitable  by  acids.  This 
protein  or  protein  mixture,  which  has  been  called  ferratin  by  Schmiede- 
berg,3  has  been  studied  by  Wohlgemuth.4  The  quantity  of  phos- 
phorus was  3.0fi  per  cent.  As  cleavage  products  on  hydrolysis  he  found 
/-xylose,  or  at  least  a  pentose,  the  four  nuclein  bases,  and  also  arginine, 
lysine  (and  histidine?),  tyrosine,  leucine,  glycocoll,  alanine,  a-proline, 
glutamic  acid,  aspartic  acid,  phenylalanine,  oxyaminosuberic  acid,  and 
oxydiaminosebacic  acid  (see  Chapter  II).  The  /-xylose  depends,  no 
doubt,  at  least  in  part,  upon  the  guanvlic  acid  isolated  from  the  liver, 
by  Levene  and  Mandel,5  and  the  finding  of  adenine  among  the  cleavage 
products  also  indicates  the  presence  of  a  thymonucleic  acid.  There  does 
not  seem  to  be  any  doubt  that  the  ferratin,  as  above  stated,  is  a  mixture, 
and  the  correctness  of  this  assumption  is  shown  by  the  recent  investiga- 
tions of  Scaffidi  and  Salkowski.6 


1  Pohl,  Hofmeister's  Beitrage,  7;  Dastre,  Compt.  rend.  soe.  biolog..  58. 

2  St.  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  10,  486;  Woltering,  ibid.,  21;  Spitzer, 
Pniiger's  Arch.,  67. 

'  Arch.  f.  exp.  Path.  u.  Pharm.,  33;  see  also  Vay,  Zeitschr.  f.  physiol.  Chem..  20. 

♦Wohlgemuth,  Zeitschr.  f.  physiol.  Chem.,  37,  42,  and  44,  and  Ber.  d.  d.  chem. 
Gesellsch.,  37.  See  on  liver  nucleoproteins  also  Salkowski,  Berl.  klin.  Wochenschr., 
1895;  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  19;  Blumenthal,  Zeitschr.  f.  klin.  Med., 
34. 

5  Bioch.  Zeitschr.,  10. 

•Scaffidi,  Zeitschr.  f.  physiol.  Chem.,  58;  Salkowski,  ibi>1.,  58. 


384  THE   LIVER. 

The  yellow  or  brown  pigment  of  the  liver  has  been  little  studied.  Dastre 
and  Floresco  l  differentiate,  in  vertebrates  and  certain  invertebrates,  between  a 
ferruginous  pigment  soluble  in  water,  ferrine,  and  a  pigment  soluble  in  chloroform 
and  insoluble  in  water,  chlorochrome.  They  have  not  isolated  these  pigments  in  a 
pure  condition.  In  certain  invertebrates  chlorophyll  originating  from  the  food  also 
occurs  in  the  liver. 

The  fat  of  the  liver  occurs  partly  as  very  small  globules  and  partly 
(especially  in  nursing  children  and  suckling  animals,  as  also  after  food 
rich  in  fat)  as  rather  large  fat-drops.  The  occurrence  of  a  fatty  infiltra- 
tion, i.e.,  a  transportation  of  fat  to  the  liver,  may  not  only  be  produced 
by  an  excess  of  fat  in  the  food  (Noel-Paton),  but  also  by  a  migration 
from  other  parts  of  the  body  under  abnormal  conditions,  such  as  poison- 
ing with  phosphorus,  phlorhizin,  and  certain  other  bodies  (Leo,  Lebedeff, 
Rosenfeld,  and  others2).  The  fatty  infiltration  occurring  in  poisoning, 
and  which  is  accompanied  with  degenerative  changes  in  the  cells,  may 
cause  a  diminution  in  the  amount  of  protein  and  a  rise  in  the  water  con- 
tent. If  the  amount  of  fat  in  the  liver  is  increased  by  an  infiltration,  the 
water  decreases  correspondingly,  while  the  quantity  of  the  other  solids 
remains  little  changed.  Changes  of  a  kind  may  occur,  so  that,  because 
of  the  antipathy  (Rosenfeld,  Bottazzi3)  existing  between  glycogen 
and  fat,  a  liver  rich  in  fat  is  habitually  poor  in  glycogen.  The  reverse 
occurs  after  feeding  with  carbohydrate-rich  food,  namely,  the  liver  is 
rich  in  glycogen  and  poor  in  fat. 

The  composition  of  the  liver-fat  seems  to  vary  not  only  in  different 
animals,  but  is  variable  with  differing  conditions.  Thus  Noel-Paton 
found  that  the  liver-fat  in  man  and  several  animals  was  poorer  in  oleic 
acid  and  had  a  correspondingly  higher  melting-point  than  the  fat  from 
the  subcutaneous  connective  tissue,  while  Rosenfeld4  observed  the 
opposite  condition  on  feeding  dogs  with  mutton-fat. 

Several  investigators,  Hartley,  Leathes  and  Mottram  suggested 
as  a  difference  between  the  fat  of  the  liver  and  the  connective  tissues, 
the  great  amount  in  the  first  of  unsaturated,  higher  fatty  acids.  Accord- 
ing to  Hartley  5  the  fat  of  the  pig  liver  contains  palmitic  acid,  stearic 


1  Arch,  de  Physiol.  (5),  10. 

2  Noel-Paton,  Journ.  of  Physiol.,  19;  Leo,  Zeitschr.  f.  physiol.  Chem.,  9;  Lebedeff, 
PfluRer's  Arch.,  31;  Athanasiu,  Pfliiger's  Arch.,  74;  Taylor,  Journ.  of  Exp.  Med.,  4; 
Kraus  u.  Sommer,  Hofmeister's  Beitrage,  2;  Rosenfeld,  Zeitschr.  f.  klin.  Med.,  36. 
See  also  Rosenfeld,  Erfiebnisse  der  Physiologie,  1,  Abt.  1,  and  Berl.  klin.  Wochenschr. 
1904;  Schwalbe,  Centralbl.  f.  Physiol.,  18,  p.  319;  Shibata,  Bioch.  Zeitschr.,  37. 

3  Arch.  Ital.  d.  Biol.,  48  (1908),  cited  in  Bioch.  Centralbl.,  7,  p.  833. 

*  Cited  by  Lummert,  Pfluger's  Arch.,  71.  In  regard  to  the  liver-fat  of  children, 
see  Thiemich,  Zeitschr.  f.  physiol.  Chem.,  26. 

6  Hartley,  Journ.  of  Physiol.,  38;  Leathes  and  Meyer-Wedell,  ibid.,  38;  Mottram,, 
ibid.,  38. 


PHOSPHATIDES   OF  THE   LIVER.  385 

acid,  and  oleic  acid  which  is  not  identical  with  the  ordinary  oleic  acid,  also 
linoleic  acid  and  an  acid  having  the  formula  C20H32O2.  A  part  of 
these  unsaturated  fatty  acids  are  contained  in  the  phosphatides  but  as 
the  unsaturated  acids  are  about  one-half  of  the  fatty  acids  they  must 
also  occur  in  the  fats.  The  abundant  occurrence  of  unsaturated  fatty 
acids  is  considered  by  the  above-mentioned  investigators  as  the  first 
step  in  the  cleavage  of  the  transportable  fat  from  the  fat  tissues  to  the 
liver  and  destined  for  use  in  the  body.  There  is  no  doubt  that  the  phos- 
phatides are  of  great  importance  for  this  transformation  of  the  fat. 

Phosphatides,  which  were  formerly  designated  lecithin,  and  whose 
quantity  is  generally  calculated  as  such,  also  belong  to  the  normal  con- 
stituents of  the  liver.  The  quantity  (as  lecithin)  amounts  to  over  23.5 
p.  m.  according  to  Noel-Paton.1  In  starvation  the  lecithin,  according 
bo  Xoel-Paton,  forms  the  greater  part  of  the  ethereal  extract,  while 
with  food  rich  in  fat,  on  the  contrary,  it  forms  the  smaller  part.  In 
the  liver  of  a  healthy  dog  Baskoff  2  found  84  p.  m.  phosphatides  (cal- 
culated as  lecithin)  in  the  dry  substance.  The  phosphatides  are  undoubt- 
edly of  various  kinds,  but  they  have  not  been  closely  studied.  Among 
others,  Ave  have  lecithin  and  the  so-called  jecorin.  Cholesterin  is  also  a 
constituent  of  the  liver,  although  only  in  small  quantities,  and  Kondo  3 
finds  that  cholesterin  ester  occurs  in  the  liver. 

Jecorin  was  first  found  by  Drechsel  in  the  liver  of  horses,  and  also  in  the 
liver  of  a  dolphin,  and  later  by  Baldi  in  the  liver  and  spleen  of  other  animals,  in 
the  muscles  and  blood  of  the  horse,  and  in  the  human  brain.  It  contains  sul- 
phur and  phosphorus,  but  its  constitution  is  not  positively  known.  Jecorin  dis- 
solves in  ether,  but  is  precipitated  from  this  solution  by  alcohol.  It  reduces 
copper  oxide,  and  gives  a  wine-red  coloration  with  an  ammoniacal  silver-solution. 
( hi  boiling  with  alkali  and  then  cooling  it  solidifies  to  a  gelatinous  mass.  Manasse 
has  detected  glucose  as  osazone  in  the  carbohydrate  complex  of  jecorin. 

The  statement  by  Bing  that  jecorin  is  a  combination  of  lecithin  and  glucose 
does  not  follow  from  the  analyses  of  jecorin  thus  far  known.  Jecorin  contains 
sulphur,  even  as  much  as  2.75  per  cent,  and  further  the  relation  of  P:N  in  lecithin  is 
1:1,  while  in  jecorin  it  is  quite  different,  1:  2  to  1:  0.  According  to  the  investiga- 
tions of  Baskoff  the  liver  jecorin,  prepared  according  to  Drechsel's  sugges- 
tion, and  when  it  is  so  pure  that  it  is  completely  soluble  in  ether,  and  quantitatively 
precipitated  by  alcohol  from  this  solution,  is  a  rather  constant  compound  at 
least  in  regard  to  the  X,  P  and  glucose  content.  Baskoff  found  as  average  2.55 
per  cent  X,  2.87  per  cent  P,  and  about  14  per  cent  glucose.  The  relation  P:X 
was  nearly  1 :2  and  therefore  jecorin  is  correspondingly  a  diaminomonophosphatide. 

The  variable  composition  and  divergent  properties  of  the  jecorin  isolated  and 
analyzed  by  various  investigators  4  depends,  according  to  Baskoff,  upon  imper- 


1  1.  c.     See  also  Heffter,  Arch.  f.  exp.  Path.  u.  Pharm.,  28. 

2  Zeitschr.  f.  physiol.  Chem.,  62. 

3  Bioch.  Zeitschr.,  26. 

*  Drechsel,  Ber.  d.  sachs.  Gesellsch.  d.  Wissensch.,  1886,  p.  44,  and  Zeitsch.  f.  Biol- 
ogie,  33;  Baldi,  Arch.  f.  (Anat.  u.)  Physiol.,  1887,  Suppl.,  100;  Manasse,  Zeitschr.  f. 
physiol.  Chem.,  20;  Bing,  Centralbl.  f.  Physiol.,  12,  and  Skand.  Arch.  f.  Physiol.,  9; 


386  THE  LIVER. 

I 

feet  purification.  His  investigations  do  not  give  any  explanation  for  the  quantity 
of  sulphur  and  it  is  very  probable  that  jecorin  is  only  a  mixture  of  several  bodies 
among  which  a  sulphurized  and  a  phosphorized  substance  occurs.  According 
to  Baskoff  it  is  very  probable  that  the  jecorin  is  a  decomposition  product  of  lecithin 
(or  other  phosphatides). 

Another  phosphatide,  which  does  not  reduce  directly  or  after  boiling  with  acid, 
has  been  called  heparphosphatide  by  Baskoff.  In  certain  respects  this  body  is 
similar   to  cuorin,  and   the  relation   P:N  =1.45:1,  although  it    was  not   pure. 

Among  the  extractive  substances  besides  glycogen,  which  will  be  treated 
later,  rather  large  quantities  of  the  purine  bases  occur.  Kossel  1  found 
in  1000  parts  of  the  dried  substance  1.97  p.  m.  guanine,  1.34  p.  m.  hypo- 
xanthine,  and  1.21  p.  m.  xanthine.  Adenine  is  also  contained  in  the 
liver.  In  addition  there  are  found  urea  and  uric  acid  (especially  in 
birds),  and  indeed  in  larger  quantities  than  in  the  blood,  paralactic  acid, 
choline,  leucine,  taurine,  and  cystine.  In  pathological  cases  inosite  and 
amino-acids  have  been  detected.  The  occurrence  of  bile-coloring  matters 
in  the  liver-cell  under  normal  conditions  is  doubtful;  but  in  retention  of 
the  bile  the  cells  may  absorb  the  coloring-matter  and  become  colored 
thereby. 

A  large  number  of  enzymes  are  found  in  the  liver,  such  as  catalases, 
oxidases,  aldehydases  and  hydrolytic  enzymes  of  various  kinds;  the  dias- 
tase acting  upon  glycogen,  the  lipases  and  the  different  proteolytic  enzymes. 
Nucleases  and  the  nucleic  acid  splitting  enzymes  of  different  kinds  men- 
tioned in  Chapter  II  have  been  formed  in  the  liver  and  deamidases  for 
amino-acids  as  well  as  purine  bodies  also  occur  in  the  liver.  The  last 
group  of  deamidases  show  a  different  behavior  in  regard  to  their  occurrence 
in  different  animals  and  the  same  is  true  for  the  uric  acid  forming  and 
uric  acid  destroying  enzymes  (Chapter  XIV).  We  must  also  mention  the 
arginase  which  splits  off  urea  from  arginine. 

The  proteolytic  enzymes  of  the  liver  are  of  special  interest,  especially 
in  regard  to  the  study  of  the  autolysis  of  this  organ.  The  processes 
in  the  liver  in  phosphorus  poisoning  and  in  acute  yellow  atrophy  of  the 
liver  are  considered  as  an  intravitally  increased  autolysis.  In  these 
cases  a  softening  of  the  organ  takes  place,  and  proteoses,  mono-  and 
diamino-acids,  and  other  bodies  are  produced,  which  may  also  be  found 
in  the  urine,  and  although  they  may  not  all  be  derived  from  the  liver 
(X Ei; berg  and  Richter),  they  are  at  least  in  part  derived  from  this  organ. 
Wakeman  has  found  in  phosphorus  poisoning  that  not  only  is  the  quan- 
tity of  nitrogen  markedly  diminished  in  the  liver  (of  dogs),  but  also 
that  the  quantity  of  nitrogen  of  the  hexone  bases  is  diminished,  and 


Meinertz,  Zeitschr.  f.  physiol.  Chem.,  46;  Siegfried  and  Mark,  ibid.;  Paul  Mayer, 
Bioch.  Zeitschr.,  1,  and  Baskoff,  Zeitschr.  f.  physiol.  Chem.,  57,  61,  62. 
1  Zeitschr.  f.  physiol.  Chem.,  8. 


IRON    IN   THE   LIVER.  387 

that  the  part  of  the  protein  molecule  richer  in  nitrogen  is  first  removed 
and  eliminated  under  these  conditions.  A  similar  condition  has  been 
observed  by  Wells  in  the  idiophatic,  acute  yellow  atrophy  of  the  liver. 
In  consideration  of  the  variable  results  for  the  diamino-nitrogen  even 
under  normal  conditions  (Glikin  and  A.  Loewy  1),  it  is  desirable  that 
a  greater  number  of  observations  be  made  on  this  subject.  The  increased 
consumption  of  glycogen  under  the  above-mentioned  pathological  con- 
ditions may  also  be  considered  as  an  increased  autolysis,  while  the  claim 
of  certain  observers  that  fat  is  formed  in  the  autolysis  of  the  liver  is, 
according  to  Saxl,2  to  be  considered  only  as  a  more  pronounced  appear- 
ance of  the  fat  previously  occurring  in  the  organ. 

Besides  the  above-mentioned  organic  constituents  in  the  liver  we  must 
mention  the  glucothionic  acid  found  by  Mandel  and  Levene,3  whose 
chemical  individuality  is  doubted. 

The  mineral  bodies  of  the  liver  consist  of  phosphoric  acid,  potassium, 
sodium,  alkaline  earths,  and  chlorine.  The  potassium  is  in  excess  of 
rhe  sodium.  Iron  is  a  regular  constituent  of  the  liver,  but  it  occurs 
in  very  variable  amounts.  Bunge  found  0.01-0.355  p.  m.  iron  in 
the  blood-free  liver  of  young  cats  and  dogs.  This  was  calculated  on  the 
liver  substance  freshly  washed  with  a  1-per  cent  XaCl  solution.  Cal- 
culated on  10  kilos  bodily  weight,  the  iron  in  the  liver  amounted  to  3.4- 
80.1  mg.  Recent  determinations  of  the  quantity  of  iron  in  the  liver  of 
the  rabbit,  dog,  hedge-hog,  pig,  and  man  have  been  made  by  Guille- 
monat  and  Lapicque,  and  in  rabbits  by  Scaffidi.  The  variation  was 
great  in  human  beings.  In  men  the  quantity  of  iron  in  the  blood-free 
liver  (blood-pigment  subtracted  in  the  calculation)  was  regularly  0.23 
p.  m.,  and  in  women  0.09  p.  m.  (calculated  on  the  fresh  moist  organ), 
and  this  relation  was  not  changed  after  the  twentieth  year.  Above 
0.5  p.  m.  is  considered  as  pathological.  According  to  Bielfeld,4  who 
worked  with  another  method,  an  even  greater  quantity  of  iron  occurs  in 
men. 

The  quantity  of  iron  in  the  liver  can  be  increased  by  drugs  contain- 
ing iron.  The  quantity  of  iron  may  also  be  increased  by  an  abundant 
destruction  of  red  blood-corpuscles,  which  will  result  from  the  injection 


1  Neuberg  and  Richter,  Deutsch.  rned.  Wochenschr.,  1904;  Wakeman,  Zeitschr.  f. 
physiol.  Chem.,  44;  Wells,  Journ.  of  Exper.  Med.,  9;  Glikin  and  Loewy,  Bioch.  Zeitschr., 
10. 

2  Hofmeister's  Beitrage,  10. 

3  Mandel  and  Levene,  Zeitschr.  f.  physiol.  Chem.,  45. 

4  Bunge,  Zeitschr.  f.  physiol.  Chem.,  17,  78;  Guillemonat  and  Lapicque,  Compt. 
rend,  de  soc.  bid.,  48,  with  Bailie,  ibid.,  68;  and  Arch  de  Physiol.  (5)  8;  Biefeld,  Hof- 
meister's Beitrage,  2;  see  also  Schmey,  Zeitschr.  f.  physiol.  Chem.,  39;  Scaffidi,  ibid., 
54. 


388  THE  LIVER. 

of  dissolved  haemoglobin,  in  which  process  the  iron  combinations  derived 
from  the  blood-pigments  in  other  organs,  such  as  the  spleen  and  marrow, 
also  seem  to  take  part.1  A  destruction  of  blood-pigments,  with  a  splitting 
off  of  compounds  rich  in  iron,  seems  to  take  place  in  the  liver  in  the  for- 
mation of  the  bile-pigments.  Even  in  invertebrates,  which  have  no 
haemoglobin,  the  so-called  liver  is  rich  in  iron,  from  which  Dastre  and 
Floresco  2  conclude  that  the  quantity  of  iron  in  the  liver  of  inverte- 
brates is  entirely  independent  of  the  decomposition  of  the  blood-pigment, 
and  in  vertebrates  it  is  in  part  so.  According  to  these  authors  the  liver 
has,  on  account  of  the  quantity  of  iron,  a  specially  important  oxidizing 
function,  which  they  call  the  "  fonction  martiale  "  of  the  liver. 

The  richness  in  iron  of  the  liver  of  new-born  animals  is  of  special 
interest — a  condition  which  was  shown  by  the  analyses  of  St.  Zaleski, 
but  was  especially  studied  by  Kruger  and  Meyer.  In  oxen  and  cows 
they  found  0.246-0.276  p.  m.  iron  (calculated  on  the  dry  substance), 
and  in  the  cow-foetus  about  ten  times  as  much.  The  liver-cells  of  a  calf 
a  week  old  contain  about  seven  times  as  much  iron  as  the  adult  animal; 
the  quantity  decreases  in  the  first  four  weeks  of  life,  when  it  reaches 
about  the  same  amount  as  in  the  adult.  Lapicque3  also  found  that  in 
rabbits  the  quantity  of  iron  in  the  liver  steadily  diminishes  from  the 
eighth  day  to  three  months  after  birth,  namely,  from  10  to  0.4  p.  m., 
calculated  on  the  dry  substance.  "  The  fcetal  liver-cells  bring  an  abun- 
dance of  iron  in  the  world  to  be  used  up,  within  a  certain  time,  for  a  pur- 
pose not  well  known."  A  part  of  the  iron  exists  as  phosphate,  but  the 
greater  part  is  in  combination  in  the  ferruginous  protein  bodies  (St. 
Zaleski). 

The  quantity  of  calcium  oxide  in  the  fresh,  moist  liver  of  the  horse, 
ox,  and  pig,  according  to  Toyonaga,  amounts  to  0.148-0.193  p.  m.,  or 
more  than  the  human  liver  (0.101  p.  m.  according  to  Magnus-Levy). 
The  amount  of  magnesium  oxide  was  remarkably  high,  namely,  0.168, 
0.198  and  0.158  p.  m.,  in  the  livers  of  the  horse,  ox,  and  pig,  respectively, 
but  considerably  less  than  the  human  liver  in  which  Magnus-Levy  found 
0.292  p.  m.  Kruger4  found  the  quantity  of  calcium  in  the  livers  of 
adult  cattle  and  of  calves  to  be  respectively  0.71  p.  m.  and  1.23  p.  m. 
of  the  dried  substance.  In  the  foetus  of  the  cow  it  is  lower  than  in  calves. 
During  pregnancy  the  iron  and  calcium  in  the  foetus  are  antagonistic; 


1  See  Lapicque,  Compt.  Rend.,  124,  and  Schurig,  Arch.  f.  exp.  Path.  u.  Pharm.,  41. 

2  Arcb.de  Physiol.  (5),  10. 

4  St.  Zaleski,  1.  c;  Kruger  and  collaborators,  Zeitschr.  f.  Biologie,  27;  Lapicque, 
Maly'fi  Jahresber.,  20. 

4  Kruger,  Zeitschr.  f.  Biologie,  31;  Toyonaga,  Bull,  of  the  College  of  Agriculture, 
Tokio,  6;  A.  Magnus-Levy,  Bioch.  Zeitschr.,  24. 


STORAGE    OF   PROTEIN    IN   THE   LIVER.  389 

that  is,  an  increase  in  the  quantity  of  calcium  in  the  liver  causes  a  diminu- 
tion in  the  iron,  and  an  increase  in  the  iron  causes  a  decrease  in  the  calcium. 
Copper  seems  to  be  a  physiological  constituent,  and  occurs  to  a  considerable 
extent  in  Cephalopods  (Henze  l).  Foreign  metals,  such  as  lead,  zinc, 
arsenic,  and  others  (also  iron),  are  easily  taken  up  and  combined  by  the 
liver  (Slowtzoff,  v.  Zeynek,  and  others2). 

v.  Bibra  3  found  in  the  liver  of  a  young  man  who  had  suddenly  died 
762  p.  m.  water  and  238  p.  m.  solids,  consisting  of  25  p.  m.  fat,  152  p.  m. 
protein,  gelatin-forming  and  insoluble  substances,  and  61  p.  m.  extract- 
ive substances. 

Magnus-Levy4  found  in  the  liver  of  a  healthy  suicide  606  p.  m. 
water,  394  p.  m.  solids  among  which  212.8  p.  m.  fat  occurred.  If  the 
total  nitrogen,  27  p.  m.,  is  calculated  as  protein  the  amount  would  be 
approximately  169  p.  m. 

Profitlich  5  found  68.2-75.17  per  cent  water  in  the  dog  liver  and  70.76- 
72. S6  per  cent  in  the  ox  liver.  The  relation  N :  C  in  the  fat  and  glycogen-free 
dried  substance  was  1:3.21  in  dogs  and  1:3.13  in  oxen  or  about  the  same  as  in 
flesh  (see  Chapter  XI). 

The  quantitative  composition  of  the  liver  may  show  great  varia- 
tion, depending  upon  the  kind  and  amount  of  the  food  supplied.  The 
amount  of  carbohydrate  (glycogen)  and  fat  may  vary  considerably, 
which  is  due  to  the  fact  that  the  liver  is  a  storage-organ  for  these  bodies, 
especially  for  the  glycogen. 

Based  upon  special  experiments,  Seitz  claims  that  the  liver  is  a 
storehouse  for  protein  also.  In  experiments  on  hens  and  ducks  which 
had  previously  been  starved,  he  found  that  the  liver  took  up  abundant 
protein  on  feeding  meat,  and  that  its  weight  as  compared  with  the  weight 
after  starvation  was  doubled  or  quadrupled.  As  it  is  characteristic  of 
storage  or  reserve  bodies  that  their  amount  in  the  storage-organs  on 
feeding  with  such  bodies  strongly  increases  in  percentage,  it  is  remarkable 
in  Seitz's  feeding  experiments  that  the  percentage  of  protein  in  the  liver 
did  not  increase,  but  rather  diminished  slightly.  In  this  case  we  did  not 
have  a  higher  percentage  of  protein,  but  an  increase  in  the  weight  of  the 
total  cell  mass  of  the  organ,  probably  brought  about  by  increased  work 
of  the  liver  due  to  the  protein  feeding.  The  investigations  of  Grund  6 
have  shown  that  with  protein  feeding  in  dogs,  the  relation  P:N  in  the 

1  Zeitschr.  f .  physiol.  Chem.,  33. 

2Slo\vtzoff,  Hofmeister's  Beitrage,  1;  v.  Zeynek,  see  Centralbl.  f.  Physiol.,  15. 

3  See  v.  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4.  Aufl.,  p.  711. 

4  Bioch.  Zeitschr.,  24. 

5  Pfluger's  Arch.,  119. 

6  Seitz,  Pfluger's  Arch.,  Ill;  Grund,  Zeitschr.  f.  Biol.,  54. 


390  THE  LIVER. 

liver  was  not  essentially  changed  which  speaks  against  a  simple  storage 
of  food  protein. 

Glycogen  and  its  Formation. 

Glycogen  was  first  discovered  by  Bernard.  It  is  a  carbohydrate 
closely  related  to  the  starches  or  dextrins,  with  the  general  formula 
m(C6Hio05).  Its  molecular  weight  is  unknown,  but  seems  to  be  very 
large  (Gatin-Gruzewska  and  v.  Knaffl-Lenz1).  The  largest  quan- 
tities are  found  in  the  liver  of  adult  animals,  and  smaller  quantities 
in  the  muscles  (Bernard,  Nasse).  It  is  found  in  very  small  quantities 
in  nearly  all  tissues  of  the  animal  body.  Its  occurrence  in  lymphoid 
cells,  blood,  and  pus  has  been  mentioned  in  a  previous  chapter,  and  it 
seems  to  be  a  regular  constituent  of  all  cells  capable  of  development. 
Glycogen  was  first  shown  to  exist  in  embryonic  tissues  by  Bernard 
and  Kuhne,  and  it  seems  on  the  whole  to  be  a  constituent  of  tissues 
in  which  a  rapid  cell  formation  and  cell  development  are  taking  place. 
It  is  also  present  in  rapidly  forming  pathological  tumors  (Hoppe-Seyler). 
Some  animals,  as  certain  mussels  (Bizio),  Taenia  and  Ascaridae  (Wein- 
land  2),  are  very  rich  in  glycogen.  Glycogen  also  occurs  in  the  vegetable- 
kingdom,  especially  in  many  fungi. 

The  quantity  of  glycogen  in  the  liver,  as  also  in  the  muscles,  depends 
essentially  upon  the  food.  In  starvation  it  disappears  almost  com- 
pletely after  a  short  time,  but  more  rapidly  in  small  than  in  large  animals, 
and  it  disappears  earlier  from  the  liver  than  from  the  muscles.  As 
shown  by  C.  Voit,  Kulz  and  especially  by  Pfluger,3  it  never  entirely 
disappears  in  starvation,  as  a  re-formation  of  glycogen  always  takes 
place.  After  partaking  of  food,  especially  such  as  is  rich  in  carbo- 
hydrates, the  liver  becomes  rich  again  in  glycogen,  the  greatest  incre- 
ment occurring  14  to  16  hours  after  eating  (Kulz).  The  quantity  of 
liver-glycogen  may  amount  to  120-160  p.  m.  after  partaking  of  large 
quantities  of  carbohydrates,  and  in  dogs  which  had  been  especially 
fed  for  glycogen  Schondorff  and  Gatin-Gruzewska  found  still  higher 
results,  even  more  than  180  p.  m.  Ordinarily  it  is  considerably  less, 
namely,  12-30  to  40  p.  m.  The  highest  amount  of  glycogen  in  the  liver 
thus  far  observed  was  201.6  p.  m.,  found  by  Mangold  4  in  the  frog.     The 

1  Gatin-Gruzewska,  Pfluger's  Arch.,  103;  v.  Knaffl-Lenz,  Zeitschr.  f.  physiol.  Chem., 
4«. 

2  Zeitschr.  f.  Biologie,  41.  The  extensive  literature  on  glycogen  may  be  found  in 
E.  Pfliiger,  Glykogen,  2.  Aufl.,  Bonn,  1905;  and  in  Cremer,  "  Physiol,  des  Glykogens," 
in  Ergebnisse  der  Physiologic,  1,  Abt.  1.  In  the  following  pages  we  shall  refer  to  these^ 
works. 

1  Pfluger's  Arch.,  119,  which  contains  the  literature. 
*Itnd.,  121 


GLYCOGEN.  391 

shark,  whose  liver  is  very  rich  in  fat,  even  though  well  nourished,  only 
has  comparatively  low  values  for  the  glycogen  in  the  liver,  9.3-23.8 
p.  m.  (Bottazzi  J).  According  to  Cbemeb  the  quantity  of  glycogen  in 
plants  (yeast-cells)  is,  as  in  animals,  dependent  upon  the  food.  He 
finds  that  the  yeast-cells  contain  glycogen,  which  disappears  from  the 
cells  in  the  auto-fermentation  of  the  yeast,  but  reappears  on  the  intro- 
duction of  the  cells  into  a  sugar  solution. 

The  quantity  of  glycogen  of  the  liver  (and  also  of  the  muscles)  is 
also  dependent  upon  rest  and  activity,  because  during  rest,  as  in  hiberna- 
tion, it  increases,  and  during  work  it  diminishes.  Iyulz  has  shown  that 
by  hard  work  the  quantity  of  glycogen  in  the  liver  (of  dogs)  is  reduced 
to  a  minimum  in  a  few  hours.  The  muscle-glycogen  does  not  diminish 
to  the  same  extent  as  the  liver-glycogen.  Kulz,  Ztjntz  and  Vogelius, 
Frentzel,  and  others  have  been  able  to  render  rabbits  and  frogs  nearly 
glycogen-free  by  suitable  strychnine  poisoning.  The  same  result  is  pro- 
duced by  starvation  followed  by  hard  work.  According  to  Gatin- 
Gruzewska,2  the  liver  and  muscles  in  rabbits  can  be  made  glycogen- 
free  after  36-40  hours  by  first  starving  one  day  and  then  injecting 
adrenalin. 

Glycogen  forms  an  amorphous,  white,  tasteless,  and  non-odorous  powder. 
When  perfectly  pure,  and  by  proper  alcohol  precipitation,  it  can  be  obtained 
as  rods  or  prisms  which  look  like  crystals  (Gatin-Gruzewska).  It 
gives  an  opalescent  solution  with  water  which,  when  allowed  to  evaporate 
on  the  water-bath,  forms  a  pellicle  over  the  surface  that  disappears  again 
on  cooling.  It  is  undecided  whether  we  here  have  a  true  solution  or 
not.  Like  other  colloids,  glycogen  in  water  under  the  influence  of  the 
electric  current  migrates  to  the  anode,  on  which  it  collects  (Gatin- 
Gruzewska).  According  to  Bottazzi,3  who  obtained  the  same  results, 
a  little  acid  or  a  little  alkali  modify  the  results  so  that  the  glycogen  becomes 
isoelectric.  Its  aqueous  solution  is  dextrorotatory,  and  Htjppert  found 
it  to  be  (tt)D  =  +196.63°.  Gatin-Grtjzewska  has  recently  obtained 
the  same  result  by  using  a  perfectly  pure  solution  of  glycogen.  A 
solution  of  glycogen,  especially  on  the  addition  of  NaCl,  is  colored  wine- 
red  by  iodine.  It  may  hold  cupric  hydroxide  in  solution  in  alkaline 
liquids,  but  does  not  reduce  it.  A  solution  of  glycogen  in  water  is  not 
precipitated  by  potassium-mercuric  iodide  and  hydrochloric  acid,  but  is 
precipitated  by  alcohol  (on  the  addition  of  NaCl  when  necessary),  or 


1  Arch.  Ital.  d.  Biol.,  48;  cited  in  Bioch.  Centralbl.,  7,  833. 

2  Compt.  Rend.,  142. 

3  Bottazzi,  Chem.  Centralbl.  1009  p.  1423;  Bottazzi  and  d'Errico  (Pfluger's  Arch., 
115)  have  investigated  the  viscosity,  the  electrical  conductivity  and  the  freezing-point 
of  glycogen  solutions  at  different  concentrations. 


392  THE  LIVER. 

ammoniacal  basic  lead  acetate.  An  aqueous  solution  of  glycogen  made 
alkaline  with  caustic  potash  (15  per  cent  KOH)  is  completely  precipitated 
by  an  equal  volume  of  96  per  cent  alcohol.  Tannic  acid  also  precipitates 
glycogen.  It  gives  a  Avhite  granular  precipitate  of  benzoyl-glycogen 
with  benzoyl  chloride  and  caustic  soda.  Glycogen  is  completely  pre- 
cipitated by  saturating  its  solution  at  ordinary  temperatures  with  magne- 
sium or  ammonium  sulphate.  It  is  not  precipitated  by  sodium  chloride, 
or  by  half  saturation  with  ammonium  sulphate  (Nasse,  Neumeister, 
Halliburton,  Young1).  On  boiling  with  dilute  caustic  potash  (1-2 
per  cent)  the  glycogen  may  be  more  or  less  changed,  especially  if  it  has 
been  previously  exposed  to  the  action  of  acid  or  to  Brucke's  reagent 
(see  below)  (Pfluger).  On  boiling  with  stronger  caustic  potash  (even 
of  36  per  cent)  it  is  not  injured  (Pfluger).  By  diastatic  enzymes 
glycogen  is  converted  into  maltose  or  glucose,  depending  upon  the  nature 
of  the  enzyme.  It  is  transformed  into  glucose  by  dilute  mineral  acids. 
According  to  Tebb  2  various  dextrins  appear  as  intermediary  steps  in 
the  saccharification  of  glycogen,  depending  on  whether  the  hydrolysis 
is  caused  by  mineral  acids  or  enzymes.  The  glycogen  from  various 
animals  and  different  organs  is  the  same  according  to  Pfluger.3  Nor  has 
it  been  decided  whether  all  the  glycogen  in  the  liver  occurs  as  such  or 
whether  it  is  in  part  combined  with  protein  (Pfluger-Nerking).  The 
investigations  of  Loeschcke  4  have  shown  that  we  have  no  positive 
reasons  for  this  assumption. 

The  preparation  of  pure  glycogen  (most  easily  from  the  liver)  is 
generally  performed  by  the  method  suggested  by  Brucke,  of  which  the 
main  points  are  the  following:  Immediately  after  the  death  of  the  animal 
the  liver  is  thrown  into  boiling  water,  then  finely  divided  and  boiled 
several  times  with  fresh  water.  The  filtered  extract  is  now  sufficiently 
concentrated,  allowed  to  cool,  and  the  proteins  removed  by  alternately 
adding  potassium-mercuric  iodide  and  hydrochloric  acid.  The  glycogen 
is  precipitated  from  the  filtered  liquid  by  the  addition  of  alcohol  until 
the  liquid  contains  60  vols,  per  cent.  By  repeating  this  and  precipitating 
the  glycogen  several  times  from  its  alkaline  and  acetic-acid  solution  it 
is  purified  on  the  filter  by  washing  first  with  60  per  cent  and  then  with 
95  per  cent  alcohol,  then  treating  with  ether,  and  drying  over  sulphuric 
acid.  It  is  always  contaminated  with  mineral  substances.  To  be  able 
to  extract  the  glycogen  from  the  liver  or,  especially,  from  muscles  and 
other  tissues  completely,  which  is  essential  in  a  quantitative  estimation, 
these  parts  must  first  be  warmed  for  two  hours  with  strong  caustic  potash 
(30  per  cent)  on  the  water-bath.     As  the  glycogen  changes  in  this  purifica- 


1  Young,  Journ.  of  Physiol.,  22,  citing  the  other  investigators. 

2  Jonrri.  of  Phyeiol.,  22. 

*  Pfluger's,  Arch.  129. 

*  Ibid.,  102. 


GLYCOGEN   FORMERS.  393 

tion,  as  suggested  by  BrxJcke,  it  is  better,  for  quantitative  determinations 
of  glycogen,  to  precipitate  it  directly  from  the  alkaline  solution  by  alcohol 
(Pfluger  1). 

The  quantitative  estimation  is  best  performed  according  to  Pfluger's 
method,  which  is  as  follows:  The  finely  divided  organ  is  heated  on 
the  water-bath  for  2-3  hours  in  the  presence  of  30  per  cent  KOH;  after 
diluting  with  water  and  filtering,  the  glycogen  is  precipitated  with 
alcohol,  and  the  redissolved  glycogen  estimated  in  part  by  the  polar- 
iscope  and  in  part  as  sugar  after  inversion.  One  part  by  weight  of  sugar 
equals  0.927  part  glycogen.  As  in  the  estimation  the  prescribed  direc- 
tions must  be  exactly  followed,  we  must  refer  to  the  original  work  of 
Pfluger  for  the  details  of  the  method.  Other  methods  of  estimating 
glycogen,  such  as  those  of  Brucke-Kulz,  Pavy,  and  Austin,  are  described 
in  Pfluger's  Archiv.  96.     Also  compare  the  recent  works  of  Pfluger.2 

Numerous  investigators  have  endeavored  to  determine  the  origin 
of  glycogen  in  the  animal  body.  It  is  positively  established  by  the 
unanimous  observations  of  many  investigators3  that  the  varieties  of 
sugars  and  their  anhydrides,  dextrins  and  starches,  have  the  property  of 
increasing  the  quantity  of  glycogen  in  the  body.  The  action  of  inulin 
seems  to  be  somewhat  uncertain.4  The  statements  are  questioned  in 
regard  to  the  action  of  the  pentoses.  Cremer  found  that  in  rabbits  and 
hens  various  pentoses,  such  as  rhamnose,  xylose,  and  arabinose,  have  a 
positive  influence  on  the  glycogen  formation,  and  Salkowski  obtained 
the  same  result  on  feeding  /-arabinose.  Frentzel,  on  the  contrary, 
found  no  glycogen  formation  on  feeding  xylose  to  a  rabbit  which  had 
previously  been  made  glycogen-free  by  strychnine  poisoning,  and  Neu- 
berg  and  Wohlgemuth  5  obtained  similar  negative  results  on  feed- 
ing rabbits  with  d-  and  r-arabinose.  In  general  we  can  for  the  present 
accept  the  view  that  the  pentoses  are  not  direct  glycogen  formers. 

The  hexoses,  and  the  carbohydrates  derived  therefrom,  do  not  all 
possess  the  ability  cf  forming  or  accumulating  glycogen  to  the  same 
extent.  Thus  C.  Voir  6  and  his  pupils  have  shown  that  glucose  has  a 
more  powerful  action  than  cane-sugar,  while  milk-sugar  is  less  active 
(in  rabbits  and  hens)  than  glucose,  fructose,  cane-sugar,  or  maltose. 

The  following  substances  when  introduced  into  the  body  also  increase  the 
quantity  of  glycogen  in  the  liver:   Glycerin,  gelatin,  arbutin,  and  likewise,  accord- 

*See  also  the  method  suggested  by  Gautier,  Compt.  Rend.,  129. 

2  Pfluger's  Arch.,  103,  104,  121  and  especially  129. 

J  In  reference  to  the  literature  on  this  subject,  see  E.  Kiilz,  Pfluger's  Arch.,  24, 
and  Ludwig,  Festschrift,  1891;  also  the  cited  works  of  Pfluger  and  Cremer,  foot-note 
2,  p.  390. 

*  See  Miura,  Zeitschr.  f.  Biologie,  32,  and  Nakaseko,  Amer.  Journ.  of  Physiol.,  4. 

6  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  32;  Neuberg  and  Wohlgemuth,  ibid.,  35. 
See  also  Pfluger,  1.  c,  and  Cremer,  1.  c. 

6  Zeitschr.  f.  Biologie,  28. 


394  THE  LIVER. 

ing  to  the  investigations  of  Kulz,  erythrite,  quercite,  dulcite,  mannite,  inosite, 
ethylene  and  propylene  glycol,  glucuronic  anhydride,  saccharic  acid,  mucic  acid, 
sodium  tartrate,  saccharin,  isosaccharin,  and  urea.  Ammonium  carbonate,  gly- 
cocoll;  and  asparagine  may  similarly,  according  to  Rohmann,  cause  an  increase 
in  the  amount  of  glycogen  in  the  liver.  Nebelthau  finds  that  other  ammonium 
salts  and  some  of  the  amides,  as  well  as  certain  narcotics,  hypnotics,  and  antipyretics, 
produce  an  increase  in  the  glycogen  of  the  liver.  This  action  of  the  antipyretics 
(especially  antipyrine)  had  been  shown  by  Lepine  and  Porteret.1 

The  fats,  according  to  Bouchard  and  Desgrez,  increase  the  glycogen 
content  of  the  muscles  but  not  of  the  liver,  while  Cotjvreur  believes  that 
the  glycogen  is  increased  at  the  expense  of  the  fat  in  the  silkworm  larva, 
but  these  statements  have  been  shown  to  be  incorrect  by  the  recent 
investigations  of  Kotake  and  Sera.2  In  general  it  is  believed  that  fat 
does  not  increase  the  amount  of  glycogen  in  the  liver  or  in  the  animal 
body,  although  a  carbohydrate  formation  from  glycerin,  but  not  a  gly- 
cogen formation,  is  probable. 

The  question  whether  the  proteins  have  the  ability  to  increase  the 
glycogen  content  of  the  liver  or  the  animal  body  has  been  long  disputed. 
The  feeding  experiments  with  meat  or  with  pure  proteins  by  older  exper- 
imenters, such  as  Naunyn,  v.  Mering  and  E.  Kulz  seem  to  show  an 
ability.  But  the  proof  of  these  investigations  has  been  strongly  dis- 
puted by  Pfluger  and  later  investigations  of  Schondorff,  Blumenthal 
and  Wohlgemuth,  as  also  those  of  Bendix  and  Stookey3  yield  contradic- 
tory results.  These  investigations  have  really  only  historical  interest, 
since  now  a  carbohydrate  formation  as  well  as  a  glycogen  formation  from 
proteins  have  been  positively  observed. 

If  the  question  is  raised  as  to  the  action  of  the  various  bodies  on 
the  accumulation  of  glycogen  in  the  liver,  it  must  be  recalled  that  a  forma- 
tion of  glycogen  takes  place  in  this  organ,  as  well  as  a  consumption  of  the 
same.  An  accumulation  of  glycogen  may  be  caused  by  an  increased 
formation  of  glycogen,  but  also  by  a  diminished  consumption,  or  by  both. 

It  is  not  known  how  the  various  bodies  above  mentioned  act  in  this 
regard.  Certain  of  them  probably  have  a  retarding  action  on  the  trans- 
formation of  glycogen  in  the  liver,  while  others  perhaps  are  more  com- 
bustible, and  in  this  way  protect  the  glycogen.  Some  probably  excite 
the  liver-cells  to  a  more  active  glycogen  formation,  while  others  yield 
material  from  which  the  glycogen  is  formed,  and  are  glycogen-formers 


1  Rohmann,    Pfliiger's  Arch.,  39;  Nebelthau,  Zeitschr.  f.  Biologie,  28;  Lepine  and 
Porteret,  Compt.  Rend.,  107. 

2  Bouchard  et  Desgrez,  Compt.  Rend.,  130;  Couvreur,  Compt.  rend,  de  soc.  biol., 
47;  Kotake  and  Sera  Zeitschr.  f.  physiol.  Chem.,  62. 

Bee  the  work  on  glycogen  by  Pfluger  and  also  Schondorff,  Pfliiger's  Arch.,  82  and  88; 
Blumenthal  and  Wohlgemuth,  Berl.  klin.  Wochenschr.,  1901;  Bendix,  Zeitschr.  f.  physiol. 
Chem.,  32  and  34;  Stookey,  Amer.  Journ.  of  Physiol.,  9. 


FORMATION  OF  GLYCOGEN.  395 

in  the  strictest  sense  of  the  word.  The  knowledge  of  these  last-mentioned 
bodies  is  of  the  greatest  importance  in  the  question  as  to  the  origin  of 
glycogen  in  the  animal  body,  and  the  chief  interest  attaches  to  the 
question:  To  what  extent  are  the  two  chief  groups  of  food,  the  proteins 
and  carbohydrates,  glycogen-formers? 

The  great  importance  of  the  carbohydrates  in  the  formation  of  gly- 
cogen has  given  rise  to  the  opinion  that  the  glycogen  in  the  liver  is  pro- 
duced from  sugar  by  a  synthesis  in  which  water  separates  with  the  for- 
mation of  an  anhydride  (Luchsinger  and  others).  This  theory  (anhy- 
dride theory)  has  found  opponents  because  it  neither  explains  the  forma- 
tion of  glycogen  from  such  bodies  as  proteins,  carbohydrates,  gelatin, 
and  others,  nor  the  circumstance  that  the  glycogen  is  always  the  same, 
independent  of  the  properties  of  the  carbohydrate  introduced,  whether 
it  is  dextrogyrate  or  levogyrate.  This  last  circumstance  does  not  now 
present  any  special  difficulty,  since  we  know  that  the  simple  sugars  can 
easily  be  transformed  into  each  other.  It  was  formerly  the  opinion  of 
many  investigators  that  all  glycogen  is  formed  from  protein,  and  that 
this  splits  into  two  parts,  one  containing  nitrogen  and  the  other  being 
free  from  nitrogen;  the  latter  is  the  glycogen.  According  to  these 
views,  the  carbohydrates  act  only  in  that  they  spare  the  protein  and 
the  glycogen  produced  therefrom  (sparing  theory  of  Weiss,  Wolffberg, 
and  others  1). 

In  opposition  to  this  theory  C.  and  E.  Voit  and  their  pupils  have 
shown  that  the  carbohydrates  are  "  true  "  glycogen-formers.  After  par- 
taking of  large  quantities  of  carbohydrates,  the  amount  of  glycogen  stored 
up  in  the  body  is  sometimes  so  great  that  it  cannot  be  covered  by  the 
protein?  decomposed  during  the  same  time,  and  in  these  cases  a  gly- 
cogen formation  from  the  carbohydrates  must  be  admitted.  According 
to  Cremer  only  the  fermentable  sugars  of  the  six  carbon  series  or  their 
di-  and  polysaccharides  are  true  glycogen-formers.  For  the  present,  only 
glucose,  fructose,  and  to  a  much  less  degree  galactose  (Weinland2), 
and  perhaps  also  e?-mannose  (Cremer)  are  designated  as  true  glycogen- 
formers.  Other  monosaccharides  may  indeed,  according  to  Cremer, 
influence  the  formation  of  glycogen,  but  they  are  not  converted  into 
glycogen,  and  hence  are  called  only  pseudoglycogen-formers. 

The  poly-  and  disaccharides  may,  after  a  cleavage  into  the  cor- 
responding fermentable  monosaccharides,  serve  as  glycogen-formers. 
This  is  true  for  at  least  cane-sugar  and  milk-sugar,  which  must  first 


1  In  regard  to  these  two  theories,  see  especially  Wolffberg,  Zeitschr.  f .  Biologie,  16. 

1  E.  Voit,  Zeitschr.  f.  Biologie,  25,  543,  and  C.  Voit,  ibid.,  2S.  See  also  Kausch 
and  Socin,  Arch.  f.  exp.  Path.  u.  Pharm.,  31;  Weinland,  Zeitschr.  f.  Biologie,  40  and 
38;  Cremer,  ibid.,  42,  and  Ergebnisse  der  Physiol.,  1. 


39G  THE  LIVER. 

be  inverted  in  the  intestine.  These  two  varieties  of  sugar,  therefore, 
cannot,  like  glucose  and  fructose,  serve  as  glycogen-formers  after  sub- 
cutaneous injection,  but  reappear  almost  entirely  in  the  urine  (Dastre, 
Fr.  Voit).  Maltose,  which  is  inverted  by  an  enzyme  present  in  the  blood, 
passes  only  to  a  slight  extent  into  the  urine  (Dastre  and  Bourquelot 
and  others),  and  it  can,  like  the  monosaccharides,  even  after  subcuta- 
neous injection,  be  used  in  the  formation  of  glycogen  (Fr.  Voit).1  Of 
the  disaccharides  the  maltose  and  the  cane-sugar  are  strong  glycogen- 
formers  while  milk-sugar  has  only  a  weak  action. 

The  ability  of  the  liver  to  form  glycogen  from  monosaccharides  has 
also  been  shown  by  K.  Grube  in  a  very  interesting  and  direct  manner, 
by  perfusion  experiments  with  solutions  of  various  carbohydrates.  In 
these  perfusion  experiments  on  tortoise  livers,  glucose  produced  an 
abundant  glycogen  formation,  while  with  fructose  and  galactose  it  was 
less  abundant.  Pentoses,  disaccharides,  casein  and  amino-acids  (gly- 
cocoll,  alanine  and  leucine)  were  inactive  while  on  the  contrary  glycerin 
and  also  formaldehyde  acted  as  glycogen-formers.  The  formation  of 
glycogen  from  formaldehyde  is  disputed  by  Schondorff  and  Grube.2 

After  Pavy3  first  showed  the  occurrence  of  carbohydrate  groups  in 
ovalbumin,  other  investigators  were  able  to  split  off  glucosamine  from 
this  and  other  protein  substances  (see  Chapter  II),  and  the  question 
arose  whether  the  amino-sugar  could  serve  in  the  formation  of  glycogen. 
The  investigations  carried  out  in  this  direction  by  Fabian,  Frankel 
and  Offer,  Cathcart  and  Bial,  have  shown  that  the  glucosamine 
introduced  into  the  organism  is  in  part  eliminated  unchanged  in  the 
urine  and  has  no  glycogen-forming  action.  No  definite  conclusions 
can  be  drawn  from  this  on  the  behavior  of  the  carbohydrate  groups, 
which  exist  not  as  free  groups  but  combined  with  the  protein  molecules. 
The  investigations  of  Forschbach  on  the  behavior  of  glucosamine 
chained  to  an  acid-group  in  an  amide-like  combination,  as  well  as  the 
investigations  of  Kurt  Meyer  and  Stolte,4  have  yielded  no  proofs  for 
the  theory  that  glycogen  is  formed  from  glucosamine. 

Whether  or  not,  or  to  what  extent,  the  glucoproteins  by  their  glucosa- 
mine component  take  part  in  the  sugar  or  glycogen  formation  in  the  animal 


'Dastre,  Arch,  de  Physiol.  (5)  3,  1891;  Dastre  and  Bourquelot,  Compt.  Rend.,  98; 
Fritz  Voit,  Verhandl.  d.  Gesellsch.  f.  Morph.  u.  Physiol,  in  Miinchen,  1896,  and  Deutsch. 
Arch.  f.  klin.  Med.,  58.  In  regard  to  the  glycogen  formation  after  intravenous  injection 
of  sugar  see  Freund  and  Popper,  Bioch.  Zeitschr.,  41. 

2Pfluger's  Arch.,  138;  Grube,  ibid.,  118,  121,  122,  126  and  139. 

3  The  Physiology  of  the  Carbohydrates,  London,  1894. 

*  Fabian,  Zeitschr.  f.  physiol.  Chem.,  27;  Frankel  and  Offer,  Centralbl.  f.  Physiol., 
13;  Cathcart,  Zeitschr.  f.  physiol.  Chem.,  39;  Bial,  Berl.  klin.  Wochenschr.,  1905; 
Forschbach,  Hofmeister's  Beitrage,  8;  Meyer,  ibid.,  9;  Stolte,  ibid.,  11. 


FORMATION  OF  GLYCOGEN.  397 

body  is  difficult  to  answer  for  the  present,  as  but  little  is  known  of  the 
quantity  of  these  substances  in  the  body,  and  our  knowledge  of  the  amount 
of  carbohydrate  which  can  be  split  off  from  the  various  protein  substances 
is  also  very  meager. 

If  the  proteins  are  to  be  counted,  and  this  is  in  agreement  with  the 
generally  accepted  view,  among  those  bodies  which  increase  the  glycogen 
of  the  body,  then  Ave  must  ask  the  question:  Do  the  proteins  act  only 
indirectly  as  pseudoglycogen-formers,  or  are  they  direct  glycogen- 
formers  which  can  serve  as  material  for  the  formation  of  glycogen  or 
sugar?  This  question  stands  in  close  relation  to  the  sugar  formation 
and  sugar  elimination  in  the  various  forms  of  glycosuria,  and  will  be  best 
discussed  below  in  connection  with  the  question  of  diabetes. 

Glycogen  is  a  reserve-food  deposited,  in  the  liver  and  which,  like  other 
carbohydrates  can  be  transformed  into  fat,  and  it  is  generally  admitted 
that  such  a  fat  formation  from  glycogen  also  takes  place  in  the  liver. 
There  is  no  doubt  that  the  glycogen  deposited  in  the  liver  is  formed  in 
the  liver-cells  from  the  sugar;  but  where  does  the  glycogen  existing  in 
the  other  organs,  such  as  the  muscles,  originate?  Is  the  glycogen  of  the 
muscles  formed  on  the  spot  cr  is  it  transmitted  to  the  muscles  by  the  blood? 
These  questions  cannot  at  present  be  answered  with  certainty,  and  the 
investigations  on  this  subject  by  different  experimenters  have  given 
varying  results.  The  experiments  of  Iyulz,1  in  which  he  studied  the 
glycogen  formation  by  passing  blood  containing  cane-sugar  through 
the  muscle,  have  led  to  no  conclusive  results,  while  the  perfusion  exper- 
iments of  Hatcher  and  Wolff  with  glucose  seem  to  indicate  a  glycogen 
formation  from  sugar  in  the  muscles.  The  investigations  of  de  Filippi  2 
on  dogs  with  so-called  Eck's  fistula  also  show  a  glycogen  formation  from 
sugar  in  the  muscles.  In  the  Eck  fistula  operation  the  portal  vein  is 
ligated  near  the  liver  hilus  and  sewed  to  the  inferior  vena  cava  and  an 
opening  established  between  the  two  veins  so  that  the  portal  blood  flows 
directly  into  the  vena  cava  without  passing  through  the  liver.  In 
well-nourished  animals,  operated  upon  in  this  manner,  the  livers  had  the 
same  properties  as  those  from  starving  animals,  while,  on  the  contrary, 
the  muscles  contained  quantities  of  glycogen  which  corresponded  to 
those  found  in  a  normal  over-fed  dog. 

If  it  be  true  that  the  blood  and  lymph  contain  a  diastatic  enzyme 
which  transforms  glycogen  into  sugar,  and  also  that  the  glycogen  regularly 
occurs  in  the  form-elements  and  is  not  dissolved  in  the  fluids,  it  seems 
probable  that  the  glycogen  in  solution  is  not  transmitted  by  the  blood  to 


1  See  Minkowski  and  Laves,  Arch.  f.  exp.  Path.  u.  Pharra.,  23;   Kiilz,  Zeitschr.  f. 
Biologie,  27;  Hatcher  and  Wolff,  Journ.  of  Biol.  Chern.,  3. 

2  Zeitschr.  f.  Biol.,  49  and  50. 


398  THE  LIVER. 

the  organs,  but  perhaps  more  likely,  if  the  leucocytes  do  not  act  as  car- 
riers, it  is  formed  on  the  spot  from  the  sugar.1  The  glycogen  formation 
seems  to  be  a  general  function  of  the  cells.  In  adults,  the  liver,  which 
is  very  rich  in  cells,  has  the  property,  on  account  of  its  anatomical  posi- 
tion, of  transforming  large  quantities  of  sugar  into  glycogen. 

This  glycogen,  which  is  deposited  in  the  liver  as  reserve-food,  in  order 
that  it  can  be  useful  to  the  body,  must  at  least  in  greater  part  be  trans- 
formed into  sugar  and  supplied  to  the  various  organs  by  the  blood.  The 
question  now  arises  whether  there  is  any  foundation  for  the  statement 
that  the  liver  glycogen  is  transformed  into  sugar. 

As  first  shown  by  Bernard  and  redemonstrated  by  many  inves- 
tigators, the  glycogen  in  a  dead  liver  is  gradually  changed  into  sugar, 
and  this  sugar  formation  is  caused,  as  Bernard  supposed  and  then  shown 
by  numerous  investigators  by  a  diastatic  enzyme  whose  relation  to  the 
diastatic  enzyme  of  the  blood  is  not  quite  clear.2 

This  post-mortem  sugar  formation  led  Bernard  to  the  assump- 
tion of  the  formation  of  sugar  from  glycogen  in  the  liver  during  life. 
Bernard  suggested  the  following  arguments  for  this  theory:  The  liver 
always  contains  some  sugar  under  physiological  conditions,  and  the 
blood  from  the  hepatic  vein  is  always  somewhat  richer  in  sugar  than  the 
blood  from  the  portal  vein.  Bernard's  views  found  in  Seegen  an  active 
supporter,  as  he  tried  to  show  by  numerous  experiments  the  physio- 
logical sugar  content  of  the  liver  as  well  as  the  high  sugar  content  of  the 
blood  of  the  liver  veins.  On  the  other  hand  the  correctness  of  the 
observations  of  Bernard  and  Seegen  is  disputed  by  many  investigators 
such  as  Pavy,  Ritter,  Schiff,  Eulenberg,  Lussana,  Mosse,  N.  Zuntz 
and  others,3  and  in  regard  to  the  sugar  content  in  the  two  kinds  of 
blood  we  have  come  to  the  general  conclusion  that  when  only  the  stasis 
and  other  disturbing  influences  of  the  operation  are  prevented,  the  blood 
of  the  liver  veins,  if  at  all,  is  only  slightly  richer  in  sugar  than  the  blood 
of  the  portal  vein.4 

The  circumstance  that  the  blood-sugar  rapidly  sinks  to  |-f  of  its 
original  quantity,  or  even  disappears  when  the  liver  is  cut  out  of  the 
circulation,  indicates  a  vital  formation  of  sugar  in  the  liver  (Seegen, 
Bock  and  Hoffmann,  Kaufmann,  Pavy  and  others).     In  geese  whose 

1  See  Dastre,  Compt.  rend,  de  soc.  biol.,  47,  280,  and  Kaufmann,  ibid.,  316. 
2R6hmann,  Verh.  d.  Ges.  deutsch.    Naturf.  u.  Aerzte.  Breslau,  1903;  Borchardt, 
Pfluger's  Arch.,  100;  Zegla,  Bioch.  Zeitschr.,  16;  E.  Starkenstein,  ibid.,  24. 

3  In  regard  to  the  literature  on  sugar  formation  in  the  liver  see  Bernard,  Lecons  sur 
le  diabete,  Paris,  1877;  Seegen,  Die  Zuckerbildung  im  Tierkorper,  2.  Aufl.  Berlin, 
1900;  M.  Bial,  Pfluger's  Arch.,  55,  434. 

4  Seegen,  Die  Zuckerbildung,  etc.,  and  Centralbl.  f.  Physiol.,  10,  497  and  822; 
Zuntz,  ibid.,  561;  Mosse,  Pfluger's  Arch.,  63;  Bing,  Skand.  Arch.  f.  Phy.siol.,  9. 


SUGAR  FORMATION  FROM  GLYCOGEN.         39$ 

livers  wire  removed  from  the  circulation,  Minkowski  found  no  sugar 
in  the  blood  after  a  few  hours.  On  removing  the  liver  from  the 
circulation  by  tying  all  the  vessels  to  and  from  the  organ,  the  quan- 
tity of  sugar  in  the  blood  is  not  increased  (Schenck1).  An  important 
proof  of  the  possibility  of  a  vital  formation  of  sugar  from  the  liver  gly- 
cogen lies  in  the  fact  that  we  shall  learn  below  of  certain  poisons  and 
operative  changes  which  may  cause  an  abundant  elimination  of  sugar, 
but  only  when  the  liver  contains  glycogen. 

A  vital  formation  of  sugar  from  the  liver  glycogen  is  now  generally 
accepted.  Most  investigators  consider  this  as  an  enzymotic  transforma- 
tion of  the  glycogen  by  means  of  the  liver  diastase,  while  certain  inves- 
tigators such  as  Dastre,  Noel-Paton,  E.  Cavazzani,  McGuigan  and 
Brooks  2  and  others  explain  it  by  a  special  activity  of  the  protoplasm. 
Bang  3  has  studied  the  formation  of  sugar  in  frogs'  livers,  which  had  not 
appreciably  changed  in  weight  in  a  Ringer's  solution  which  was  isotonic 
with  the  frog  blood  and  which  correspondingly  had  retained  their  vital 
properties.  This  sugar  formation  does  not  depend  upon  a  protoplasmic 
activity  but  is  of  an  enzymotic  nature.  It  is  caused  by  a  diastase^ 
which  in  Rana  esculenta  occur  in  great  part  in  a  latent,  inactive  form 
due  to  the  inhibitory  action  of  the  liver  lipoids.  Common  salt  is  espe- 
cially important  as  an  activator  for  this  enzyme.  The  surviving  frog 
liver  is  stimulated  to  a  strong  sugar  production  by  adrenalin,  and  this 
sugar  formation  is  also  of  an  enzymotic  nature.  The  action  of  the 
adrenalin  consists  in  an  activation  of  the  liver  diastase,  brought  about 
in  various  ways. 

The  relation  of  the  sugar  eliminated  in  the  urine  under  certain 
conditions,  such  as  in  diabetes  mellitus,  certain  intoxications,  lesions 
of  the  nervous  system,  etc.,  to  the  glycogen  of  the  liver  is  also  an  important 
question. 

It  does  not  enter  into  the  plan  and  scope  of  this  book  to  discuss  in 
detail  the  various  views  in  regard  to  glycosuria  and  diabetes.  The 
appearance  of  glucose  in  the  urine  is  a  symptom  which  may  have  essen- 
tially different  causes,  depending  upon  different  circumstances.  Only 
a  few  of  the  most  important  points  will  be  mentioned. 

The  blood  always  contains  about  the  average  of  1  p.  m.,  while  the 
urine  has  in  it  at  most  only  traces  of  glucose.     When  the  quantity  of 

'Seegen,  Bock,  and  Hoffmann,  see  Seegen,  1.  c;  Kaufmann,  Arch,  de  Physiol.  (5), 
8;  Tangl  and  Harley,  Pfluger's  Arch.,  61;  Pavy,  Journ.  of  Physiol.,  29,  Minkowski, 
Arch.  f.  exp.  Path.  u.  Pharm.,  21;  Schenck,  Pfluger's  Arch.,  57. 

2  See  Dastre,  Noel-Paton,  Cavazzani  and  their  work  cited  in  Pick,  Hofmeister's- 
Beitrage,  3,  and  McGuigan  and  Brooks,  Amer.  Journ.  of  Physiol.,  18;  R.  G.  Pearce,. 
ibid.,  25. 

8  Bioch.  Zeitschr.,  49. 


400  THE  LIVER. 

sugar  in  the  blood  rises  above  this  average,  sugar  passes  into  the  urine, 
sometimes  even  with  slight  rise  and  in  other  cases  with  stronger  rise. 
The  kidneys  have  the  property  to  a  certain  extent  of  preventing  the 
passage  of  blood-sugar  into  the  urine;  and  it  follows  from  this  that  an 
elimination  of  sugar  in  the  urine  may  be  caused  partly  by  a  reduction 
or  suppression  of  this  above-mentioned  activity,  and  partly  also  by  an 
abnormal  increase  of  the  quantity  of  sugar  in  the  blood. 

The  first  seems,  according  to  v.  Mering  and  Minkowski,  and  others 
to  be  the  case  in  phlorhizin  diabetes,  v.  Mering  found  that  a  strong 
glycosuria  appears  in  man  and  animals  on  the  administration  of  the 
glucoside  phlorhizin.  The  sugar  eliminated  is  not  derived  from  the 
glucoside  alone.  It  is  formed  in  the  animal  body,  and  in  fact  from  the 
carbohydrates,  or  as  generally  admitted  on  prolonged  starvation,  from 
the  protein  substances  of  the  body  (Lusk).  The  quantity  of  sugar  in 
the  blood  is  not  increased,  but  rather  diminished,  in  phlorhizin  diabetes 
(Minkowski),  which  does  not  indicate  increase  in  the  sugar  production 
but  rather  an  increased  excretion  of  the  sugar  by  the  kidneys.  The 
fact  that  after  extirpation  of  the  kidney  in  phlorhizin  diabetes  no  rise 
in  the  blood-sugar  is  observed,  and  that  after  the  injection  of  phlorhizin 
in  the  renal  artery  of  one  side  the  urine  secreted  by  this  kidney  contains 
sugar  sooner  and  more  abundantly  than  the  urine  from  the  other  kidney 
(Ztjntz),  tends  to  favor  this  view.  The  experiments  especially  performed 
by  Pavt,  Brodie,  and  Siau  upon  blood  containing  phlorhizin  and  sur- 
viving kidneys  also  indicate  the  same,  namely,  that  the  phlorhizin  acts 
upon  the  kidneys  and  the  researches  of  Erlandsen  also  lead  to  the  same 
conclusion.  He  found  that  on  combining  the  phlorhizin  action  with 
bleeding  that  the  glycosuria  was  increased  while  after  bleeding  alone 
without  phlorhizin  poisoning  the  hyperglycemia  was  absent.  While 
v.  Mering  and  others  believe  in  an  increased  permeability  of  the  kidneys 
for  sugar,  produced  by  the  phlorhizin  Lepine  *  is  of  the  view  that  the 
phlorhizin  causes  a  formation  of  glucose  from  the  virtual  sugar  in  the 
kidneys.  Pavy  is,  on  the  contrary,  of  the  opinion  that  the  kidneys, 
under  the  influence  of  the  phlorhizin,  split  off  sugar  from  a  substance 


1  In  regard  to  the  literature  on  phlorhizin  diabetes  see  v.  Mering,  Zeitschr.  f.  klin. 
Med.,  14  and  16;  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  31;  Moritz  and  Prausnitz, 
Zeitschr.  f.  Biologie,  27  and  29;  Kiilz  and  Wright,  ibid.,  27,  181;  Cremer  and  Rioter, 
ihvL,  28  and  29;  Contejean,  Compt.  rend,  de  soc.  biol.,  48;  Lusk,  Zeitschr.  f.  Biologie, 
36  and  42;  Levene,  Journal  of  Physiol.,  17;  Pavy,  ibid.,  20,  and  with  Brodie  and  Siau, 
29;  Arteaga,  Amer.  Journ.  of  Physiol.,  6;  O.  Loewi,  Arch.  f.  exp.  Path.  u.  Pharm.,  47; 
X.  Zuntz,  Arch.  f.  (Anat.  u.)  Physiol.,  189.5;  Stiles  and  Lusk,  Amer.  Journ.  of  Physiol., 
10:  Lusk,  ibid.,  22;  Cremer,  Ergebnisse  der  Physiol.,  1,  Abt.  1;  Erlandsen,  Bioch. 
Zeitschr.,  23  and  24;  I.epine,  Compt.  rend.  soc.  biol.,  68;  Lusk,  Ergebnisse  der  Physiol., 
Bd.  12,  315-392,  and  the  monographs  upon  diabetes. 


GLYCOSURIAS.  401 

•circulating  in  the  blood,  perhaps  from  a  protein  with  loosely  combined 
•carbohydrate  groups. 

Grube  from  experiments  upon  the  surviving  tortoise  liver  has  made  the 
suggestion  that  it  is  not  the  kidneys  which  are  first  attacked  by  the  phlorhizin 
action  in  phlorhizin  glycosuria  but  the  liver.  Important  experimental  evidence 
against  this  view  has  been  raised  by  Schondorff  and  Suckrow.1 

Another  form  of  glycosuria  which  according  to  certain  investigators 
is  to  be  connected  with  a  changed  permeability  of  the  kidneys  (Under- 
hill  and  Closson)  is  the  glycosuria  first  observed  by  Bock  and  Hoff- 
mann after  the  intravascular  injection  of  large  quantities  of  a  1-per 
cent  salt  solution,  which  is  also  of  great  interest  because,  as  shown  by 
Martin  Fischer,2  it  can  be  again  arrested  by  an  injection  of  a  salt  solu- 
tion containing  CaCUj.  There  are  investigators  who  attempt  to  connect 
this  glycosuria  with  the  adrenals  and  a  hyperglycemia. 

With  the  exception  of  these  two  forms  of  glycosuria,  the  phlorhizin 
diabetes  and  the  salt-glycosuria,  and  also  the  glycosuria  produced  by 
certain  kidney  poisons,  all  other  forms  of  glycosuria  or  diabetes,  as  far 
as  known  at  present,  depend  on  a  hyperglycemia. 

A  hyperglycemia  may  be  caused  in  various  ways.  It  may  be  caused, 
for  example,  by  the  introduction  of  more  sugar  than  the  body  can  destroy. 

The  ability  of  the  animal  body  to  assimilate  the  different  varieties 
of  sugar  has  naturally  a  limit.  If  too  much  sugar  is  introduced  into  the 
intestinal  tract  at  one  time,  so  that  the  so-called  assimilation  limit 
(see  Chapter  VIII,  on  absorption)  is  overreached,  then  the  excess  of 
absorbed  sugar  passes  into  the  urine.  This  form  of  glycosuria  is  called 
alimentary  glycosuria,3  and  is  caused  by  the  passage  of  more  sugar  into 
the  blood  than  the  liver  and  other  organs  can  destroy. 

As  the  liver  cannot  transform  into  glycogen  all  the  sugar  which  comes 
to  it  in  these,  to  a  certain  extent  physiological,  alimentary  glycosurias, 
it  is  possible  that  a  glycosuria  may  also  be  produced  under  pathological 
conditions,  even  by  a  moderate  amount  of  carbohydrate  (100  grams 
glucose),  which  a  healthy  person  could  overcome.  This  is  true,  among 
other  cases,  in  various  affections  of  the  cerebral  system  and  in  certain 
chronic    poisonings.     Certain    observers    include    the    lighter    forms    of 

1  Grube,  Pfltiger's  Arch.,  128;  Schondorff  and  Suckrow,  ibid.,  138.  See  also  the 
opposed  view  of  Underhill,  Journ.  of  biol.  Chem.,  13. 

2  Bock  and  Hoffmann,  Arch.  f.  (Anat.  u.)  Physiol.,  1871;  M.  Fischer,  University 
of  California  publications  Physiol.,  1903  and  1904,  and  Pfliiger's  Arch.,  106  and  109; 
Underhill  and  Closson,  Amer.  Journ.  of  Physiol.,  15,  and  Journ.  of  Biol.  Chem.,  4. 

3  In  regard  to  alimentary  glycosuria  see  Moritz,  Arch.  f.  klin.  Med.,  46,  which  also 
contains  the  earlier  literature;  B.  Rosenberg,  Ueber  das  Vorkommen  der  alimentaren 
Glykosurie,  etc.  (Inaug.-Dissert.  Berlin,  1897);  van  Oondt,  Munch,  med.  Wochen- 
schr.,  1898;  v.  Noorden,  Die  Zuckerkrankheit,  3.  Aufl.,  1901. 


402  THE  LIVER. 

diabetes,  where  the  sugar  disappears  from  the  urine  when  the  carbohy- 
drates are  cut  off  as  much  as  possible  from  the  food,  in  this  class  of  gly- 
cosuria. 

A  hyperglycemia  which  passes  into  a  glycosuria  may  also  be  brought 
about  by  an  excessive  or  sudden  formation  of  sugar  from  the  glycogen 
and  other  substances  within  the  animal  body. 

To  this  group  of  glycosurias  belongs,  it  seems,  the  adrenalin  glycosuria, 
in  which  an  increased  mobilization  of  the  carbohydrate  occurs,  espe- 
cially the  liver  glycogen.  Several  circumstances  indicate  this  origin 
of  the  sugar.  Thus,  after  adrenalin  injection  the  glycogen  disappears 
from  the  liver  and,  according  to  Michaud,1  adrenalin  is  without  action 
in  dogs  with  Eck  fistula.  The  activity  of  the  adrenalin  in  starving 
animals  whose  livers  are  very  poor  in  glycogen  speaks  for  the  possibility 
that  the  sugar  also  may  in  part  have  another  origin  than  that  from  the 
liver  glycogen. 

Adrenalin  glycosuria  takes,  to  a  certain  degree,  a  central  position  and 
as  such  a  glycosuria  we  consider  also  several  other  forms  of  gl}'cosuria 
caused  by  hyperglycemia.  This  is  for  example  the  case  with  the  gly- 
cosuria after  Bernard's  sugar  puncture  or  piqUre.  That  the  glycosuria, 
produced  after  piqure  is  due  to  an  increased  transformation  of  the  gly- 
cogen, follows  from  the  fact  that  no  glycosuria  appears,  under  the  above- 
mentioned  circumstances,  when  the  liver  has  been  previously  made  free 
from  glycogen  by  starvation  or  other  means.  The  close  relation  of 
this  form  of  hyperglycemia  and  glycosuria  to  the  adrenals  follows  from 
the  fact  that  the  sugar  puncture  is  without  action  after  the  extirpation 
of  the  two  adrenals.  In  rats,  Schwarz  found,  after  such  a  double  extirpa- 
tion of  the  adrenals,  that  the  liver  was  glycogen  free  and  he  considers 
this  lack  of  glycogen  as  the  cause  for  the  inaction  of  the  piqure  under 
these  conditions.  According  to  Kahn  and  Starkenstein  2  the  conditions 
must  be  different,  as  they  found  in  rabbits  who  remained  alive  a  year 
after  the  total  extirpation  of  the  adrenals,  that  the  liver  had  a  normal 
amount  of  glycogen  and  that  the  sugar  puncture  nevertheless  was  with- 
out action.     Adrenalin  caused  glycosuria  in  such  animals. 

It  is  generally  admitted  that  the  stimulation  which  the  sugar  center 
in  the  fourth  ventricle  exerts,  through  the  sympathetic  nerve  reaches  to 
the  adrenals  and  causes  a  secretion  of  adrenalin,  which  increases  the  sugar 
formation.  Certain  circumstances,  for  example,  that  a  glycosuria  can  be 
brought  about  in  starving  animals,  in  which  the  piqure  is  without  action, 
by  adrenalin,  make  the  mechanism  of  this  glycosuria  somewhat  uncer- 


1Verhandl.  d.  deutsch.  Kongr.  f.  inn.  Med.  Wiesbaden,  1911. 
*  Schwarz,  Pfliiger's  Arch.,   134;  Kahn  and  Starkenstein,  ibid.,  139;  Kahn,  ibid.T 
140;  Starkenstein,  Arch.  f.  exp.  Path.  u.  Therap.,  10. 


GLYCOSURIAS.  403 

tain.  Under  all  circumstances  the  sugar  puncture  glycosuria  stands  in 
close  relation  to  the  adrenals  and  is  generally  considered  as  an  adrenalin- 
glycosuria.  The  same  is  true  for  the  glycosuria  after  splanchnic  stimula- 
tion and  probably  for  several  other  forms  of  glycosuria.  In  the  gly- 
cosuria produced  by  stimulation  of  the  central  vagus,  according  to 
Bang,  Ljungdahl  and  Bohm,1  the  hyperglycemia  (in  rabbits)  depends 
upon  an  increased  destruction  of  the  glycogen  of  the  muscles  and  not  of 
the  liver. 

Many  investigators  consider  the  glycosuria  appearing  after  the  occur- 
rence of  dyspncc,2  produced  in  various  ways,  and  also  after  certain  poisons 
such  as  carbon  monoxide,  curare,  ether,  chloroform,  strychnine,  morphine, 
piperidin  and  others  as  adrenalin  glycosurias.  That  also  in  many  of 
such  cases  the  glycosuria  is  brought  about  by  an  increased  glycogen 
destruction  is  not  doubted.  In  certain  cases,  as  in  carbon  monoxide 
poisoning,  a  formation  of  sugar  has  been  claimed  from  protein,  because 
Straub  and  Rosenstein3  found  that  this  glycosuria  only  occurred  in 
those  animals  that  had  a  sufficient  quantity  of  protein  at  their  disposal. 
Protein  starvation  and  simultaneous  abundant  carbohydrate  supply  cause  a 
disappearance  of  this  glycosuria. 

A  hyperglycemia  and  glycosuria  may  also  be  caused  by  a  decreased 
ability  of  the  animal  to  consume  or  to  utilize  the  sugar  or  to  transform 
it  into  glycogen.  In  this  case  the  sugar  must  accumulate  in  the  blood, 
and  the  formation  of  severe  cases  of  diabetes  mellitus  is  now  generally 
explained  by  this  process. 

The  inability  of  diabetics  to  destroy  .or  consume  the  sugar  does  not 
seem  to  be  connected  with  any  decrease  in  the  oxidative  energy  of  the 
cells.  The  oxidative  processes  are  not  generally  diminished  in  diabetes 
(Schultzen,  Nencki  and  Sieber),  and  this  has  recently  been  sub- 
stantiated by  Baumgarten.4  This  latter  investigator  made  experiments 
with  several  bodies  which  on  account  of  their  aldehyde  nature  were 
closely  related  to  sugar  or  were  cleavage  or  oxidation  products  of  it, 
namely,  glucuronic  acid,  d-gluconic  acid,  d-saccharic  acid,  glucosamine, 


1  Hofmeister's  Bietrage,  10. 

2  On  the  importance  of  the  oxygen  and  the  carbon  dioxide  content  of  the  blood 
for  the  non-appearance  or  appearance  of  glycosuria  see  Underhill,  Journ.  of  biol.  Chem., 
1;  Penzoldt  and  Fleischer,  Virchow's  Arch.,  87;  Sauer,  Pfluger's  Arch.,  49,  425,  426; 
Macleod,  Amer.  Journ.  of  Physiol.,  19,  with  Briggs,  Cleveland  Med.  Journ.,  1907; 
Eddie,  Bioch.  Journ.,  1,  with  Moore  and  Roaf,  ibid.,  5;  Henderson  and  Underhill, 
Amer.  Journ.  of  Physiol.,  28. 

3  Straub,  Arch.  f.  exp.  Path.  u.  Pharm.,  38;  Rosenstein,  ibid.,  40. 

4 Schultzen,  Berl.  klin.  Wochenschr.,  1872;  Nencki  and  Sieber,  Journ.  f.  prakt. 
Chem.  (N.  F.),  26,  35;  Baumgarten,  "  Ein  Beitrag  zur  Zenntniss  des  Diabetes  mel- 
litus," Habilitationschrift,  also  Zeitschr.  f.  exp.  Path.  u.  Therap.,  2,  1905. 


404  THE  LIVER. 

mucic  acid,  and  others,  and  he  found  that  diabetics  destroyed  or  burned 
these  bodies  to  the  same  extent  as  healthy  individuals.  Besides  this 
it  must  be  remarked  that  the  two  varieties  of  sugar,  glucose  and  fructose, 
which  are  oxidized  with  the  same  readiness,  act  differently  in  diabetics. 
According  to  Rulz  and  other  investigators  fructose  is,  contrary  to 
glucose,  utilized  to  a  great  extent  in  the  organism,  but  this  in  man  is, 
not  always  the  case  or  at  least  to  a  less  extent  than  in  certain  animals. 
In  animals  with  pancreas  diabetes  (see  below)  fructose x  may  cause 
a  deposition  of  glycogen  in  the  liver  while  with  glucose  this  does  not 
occur.  The  combustion  of  protein  and  fat  takes  place  as  in  healthy 
subjects,  and  the  fat  is  completely  burned  into  carbon  dioxide  and  water. 
In  this  diabetes  the  ability  of  the  cells  to  utilize  the  glucose  suffers  diminu- 
tion, and  the  explanation  of  this  has  been  sought  in  the  fact  that  the  glu- 
cose is  not  previously  split  before  combustion. 

,     ■       c°2 
The  variation  in  the  respiratory  quotient,  i.e.,  the  relation  — — ,  seems 

to  show  an  insufficiency  of  the  glucose  combustion  in  the  tissues  in 
diabetes.  As  will  be  thoroughly  explained  in  a  subsequent  chapter,  this 
quotient  is  greater  the  more  carbohydrates  are  burned  in  the  body,  and 
it  is  correspondingly  smaller  when  protein  and  fat  are  chiefly  burned. 
The  investigations  of  Leo,  Hanriot,  Weintraud  and  Laves,2  and 
others  have  shown  that  in  severe  cases  of  diabetes,  in  the  starving  con- 
dition, the  low  quotient  is  not  raised  after  partaking  of  glucose,  as  in 
healthy  individuals,  but  that  it  is  raised  after  feeding  fructose,  which  is 
also  of  value  to  diabetics. 

The  poverty  of  the  organs  and  tissues  of  diabetics  in  glycogen  indicates 
that  the  glycogen  in  them  is  more  abundantly  transformed  into  sugar. 
From  what  has  been  said  above  in  regard  to  the  different  behavior  of 
fructose  and  glucose  in  the  glycogen  formation  in  diabetes,  indicates  that 
in  diabetes,  also  an  inability  of  the  body  to  transform  glucose  into  glycogen 
exists  and  that  the  lack  of  glycogen  may  come  about  in  this  way. 

Indeed  it  has  been  suggested  that  a  preliminary  transformation  of 
glucose  into  glycogen  is  necessary  before  it  can  be  burned  in  the  animal  body. 
This  assumption  is  without  foundation,  at  least  for  the  glycogen  for- 
mation in  the  liver,  as  the  animal  body  as  is  shown  with  experiments 
on  dogs,  can  assimilate  and  burn  considerable  quantities  of  carbohy- 
drates even  after  the  liver  is  excluded  (Wehrle,  Verz ar  3) .     The  admitted 


1  Kiilz,  Beitrage  zur  Path.  u.  Therap.  des  Diabetes  mellitus  (Marburg,  1874),  1; 
Weintraud  and  Laves,  Zeitsehr.  f.  physiol.  Chem.,  19;  Haycraft,  ibid.;  Minkowski, 
Arch.  f.  exp.  Path.  u.  Pharm.,  31. 

2  See  v.  Noorden,  Die  Zuckerkrankheit,  3.  Aufl.,  1901. 

3  Wehrle,  Bioch.  Zeitsehr.,  34;  Verzar,  ibid.,  34. 


PANCREAS  DIABETES.  405 

ability  of  the  liver  in  diabetes  to  use  fructose  and  not  glucose  in  the 
formation  of  glycogen  is,  according  to  E.  Neubauer,1  not  characteristic 
for  diabetes,  because  it  also  occurs  in  phosphorus  poisoning.  Whether 
the  different  behavior  of  the  two  kinds  of  sugar  actually  depends  upon 
a  diminished  ability  of  the  liver  in  diabetes  to  form  glycogen  from  glucose 
or  to  another  unknown  circumstance  has  not  been  sufficiently  proved. 
In  experiments  on  tortoise  livers,  by  perfusion  of  Ringer's  solution  con- 
taining sugar,  Nishi  2  found  that  the  livers  of  diabetic  animals  formed 
as  much  glycogen  as  the  livers  of  normal  animals.  These  results,  which 
cannot  be  applied  to  other  animals,  require  at  least  further  investiga- 
tion. 

The  relation  of  the  pancreas  to  diabetic  glycosuria  is  of  the  greatest 
importance  for  its  proper  understanding. 

The  investigations  of  Minkowski,  v.  Mering,  Dominicis,  and  later 
of  many  other  investigators,3  show  that  a  true  diabetes  of  a  severe 
kind  is  caused  by  the  total  or  almost  total  extirpation  of  the  pancreas 
of  many  animals,  especially  dogs.  As  in  man  in  severe  forms  of  diabetes, 
so  also  in  dogs  with  pancreatic  diabetes,  an  abundant  elimination  of 
sugar  takes  place  even  on  the  complete  exclusion  of  carbohydrates  from 
the  food. 

Artificial  pancreas  diabetes  may  indeed  also  in  other  respects  present 
the  same  picture  as  diabetes  in  man,  but  there  exist  important  differences 
between  these  two.4  It  is  generally  accepted  that  in  pancreas  diabetes 
a  diminished  consumption  exists,  i.e.,  diminished  utilization,  which 
does  not  exclude  an  increased  sugar  formation  from  other  bodies  not 
carbohydrates. 

Many  important  observations  show  that  a  close  relation  exists  between 
the  liver  and  pancreas  diabetes.  Pfluger  has  also  especially  shown 
that  in  diabetes  produced  by  Sandmeyer's  method  (partial  extirpa- 
tion with  subsequent  destruction  of  the  remains  of  the  gland  in  the  abdom- 
inal cavity,  when  the  animal  remains  alive  for  a  longer  time  than  after 
total  extirpation)  the  liver  does  not  lose  weight,  although  the  total  weight 
of  the  animal  diminishes  greatly,  while  in  starvation  without  diabetes 


1  Arch.  f.  exp.  Path.  u.  Pharm.,  61. 

2  Ibid.,  62. 

3  See  Minkowski,  Untersuchungen  liber  Diabetes  mellitus  nach  Exstirpation  des 
Pankreas  (Leipzig,  1S93);  v.  Noorden,  Die  Zuckerkrankheit  (Berlin,  1901),  which 
contains  a  very  complete  index  of  the  literature.  In  regard  to  diabetes  see  also  CI. 
Bernard,  I.econs  sur  le  diabete  (Paris),  Seegen,  Die  Zuckerbildung  im  Thierkorper 
(Berlin,  1890),  and  Pfluger,  Des  Glykogen,  2.  Aufl.,  1905,  and  especially  v.  Xoorden's 
Hanb.  d.  Pathol,  des  Stoffwechsels,  2.  Aufl.,  1907,  Bd.  2,  Chapter  I. 

4  See  Falta  "  Ueber  den  Eiweissumsatz  beim  Diabetes  mellitus."  Berl.  klin.  Woch- 
enschr.,  1908,  and  Zeitschr.  f.  klin.  Med.,  66;  Gigon,  Deutsch.  Arch.  f.  klin.  Med.,  97. 


406  THE  LIVER. 

the  liver  loses  weight  more  than  the  other  parts  of  the  body.  Pfluger 
concludes  from  this  that  the  liver  in  diabetes  works  actively,  and  is  the 
most  important  seat  of  production  of  diabetic  sugar. 

Pfluger  has  found  that  in  frogs  the  total  extirpation  of  the  duodenum 
causes  a  strong  and  continuous  glycosuria  and  based  upon  his  investigations 
and  those  of  other  investigators,  he  believes  that  a  certain  relation  exists  between 
the  duodenum  and  pancreas  diabetes.  The  question  as  to  the  occurrence  of  a 
duodenal  diabetes  has  been  the  subject  of  numerous  investigations  but  the  works 
-of  Ehrmann,  Minkowski  and  Rosenberg  x  show  that  such  a  view  is  untenable. 

There  does  not  seem  to  be  any  doubt  as  to  the  existence  of  a  certain 
relationship  between  the  pancreas  to  the  adrenals  and  adrenalin  gly- 
cosuria. The  glycosuric  action  of  adrenalin  could  be  prevented  by 
Zuelzer  by  the  injection  of  pancreas  extracts,  and  this  statement  is 
confirmed  by  Frugoni  by  experiments  with  pancreatic  juice  or  pancreatic 
extracts,  v.  Furth  and  Schwarz  2  have  confirmed  the  correctness  of 
.Zuelzer's  statement  but  dispute  the  fact  that  we  are  here  dealing  with 
an  antagonistic  hormone  action  as  they  have  obtained  similar  results  also 
with  other  bodies,  for  example  with  turpentine. 

Very  stimulating  views  on  the  relationship  of  pancreas  diabetes 
to  the  adrenals  and  the  thyroids  have  been  given  by.  Falta,  Eppinger 
and  Rudinger.3  According  to  these  investigators  a  reciprocal  retarda- 
tion exists  between  the  pancreas  and  thyroid  as  between  the  pancreas 
and  the  adrenals  while  a  mutual  accelerating  action  exists  between  the 
thyroids  and  the  adrenals.  Tn  depancreatized  dogs  the  retarding  action 
of  the  pancreas  upon  the  thyroids  is  removed,  and  in  this  way  we  explain 
the  strong  increase  in  the  protein,  fat  (Mohr)  and  salt-metabolism 
(Falta  and  Whitney  4)  observed  in  pancreas  diabetes.  By  the  removal 
of  the  retarding  action  of  the  pancreas  upon  the  adrenals,  the  mobiliza- 
tion of  the  carbohydrates  by  means  of  the  adrenalin  is  increased,  and 
herein,  as  well  as  the  diminished  sugar  utilization,  lies  the  reason  for  the 
strong  elimination  of  sugar.  The  relations  between  the  above  three 
glands  is  still  further  described  by  the  above-mentioned  authors,  but  we 
cannot  enter  more  into  detail  in  regard  to  the  interesting  question, 
which  requires  further  study. 

The  conditions  in  pancreas  diabetes  are  certainly  very  complicated, 
and  the  reasons  for  this  are  still  very  uncertain.   Most  investigators  are  of 

1  Rosenberg,  Bioch.  Zeitschr.,  18,  which  contains  the  literature. 

2  Frugoni,  Berl.  klin.  Wochenschr.,  45,  1908;  v.  Furth  and  Schwarz,  Bioch.  Zeitschr., 
31. 

3  Eppinger,  Falta  and  Rudinger,  Zeitschr.  f.  klin.  Med.,  66,  which  also  contains  the 
literature  on  adrenalin  diabetes. 

4  Mohr,  Zeitschr.  f.  exp.  Path.  u.  Therap.,  4;  Falta  and  Whitney,  Hofmeister's 
Beitrage,  11. 


PANCREAS   DIABETES.     GLYCOLYSIS.  407 

the  opinion  that  we  arc  here  dealing  with  the  abolition  of  one  or  more 
bodies  which  are  considered  as  products  of  the  internal  secretion  of  the 
glands  (hormones  according  to  Starling)  and  which  in  an  unknown 
manner  regulate  the  sugar  destruction  or  carbohydrate  metabolism. 

The  assumption  of  an  internal  secretion  is  based  on  the  investiga- 
tions of  Minkowski,  H£don,  Lanceraux,  Thiroloix,  and  others l 
upon  the  action  of  the  subcutaneous  transplantation  of  the  gland. 
According  to  these  investigations  a  subcutaneously  transplanted  piece 
of  the  gland  can  completely  perform  the  functions  of  the  pancreas  as 
to  the  sugar  exchange  and  the  sugar  elimination,  because  on  the  removal 
of  the  intra-abdominal  piece  of  gland,  the  animal  in  this  case  does  not 
become  diabetic,  but  if  the  subcutaneously  embedded  piece  of  pancreas 
is  subsequently  removed,  an  active  elimination  of  sugar  appears  immedi- 
ately. As  this  occurs  also  on  completely  cutting  off  the  nerve  supply, 
it  is  explained  by  the  assumption  of  a  formation  of  a  special  product 
in  the  gland,  which  passes  into  the  blood;  on  the  other  hand  Zuelzer, 
Dohm  and  Marxer  2  have  made  preparations  from  the  pancreas  which, 
in  dogs  as  well  as  in  man,  cause  a  diminution  in  the  elimination  of  sugar 
(and  acetone  bodies)  in  diabetes  and  an  improvement  in  the  general  con- 
dition. 

This  internal  secretion  of  the  pancreas  has  in  recent  times  been  sup- 
posed to  be  connected  with  the  so-called  islands  of  Langerhans;  but  no 
positive  results  have  been  obtained  in  this  connection.  Nor  are  we 
acquainted  with  the  kind  of  active  substance  here  formed. 

The  glycolytic  property  of  the  blood  as  shown  by  Lupine  was  con- 
sidered for  a  time  to  be  due  to  a  glycolytic  enzyme  formed  in  the  pancreas, 
and  pancreas  diabetes  used  to  be  explained  by  the  fact  that  the  action 
of  this  enzyme  was  removed  when  the  gland  was  extirpated.  This 
glycolysis  is  net  sufficient,  even  if  it  is  derived  from  the  pancreas,  to 
explain  the  transformation  of  the  large  quantity  of  sugar  in  the  body, 
and  for  the  destruction  of  sugar  we  are  also  obliged  to  accept  a  glycolysis 
in  the  organs  and  tissues.  Opinions  in  regard  to  this  glycolysis  differ 
in  certain  points.  According  to  one  view  (Siitzer  and  others)  special 
oxidases  are  active  in  the  glycolysis,  while  another  (Stoklasa  3)  con- 
considers  the  glycolysis  as  analogous  to  alcoholic  fermentation,  where  we 
have  processes  brought  on  by  special  tissue  zymases,   in  which  lactic 


1  See  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  31;  Hedon,  Diabete  pancreatique, 
Travaux  de  Physiologic  (Laboratoire  de  Montpellier,  1898),  and  the  works  on  diabetes. 

2  Deutsch.  med.  Wochenschr.,  1908. 

3  Hofmeisters  Beitrage,  3,  Centralbl.  f.  Physiol.,  16,  17,  18;  Ber.  d.  d.  chem.  Ge- 
sellsch.,  38;  also  with  Czerny,  ibid.,  36;  with  Jelinek,  Simacgk  and  Vitek,  Pfluger's 
Arch.,  101. 


408  THE  LIVER. 

acid  is  an  intermediary  step.  Many  x  objections  have  been  advanced 
against  the  view  of  Stoklasa  that  in  animal  as  well  as  in  plant  tissues, 
in  anaerobic  respiration,  an  alcoholic  fermentation  may  occur  as  this 
observed  action  of  the  tissues  could  only  be  brought  about  by  the  presence 
of  micro-organisms. 

That  lactic  acid  can  be  an  intermediary  step  in  the  destruction  of 
sugar  in  the  animal  body  cannot  be  denied.  On  the  contrary  it  follows 
from  several  circumstances  which  will  be  mentioned  in  Chapter 
X.  (muscle)  on  the  origin  of  lactic  acid  that  such  a  condition  exists 
and  the  following  observations  of  A.  R.  Mandel  and  Lusk  2  on  the 
relation  of  lactic  acid  to  diabetes  indicate  the  same.  These  exper- 
imenters showed  after  phosphorus  poisoning  in  dogs,  that  the  blood  and 
urine  contained  abundance  of  lactic  acid,  and  on  producing  phlorhizin- 
diabetes  it  disappeared  from  these  fluids,  and  also  that  phosphorus  poison- 
ing does  not  cause  a  lactic  acid  formation  in  dogs  with  phlorhizin- 
diabetes.  Although  it  is  difficult  to  give  a  satisfactory  interpretation 
of  these  observations,  it  is  still  very  probable  that  in  the  elimination 
of  the  sugar  in  phlorhizin-diabetes  a  mother-substance  of  the  lactic  acid 
is  lost. 

We  do  not  agree  as  to  the  ways  and  means  which  bring  about 
the  so-called  glycolysis,  and  another  disputed  question  is  whether  the 
glycolysis  can  be  produced  by  one  organ  or  only  by  the  combined  action 
of  several  organs.  Cohnheim  3  found  that  a  cell-free  fluid  can  be  obtained 
from  a  mixture  of  pancreas  and  muscle,  which  destroys  glucose,  while 
the  pancreas  alone  does  not  have  this  action,  and  the  muscle  only  to  a 
slight  extent.  The  pancreas  does  not  contain,  according  to  Cohnheim, 
a  glycolytic  enzyme,  but  a  substance  resistant  to  boiling  temperatures, 
which  is  soluble  in  water  and  alcohol,  and  which,  like  an  amboceptor, 
activates  a  glycolytic  proenzyme  which  exists  in  the  muscle  fluid,  but 
which  is  inactive  alone  and  which  retards  glycolysis  when  it  exists  in 
excess. 

The  statements  of  Cohnheim  have  been  disputed,  and  recently  Levene 
and  Meyer4  have  shown  that  we  are  not  here  dealing  with  a  disap- 
pearance of  glucose  by  glycolysis,  but  more  likely  with  a  disappearance 


1  See  the  works  of  O.  Cohnheim,  Zeitschr.  f.  physiol.  Chem.,  39,  42,  43;  Batelli, 
Compt,  rend.,  137;  Portier,  Compt.  rend.  soc.  biol.,  57;  Harden  and  Maclean,  Journ. 
of  Physiol.,  42  and  43. 

2  Amer.  Journ.  of  Physiol.,  16. 

3  Cohnheim,  Zeitschr.  f.  physiol.  Chem.,  39,  42,  43,  and  47. 

4  Stocklasa  and  collaborators,  Centralbl.  f.  Physiol.,  17,  and  Ber.  d.  d.  chem. 
Gesellsch.,  36  and  38;  Feinschmidt,  Hofmeister's  Beitrage,  4;  Hirsch,  ibid.;  Claus  and 
Embden,  ibid.,  6;  Arnheim  and  Rosenbaum,  Zeitschr.  f.  physiol.  Chem.,  40;  Braun- 
stein,  Zeitschr.  f.  klin.  Med.,  51;  Levene  and  Meyer,  Journ.  of  biol.  Chem.,  9. 


ORIGIN  OF  THE  SUGAR.  409 

due  to  synthesis,  where  a  disaccharide  is  formed.  According  to  J.  de 
Mkyku  ■  neither  the  pancreas  nor  the  tissues  as  a  whole  contain  any 
glycolytic  enzymes.  According  to  him  only  the  blood  has  a  glycolytic 
action,  and  this  action  is  supported  by  a  body  acting  as  an  amboceptor 
and  produced  in  the  pancreas.  Our  knowledge  as  to  the  existence  of  the 
glycolysis  and  the  mode  of  action  of  the  pancreas  in  the  metabolism  of 
sugar  in  the  animal  body  is  very  meager  and  incomplete. 

Where  does  the  sugar  eliminated  in  diabetes  originate?  Does  it 
depend  entirely  upon  the  carbohydrates  of  the  food  or  the  store  of  car- 
bohydrates in  the  body,  or  has  the  body  the  power  of  producing  sugar 
from  other  material?  To  Luthje  belongs  the  credit  for  positively 
deciding  this  question.  He  has  made  experiments  on  dogs  with  pan- 
creas  diabetes,  in  which  on  a  protein  diet  free  from  carbohydrates  so  much 
sugar  was  eliminated  that  it  could  not  possibly  be  accounted  for  by 
the  store  of  glycogen  or  other  carbohydrate-containing  substances  in  the 
body.  Similar  experiments  were  also  performed  later  by  Pfluger,2 
with  the  results  that  the  power  of  the  animal  body  to  produce  sugar  from 
non-carbohydrate  material  is  now  definitely  proved. 

Is  this  sugar  produced  from  protein  or  fat,  or  from  both?  This  ques- 
tion so  far  has  not  been  answered,  and  it  is  the  subject  of  continuous 
dispute.  It  is  not  possible  to  enter  into  an  exhaustive  and  detailed 
discussion  of  the  question  in  a  text-book,  and  we  will  only  mention, 
briefly,  certain  of  the  most  important  observations  and  historical  points. 

The  largest  amount  of  sugar  which  we  can  obtain  theoretically  from 
protein  is  8  grams  of  sugar  from  1  gram  of  protein  nitrogen,  if  we  admit 
that  all  the  carbon  of  the  protein,  with  the  exception  of  that  necessary 
to  form  ammonium  carbonate,  is  used  for  the  formation  of  sugar.  These 
results  are  still  somewhat  too  high  for  the  average  carbon  and  nitrogen 
content  of  the  proteins  and  the  values  D:N  =  6.6  is  probably  more  correct.3 
The  actual  relation  between  glucose  and  nitrogen  in  the  urine,  i.e., 
the  quotient  D:  N,  has  been  repeatedly  determined  in  various  forms  of 
diabetes,  and  in  depancreatized  dogs  it  is  generally  2.8  and  in  starving 
dogs  or  dogs  fed  with  protein  and  poisoned  with  phlorhizin  it  is  equal  to 
3.65  (Lusk).  It  may  undergo  considerable  variation,  and  in  certain 
cases  it  may  indeed  be  lower  than  1  as  well  as  higher  than  8,  and  high 
results  have  been  repeatedly  obtained  in  cases  of  human  diabetes.  From 
these  quotients  conclusions  have  been  drawn  as  to  the  amount  of  sugar 


1  Cited  from  Centralbl.  f.  Physiol.,  20  and  23.  See  also  Lepine,  Etat  actuel  de  la 
question  de  la  Glycolyse,  La  semaine  medicale,  1911. 

'Luthje,  Deutsch.  Arch.  f.  klin.  Med.,  "9,  and  Pfluger's  Arch.,  106;  Pfluger,  Pflii- 
ger's  Arch.,  10S. 

*  See  Falta,  Zeitschr.  f.  klin.  Med.,  65;  see  also  Gigon,  Deutsch.  Arch.  f.  klin.  Med.,  97. 


410  THE  LIVER. 

formed,  as  well  as  the  origin  of  the  sugar,  but  according  to  the  views  of 
Hammarsten  such  conclusions  are  mostly  very  uncertain.  The  sugar 
eliminated  by  the  urine  represents  the  difference  between  the  total  sugar 
production  of  the  body  and  the  quantity  of  sugar  burned  or  utilized. 
Only  under  the  supposition  that  the  body  cannot  burn  or  utilize  any 
sugar,  is  the  sugar  of  the  urine  a  measure  of  the  quantit}^  produced, 
and  this  seems  to  be  the  case  in  phlorhizin  diabetes  ;  but  it  is 
difficult  to  decide  how  these  suppositions  apply  to  the  different  forms 
of  diabetes.  Still  several  observations  seem  to  show  that  in  the  different 
forms  of  diabetes  variable  amounts  of  the  sugar  are  burned,  and  only 
in  special  cases  can  we  draw  approximately  accurate  conclusions. 

The  property  of  protein  of  increasing  the  elimination  of  sugar  is 
considered  as  an  important  proof  of  the  formation  of  sugar  from  protein. 
In  this  regard  those  experiments  are  of  special  interest  in  which  the 
diabetic  animal  is  allowed  to  starve  until  the  urine  is  poor  in  sugar  or 
indeed  free  from  sugar,  and  then  on  feeding  with  protein,  an  abundant 
elimination  of  sugar  is  produced.  If  we  do  not  accept  the  view  in  this 
case  that  the  protein,  but  rather  the  fat,  was  the  material  from  which 
the  sugar  was  produced,  still  we  must  admit  either  of  a  sugar-sparing 
action  due  to  protein  or  of  a  strong  sugar  formation  from  fat,  incited 
by  the  protein. 

A  sparing  in  the  sense  that  the  protein  is  oxidized  instead  of  the  sugar, 
and  in  this  manner  protects  it,  is  naturally  possible  only  under  the  sup- 
position that  the  body  can  burn  &t  least  a  part  of  the  sugar,  otherwise 
there  would  be  nothing  to  spare  and  nothing  to  protect  from  burning. 
The  assumption  of  such  an  indirect  action  of  proteins  is  difficult  to  recon- 
cile with  the  common  view  of  the  inability  of  the  body  to  burn  sugar 
in  diabetes.  Luthje  l  has  communicated  one  experiment  among  others, 
in  which  a  dog  with  pancreas  diabetes,  ariose  weight  before  starvation 
was  18  kilos,  with  nineteen  days'  starvation  eliminated  an  average  of 
10.4  grams  sugar  for  the  last  six  days  of  starvation.  By  exclusive  pro- 
tein feeding  the  quantity  of  sugar  per  day  could  be  raised  to  a  maximum 
of  123.6  grams,  and  as  average  it  was  97.5  grams  for  the  ten  protein 
days.  The  protein,  therefore,  had  protected  daily  an  average  of  87 
grams  sugar  from  burning,  which  is  hardly  possible;  and  if  in  the  diabetic 
animal  we  admit  of  this  considerable  power  of  burning  sugar,  the  quotient 
D :  N  becomes  valueless  as  a  measure  of  the  quantity  of  sugar  formed. 

If,  on  the  contrary,  we  admit  of  an  indirect  action  of  proteins  in 
that  they  incite  a  sugar  formation  from  fat,  perhaps  by  a  certain  very 
important  increase  in  the  activity  of  the  liver,  we  are  opposed  by  the 
great  difficulty  that,  according  to  known  laws;  of  metabolism,  the  pro- 

1  Deutsch.  Arch.  f.  klin.  Med.,  79. 


SUGAR  FORMATION  FROM  PROTEINS.  411 

triiis  do  not  raise  the  fat  metabolism,  but  rather  diminish  it.  The  pro- 
tein displaces  a  corresponding  quantity  of  fat  from  the  metabolism, 
and  if  the  fat  were  the  only  source  of  sugar  then  in  this  case  we  would 
expect  a  diminished  elimination  of  sugar  instead  of  an  increased  one. 
Nevertheless  the  above  action  of  protein  upon  sugar  elimination  is  much 
more  easily  explained  by  the  assumption  of  a  sugar  formation  from  pro- 
tein than  from  fat. 

The  action  of  monamino-acids  upon  the  carbohydrate  metabolism 
has  also  given  important  ground  for  the  assumption  cf  a  sugar  forma- 
tion from  protein.  That  a  deamidation  occurs  in  the  animal  body  was 
shown  by  the  earlier  observations  of  Baumann  and  Blendermann. 
Further  proofs  of  this  were  furnished  by  the  investigations  of  Neuberg 
and  Langstein,  where  in  feeding  experiments  with  alanine  they  found 
abundance  of  lactic  acid  in  the  urine,  and  P.  Mayer  *  observed  glyceric 
acid  in  the  urine  after  the  subcutaneous  injection  of  diaminopropionic 
acid.  As  from  amino-acids  by  deamidation  ketone  acids  or  oxyacids 
may  be  formed  (see  Chapter  XIV)  it  would  be  of  interest  to  test  the  action 
of  amino-acids  upon  the  carbohydrate  metabolism.  Several  investiga- 
tions have  been  carried  on  with  this  in  view,  such  as  those  of  Langstein 
and  Neuberg,  R.  Cohn  and  F.  Kraus,  which  have  shown  a  very  prob- 
able formation  of  carbohydrate  under  the  influence  of  amino-acids;  but 
the  investigations  of  Embden  and  Salomon,  and  of  Embden  and  Almagia 
have  positively  shown,  in  a  dog  without  a  pancreas,  that  the  amino- 
acids  can  bring  about  a  re-formation  of  carbohydrate.  Lusk  alone 
and  with  Ringer  2  have  shown  the  same  for  several  amino-acids  by 
experiments  on  dogs  poisoned  with  phlorhizin.  According  to  the  exper- 
iments and  calculations  of  the  two  last  mentioned  investigators  glycocoll 
and  alanine  can  be  completely  transformed  into  glucose.  Of  the  four 
carbon  atoms  of  aspartic  acid  and  of  the  five  carbon  atoms  of  glutamic 
acid  three  appear  as  glucose. 

The  investigations  of  Weinland  3  tend  to  prove  a  sugar  formation 
from  protein  He  studied  the  formation  of  sugar  in  the  chrysalis  pulp 
of  the  Calliphora  and  showed  that  the  sugar  formed  thereby  did  not  orig- 
inate from  the  fat,  but  that  the  protein  was  the    only   material  from 


1  Baumann,  Zeitschr.  f.  physiol.  Chem.,  4;  Blendermann,  ibid.,  6;  Neuberg  and 
Langstein,  Arch.  f.  (Anat.  u.)  Physiol.,  1903,  Suppl.;  Mayer,  Zeitschr.  f.  phvsiol.  Chem., 
42. 

2  Langstein  and  Neuberg,  1.  c;  Cohn,  Zeitschr.  f.  physiol.  Chem.,  28;  F.  Kraus, 
Berl.  klin.  Wochenschr.,  1904;  Embden  and  Salomon,  Hofmeister's  Beitrage,  5  and 
6,  and  with  Almagia,  ibid.,  7;  Lusk,  Amer.  Journ.  of  Physiol.,  22;  Ringer  and  Lusk, 
Zeitschr.  f.  physiol.  Chem.,  66. 

3  Zeitsflir.  f.  Biol.,  49  (N.  F.,  31);  with  Krummacher,  ibid.,  52. 


412  THE   LIVER. 

which  the  sugar  was  formed.  The  formation  of  sugar  from  protein  is 
now  generally  considered  as  positively  proved. 

Darin  l  has  found  with  experiments  with  phlorhizinized  dogs  that 
serine,  cysteine,  proline,  ornithine  and  arginine  yield  abundant  sugar  in 
glycosuric  animals.  Valine,  leucine,  isoleucine,  lysine,  histidine,  phenyl- 
alanine and  tryptophane  gave  relatively  little  sugar  or  none  at  all. 
The  amino-acids  with  straight  chains  (with  the  exception  of  lysine)  give 
sugar  while  those  with  branched  chains  do  not.  Proline  is  the  only 
cyclic  amino-acid,  which  yields  abundance  of  sugar.  Arginine  is  the 
only  one  with  more  than  five  carbon  atoms  which  yields  sugar  and  the 
sugar  comes  in  this  case  from  the  ornithine  components. 

If  we  assume  a  formation  of  sugar  from  fat,  we  must  differentiate 
between  the  two  components  of  neutral  fats,  that  is,  between  the  glyc- 
erin and  the  fatty  acids.  A  formation  of  sugar  from  glycerin  can 
be  considered  as  proved  by  the  investigations  of  Cremer,  and  especially 
those  of  Luthje  2  and  in  the  following  we  will  discuss  only  the  forma- 
tion of  sugar  from  the  fatty  acids. 

The  formation  of  sugar  from  fat  seems  to  occur  in  the  plant  king- 
dom, and  as  the  chemical  processes  in  the  animal  and  plant  life  are  in 
principle  the  same,  it  makes  the  possibility  of  a  sugar  formation  from 
fat  very  probable.  Such  an  origin  of  sugar  in  the  animal  body  is  accepted 
by  many  investigators,  especially  by  Pfuuger  and  several  French  observ- 
ers, among  whom  we  must  specially  mention  Chauveau  and  Kauf- 
mann.3 

When  food  as  free  from  carbohydrate  as  possible  is  taken,  the  quo- 
tient D:N  is  high,  i.e.,  higher  than  8,  as  well  as  when  the  quantity  of 
sugar  is  so  large  that  it  cannot  be  accounted  for  by  the  calculated 
protein  (and  carbohydrate)  metabolism,  then  if  the  observations  are 
otherwise  free  from  error  we  can  admit  of  a  formation  of  sugar  from  fat. 
Several  such  cases  of  diabetes  in  man  have  been  published  (Rumpf, 
Rosenqvist,  Mohr,  v.  Noorden,  Allard,  Falta  and  co-workers  and 
others),  and  also  in  animals  (Hartogh  and  Schumm4).  Although  these 
researches  are  not  fully  conclusive,  still  certain  of  them  indicate  a  prob- 
able formation  of  su«ar  from  fat.     We  also  have  several  conditions  which 


1  Journ.  of  biol.  Chem.,  14,  321. 

2  Cremer,  Sitzungsber.  d.  Ges.  f.  Morph.  u.  Physiol.  Munchen,  1902;  Luthje,  Deutsch. 
Arch.  f.  klin.  Med.,  80. 

3  Kaufmann,  Arch.  f.  Physiol.  (5),  8,  where  Chauveau's  work  is  cited. 

4  Rumpf,  Berl.  klin.  Wochenschr.,  1899;  Rosenqvist,  ibid.;  Mohr,  ibid.,  1901;  v. 
Noorden,  Die  Zuckerkrankheit,  3.  Aufl.  Berlin,  1901;  Allard,  Arch.  f.  exp.  Path.  u. 
Pharm.,  57;  Falta  and  co-workers,  Zeitschr.  f.  klin.  Med.,  66;  Hartogh  and  Schumm, 
Arch.  f.  Path.  u.  Pharm.,  45.  See  also  the  works  of  O.  Loewi,  ibid.,  47,  and  Lusk, 
Zeitschr.  f.  Biologie,  42. 


SUGAR  FORMATION  FROM   FATS.  413 

indicate  the  same,  namely,  that  in  phlorhizin  diabetes  after  the  disap- 
pearance of  the  liver-glycogen  the  fat  which  migrates  to  the  liver  serves 
as  material  for  the  formation  of  sugar  (Pfluger).  These  observations 
make  the  formation  of  sugar  from  fat  highly  probable  and  the  same  is  true 
for  the  observations  of  Junkersdorf.1  He  found  that  in  an  animal  made 
glycogen  free,  by  starvation  and  with  phlorhizin  poisoning,  that  toward 
death,  the  nitrogen  as  well  as  the  sugar  elimination  increased  but  that 
the  D :  N  ratio  was  higher  than  with  the  sugar  formation  from  protein 
alone.     His  calculations  are  not  free  from  exception. 

On  the  other  hand  there  are  many  observations  on  animals  and 
also  clinical  observations  which  oppose  the  theory  of  the  formation 
of  sugar  from  fat  in  diabetes.  Lusk  found  in  a  dog  with  phlorhizin 
diabetes  that  the  quotient  D:N  =  3.65:1  was  not  changed  on  feeding  fat, 
and  he  has  published  further  results  of  experiments  2  which  show  that 
active  muscular  work,  which  strongly  increases  the  fat  decomposition, 
does  not  change  the  quotient  in  dogs  with  phlorhizin  diabetes.  It  is 
difficult  to  draw  positive  conclusions  from  these  experiments,  still  Lusk 
seems  to  deny  the  formation  of  sugar  from  fat. 

Attempts  have  been  made  to  solve  the  question  as  to  the  material 
from  which  sugar  is  formed  by  the  determination  of  the  respiratory 
quotient  and  comparing  this  with  the  quotient  D:N.  The  calculations 
in  this  direction  have  not  led  to  positive  results.3  As  the  quotient  D :  N 
is  not  an  accurate  measure  of  the  quantity  of  sugar  formed,  and  as  we, 
as  yet,  do  not  know  the  quantity  of  oxygen  necessary  to  form  sugar 
from  protein,  Hammarsten  believes  that  it  is  just  as  impossible  to  con- 
clude from  the  respiratory  quotient  that  sugar  is  formed  from  the  fats 
as  from  the  proteins. 

We  have  no  complete  proofs  for  the  formation  of  sugar  from  fat,  still 
we  can  indicate  the  probable  proofs  therefor.  There  is  really  no  objec- 
tion from  a  theoretical  standpoint  to  the  assumption  that  the  body  has 
the  power  of  producing  sugar  from  protein  as  well  as  from  fat,  and  such 
a  power  does  not  seem  improbable. 

As  a  formation  of  sugar  from  protein  is  now  generally  considered  as 
proved,  it  follows  that  the  protein  can  yield  material  for  the  formation 
of  glycogen  and  that  it  is  a  true  glycogen-former.  Pfluger  and  Junkers- 
dorf4 have  given  direct  proof  for  this.  They  fed  a  dog,  which  had 
previously  been  made  glycogen-free  by  starvation  and  phlorhizin  injec- 


1  Pfliiger's  Arch.,  137. 

2  Amer.  Journ.  of  Physiol.,  22. 

3  Magnus-Levy,  Zeitschr.  f.  klin.  Med.,  56;  Pfliiger's  Arch.,  108;  Mohr,  Zeitschr. 
I.  exp.  Path.  u.  Therap.,  4. 

*  Pfliiger's  Arch.,  131. 


414  THE  LIVER. 

tions,  with  abundance  of  codfish  and  then  found  so  much  glycogen  (6.46 
per  cent  in  the  liver  and  1  per  cent  in  the  muscle)  that  a  re-formation  of 
glycogen  must  have  undoubtedly  occurred.  By  special  control  exper- 
iments with  fat  feeding  they  also  showed  that  the  glycogen  did  not  orig- 
inate from  the  fat  but  must  unquestionably  have  come  from  the  protein. 
Carbohydrates  and  proteins  are  without  question  true  glycogen-formers, 
while  the  question  in  regard  to  fats  is  still  open. 

The  Bile  and  Its  Formation. 

By  the  establishment  of  a  biliary  fistula,  an  operation  which  was 
first  performed  by  Schwann  in  1844  and  which  has  been  improved  lately 
by  Dastre  and  Pawlow,1  it  is  possible  to  study  the  secretion  of  the  bile. 
This  secretion  is  continuous,  but  with  varying  intensity.  It  takes 
place  under  a  very  low  pressure;  therefore  an  apparently  unimportant 
hindrance  in  the  outflow  of  the  bile,  namely,  a  stoppage  of  mucus  in  the 
exit,  or  the  secretion  of  large  quantities  of  viscous  bile,  may  cause  stagna- 
tion and  absorption  of  the  bile  by  means  of  the  lymphatic  vessels  (absorp- 
tion icterus). 

The  quantity  of  bile  secreted  in  the  twenty-four  hours  in  dogs  can  be 
exactly  determined.  The  quantity  secreted  by  different  animals  varies, 
and  the  limits  are  2.9-36.4  grams. of  bile  per  kilo  of  weight  in  the  twenty- 
four  hours.2 

The  reports  as  to  the  extent  of  bile  secretion  in  man  are  few  and 
not  to  be  depended  on.  Noel-Pa yton,  Mayo-Robson,  Hammarsten, 
Pfaff  and  Balch,  and  Brand3  found  a  variation  between  514  and 
1083  cc.  per  twenty-four  hours.  Such  determinations  are  of  doubtful 
value,  because  in  most  cases  it  follows  from  the  composition  of  the 
collected  bile  that  the  fluid  is  not  the  result  of  a  secretion  of  normal  liver 
bile. 

The  quantity  of  bile  secreted  is,  however,  as  shown  by  Stadel- 
mann,4  subject  to  such  great  variation,  even  under  physiological  con- 
ditions, that  the  study  of  those  circumstances  which  influence  the  secre- 
tion is  very  difficult  and  uncertain.  The  contradictory  statements 
by  different  investigators  may  probably  be  explained  by  this  fact. 

1  Schwann,  Arch.  f.  (Anat.  u.)  Physiol.,  1844;  Dastre.  Arch,  de  Physiol.  (5i)  2; 
Pawlow,  Ergebnisse  der  Physiol.,  1,  Abt.  1. 

2  In  regard  to  the  quantity  of  bile  secreted  in  animals  see  Heidenhain,  Die  Gallenab- 
eonderung,  in  Hermann's  Handbuch  der  Physiol.,  5,  and  Stadelmann,  Der  Icterus  und 
seine  verschiedenen  Formen  (Stuttgart,  1891). 

3  Noel-Payton,  Rep.  Lab.  Roy.  Coll.  Edinburgh,  3;  Mayo-Robson,  Proc.  Roy.  Soc, 
47;  Hammarsten,  Nova  Act.  Reg.  Soc.  Scient.  Upsala  (3),  16;  Pfaff  and  Balch,  Journ, 
of  Exp.  Med.,  1897;  Brand,  Pfluger's  Arch.,  90. 

4  Stadelmann,  Der  Icterus,  etc.,  Stuttgart,  1891. 


BILE  SECRETION.  415 

In  starvation  the  secretion  diminishes.  According  to  Lukjanow 
and  Albertoni,1  under  these  conditions  the  absolute  quantity  of  solids 
decreases,  while  the  relative  quantity  increases.  After  partaking  of 
food  the  secretion  increases  again.  The  findings  are  very  contradictory 
in  regard  to  the  time  necessary,  after  partaking  of  food,  before  the 
secretion  reaches  its  maximum.  After  a  careful  examination  and  com- 
pilation of  all  the  existing  reports,  Heidenhain  2  has  come  to  the  con- 
clusion that  in  dogs  the  curve  of  rapidity  of  secretion  shows  two  maxima, 
the  first  at  the  third  to  fifth  hour  and  the  second  at  the  thirteenth  to 
fifteenth  hour  after  partaking  of  food.  According  to  Barbera  the 
time  when  the  maximum  occurs  is  dependent  upon  the  kind  of  food. 
With  carbohydrate  food  it  is  two  to  three  hours,  after  protein  food  three 
to  four  hours,  and  with  fat  diet  it  is  five  to  seven  hours,  after  feeding. 
According  to  Loeb3  the  maximum  occurs  in  dogs  one  to  two  hours  after 
feeding  with  meat,  casein  or  gliadin. 

According  to  earlier  observations,  the  proteins  of  all  the  various 
foods  cause  the  greatest  secretion  of  bile,  while  the  carbohydrates  dimin- 
ish the  secretion,  or  at  least  excite  it  much  less  than  the  proteins.  This 
coincides  with  the  recent  observations  of  Barbera.  The  authorities 
by  no  means  agree  as  to  the  action  of  the  fats.  While  many  older 
investigators  have  not  observed  any  increase,  but  rather  the  reverse 
in  the  secretion  of  bile  after  feeding  with  fats,  the  researches  of  Barbera 
show  an  undoubted  increase  in  the  secretion  of  bile  on  fat  feeding,  greater 
even  than  after  carbohydrate  feeding.  According  to  Rosenberg  olive- 
oil  is  a  strong  cholagogue,  a  statement  which,  according  to  other  inves- 
tigators— Mandelstamm,  Doyon  and  Dufourt  ' — has  not  been  proved. 

As  Barbera  has  shown,  a  close  relation  exists  between  the  bile 
secretion  and  the  quantity  cf  urea  formed,  as  an  increase  in  the  first 
goes  hand  in  hand  with  an  increase  of  the  latter.  The  bile  is,  therefore, 
according  to  him,  a  product  of  disassimilation,  whose  quantity  rises  and 
falls  with  the  degree  of  activity  of  the  liver. 

The  question  whether  there  exists  special  medicinal  bodies,  so-called 
cholagogues,  which  have  a  specific  excitant  action  on  the  secretion  of 


lukjanow,  Zeitschr.  f.  physiol.  Chem.,  16;  Albertoni,  Recherches  sur  la  s£cr6tion 
biliaire,  Turin,  1893. 

2  Hermann's  Handb.,  5,  and  Stadelmann,  Der  Icterus,  etc. 

3  Barbera,  Centralbl.  f.  Physiol.,  12  and  16;  A.  Loeb,  Zeitschr.  f.  Biol.,  55. 

4  Barbera,  Bull,  della  scienz.  med.  di  Bologna  (7),  5,  Maly's  Jahresber.,  24,  and 
Centralbl.  f.  Physiol.,  12  and  16;  Rosenberg,  Pfluger's  Arch.,  46;  Mandelstamm.  Ueber 
den  Einfluss  einiger  Arzneimittel  auf  Sekretion  und  Zusammensetzung  der  Galle  (Dis- 
sert. Dorpat,  1890);  Doyon  and  Dufourt,  Arch,  de  Physiol,  (o),  9.  In  regard  to  the 
action  of  various  foods  on  the  secretion  of  bile  see  also  Heidenhain,  1.  c;  Stadelmann, 
Der  Icterus;  and  Barbera,  1.  c. 


416  THE  LIVER. 

bile,  has  been  answered  in  very  different  ways.  Many,  especially  the 
older  investigators,  have  observed  an  increase  in  the  bile  secretion  after 
the  use  of  certain  therapeutic  agents,  such  as  calomel,  rhubarb,  jalap, 
turpentine,  olive-oil,  etc.;  while  others,  especially  the  more  recent  inves- 
tigators, have  arrived  at  quite  opposite  results.  From  all  appearances 
this  contradiction  is  due  to  the  great  irregularity  of  the  normal  secretion, 
which  might  readily  cause  mistakes  in  tests  with  therapeutic  agents. 

Schiff's  view,  that  the  bile  absorbed  from  the  intestinal  canal  increases 
the  secretion  of  bile  and  hence  acts  as  a  cholagogue,  seems  to  be  a  pos- 
itively proved  fact  by  the  investigations  of  several  experimenters.1 
Sodium  salicylate  is  also  perhaps  a  cholagogue  (Stadelmann,  Doyon 
and  Dufourt,  Winogradow)  and  according  to  Petrowa  2  in  dogs  sodium 
benzoate,  thymole,  phenol,  menthol  and  all  such  bodies  which  are 
conjugated  to  ethereal  sulphuric  acid  in  the  animal  body,  increase  the 
secretion  of  bile. 

Acids,  and  especially,  under  normal  conditions,  hydrochloric  acid, 
seem  to  be  physiological  excitants  for  bile  secretion.  According  to 
Falloise  and  Fleig  the  acids  act  upon  the  duodenum  and  the  upper 
part  of  the  jejunum,  and  the  action  is  brought  about  by  a  secretin  forma- 
tion similar  to  the  action  of  acids  upon  the  secretion  of  pancreatic  juice 
(see  Chapter  VIII).  According  to  Falloise3  chloral  hydrate  introduced 
into  the  duodenum  causes  a  secretion  of  bile  in  an  analogous  manner,  by 
the  aid  of  a  special  chloral  secretin. 

The  bile  is  a  mixture  of  the  secretion  of  the  liver-cells  and  the  so- 
called  mucus  which  is  secreted  by  the  glands  of  the  biliary  passages 
and  by  the  mucous  membrane  of  the  gall-bladder.  The  secretion  of  the 
liver,  which  is  generally  poorer  in  solids  than  the  bile  from  the  gall- 
bladder, is  thin  and  clear,  while  the  bile  collected  in  the  gall-bladder 
is  more  ropy  and  viscous  on  account  of  the  absorption  of  water  and  the 
admixture  of  "  mucus,"  and  cloudy  because  of  the  presence  of  cells, 
pigments,  and  the  like.  The  specific  gravity  of  the  bile  from  the  gall- 
bladder varies  considerably,  being  in  man  between  1.010  and  1.040. 
Its  reaction  is  alkaline  to  litmus.  The  color  changes  in  different  animals: 
golden-yellow,  yellowish-brown,  olive-brown,  brownish-green,  grass-green 
or  bluish-green.  Bile  obtained  from  an  executed  person  immediately 
after  death  is  golden-yellow  or  yellow  with  a  shade  of  brown.     Still  cases 

1  SchifF,  Pfliiger's  Arch.,  3.  See  Stadelmann,  Der  Icterus,  and  the  dissertations  of 
his  pupils,  especially  Winteler,  "  Experimentelle  Beitrage  zur  Frage  des  Kreislaufes 
der  Galle  "  (Inaug.-Diss.  Dorpat,  1892),  and  Gartner,  "  Experimentelle  Beitrage  zur 
Physiol,  und  Path,  der  Gallensekretion  "  (Inaug.-Dis.  Jurjew,  1893);  also  Stadelmann, 
"  Ueber  den  Kreislauf  der  Galle,"  Zeitschr.  f.  Biologie,  34. 

2  Zeitschr.  f.  physiol.  Ghem.,  74  (literature).     See  also  footnote  4,  page  415. 
J  Falloise,  Bull.  Acad.  Roy.  de  Belg.,  1903;  Fleig,  ibid.,  1903. 


BILE-SALTS.  417 

occur  in  which  fresh  human  bile  from  the  gall-bladder  lias  a  green  color. 
The  ordinary  post-mortem  bile  has  a  variable  color.  The  bile  of  cer- 
tain animals  has  a  peculiar  odor;  for  example,  ox-bile  has  an  odor  of 
musk,  especially  on  warming.  The  taste  of  bile  is  also  different  in 
different  animals.  Human  as  well  as  ox-bile  has  a  bitter  taste,  with  a 
sweetish  after-taste.  The  bile  of  the  pig  and  rabbit  has  an  intensely 
persistent  bitter  taste.  On  heating  bile  to  boiling  it  does  not  coagulate. 
It  contains  (in  the  ox)  only  traces  of  true  mucin,  and  its  ropy  properties 
depend,  it  seems,  chiefly  on  the  presence  of  a  nucleoalbumin  similar  to 
mucin  (Paijkull).  The  bile  from  the  animals  investigated  by  Ham- 
marstex showed  a  similar  behavior.  Hammarstex  x  has,  on  the  con- 
trary, found  a  true  mucin  in  human  bile.  To  all  appearances  this  mucin 
originates  from  the  billiary  passages,  as  he  found  it  in  the  bile  flowing 
from  the  hepatic  duct,  and  also  because  the  mucous  membrane  of  the 
gall-bladder,  according  to  Wahlgrex,2  does  not  in  man  secrete  any 
mucin,  but  a  mucin-like  nucleoalbumin. 

The  specific  constituents  of  the  bile  are  bile-acids  combined  with  alkalies, 
bile-pigments,  and,  besides  small  quantities  of  lecithin  and  phosphatides, 
cholesterin,  soaps,  neutral  fats,  urea,  ethereal  sulphuric  acid,  traces  cf 
conjugated  glucuronic  acids,  enzymes  and  mineral  substances,  chiefly  chlorides, 
besides  phosphates  of  calcium,  magnesium,  and  iron.  Traces  of  copper 
also  occur. 

Bile-salts.  The  bile-acids,  which  thus  far  have  best  been  studied, 
may  be  divided  into  two  groups,  the  glycocholic  and  taurocholic  acid 
groups.  As  found  by  Hammarstex  3  a  third  group  of  bile-acids  occurs 
in  the  shark,  which  are  rich  in  sulphur,  and  like  the  ethereal  sulphuric 
acids  they  split  off  sulphuric  acid  on  boiling  with  hydrochloric  acid. 
All  glycocholic  acids  contain  nitrogen,  but  are  free  from  sulphur  and 
can  be  split,  with  the  addition  of  water,  into  glycocoll  (amino-acetic  acid) 
and  a  nitrogen-free  acid,  a  cholic  acid.  All  taurocholic  acids  contain 
nitrogen  and  sulphur  and  are  split,  with  the  addition  cf  water,  into 
taurine  and  a  cholic  acid.  The  reason  for  the  existence  of  different  glyco- 
cholic and  taurocholic  acids  depends  on  the  fact  that  there  are  several 
cholic  acids. 

The  conjugated  bile-acid  found  in  the  shark,  and  called  scynuwl-sulphuric  acid 
by  Hammarstex,  yields  as  cleavage  products  sulphuric  acid  and  a  non-nitrogenous 
substance,  scymnol  (C27H46O5),  which  gives  the  characteristic  color  reactions  of 
cholic  acid. 


1  Paijkull,  Zeitschr.  f.  physiol.  Chem.,  12;  Hammarsten,  1.  c,  Nova  Act.  (3),  16, 
and  Ergebnisse  der  Physiol.,  Bd.  4. 

2  Maly's  Jahresber.,  32. 

*  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  24. 


41S  THE   LIVER. 

The  different  bile-acids  occur  in  the  bile  as  alkali  salts,  generally 
the  sodium  compounds,  even  in  sea-fishes,  although  this  is  contrary  to 
the  earlier  observations  (Zanetti1).  In  the  bile  of  certain  animals  we 
find  almost  solely  glycocholic  acid,  in  others  only  taurocholic  acid,  and 
in  still  others  a  mixture  of  both  (see  below). 

All  alkali  salts  of  .the  biliary  acids  are  soluble  in  water  and  alcohol, 
but  insoluble  in  ether.  Their  solution  in  alcohol  is  therefore  precipitated 
by  ether,  and  this  precipitate,  with  proper  care  in  manipulation,  gives, 
for  nearly  all  kinds  of  bile  thus  far  investigated,  rosettes  or  balls  of  fine 
needles,  or  four-  to  six-sided  prisms  (Plattner's  crystallized  bile).  Fresh 
human  bile  also  crystallizes  readily.  The  bile-acids  and  their  salts 
are  optically  active  and  dextrorotatory.  The  salts  of  the  different  bile- 
acids  act  somewhat  differently  toward  neutral  salts.  The  alkali  salts 
of  the  ordinary  and  best-studied  bile-acids  from  man,  ox,  and  dog  are, 
according  to  Tengstrom,2  precipitated  by  ammonium  and  magnesium 
sulphates,  and  also,  in  pure  form,  by  sodium  nitrate  and  sodium  chloride 
(added  to  saturation).  Potassium  and  sodium  sulphates  do  not  precip- 
itate them.  The  alkali  salts  cannot  be  directly  precipitated  from  the 
bile  by  NaCl,  on  account  of  the  presence  of  bodies  retarding  precipita- 
tion, among  which  we  find  oil-soaps. 

The  bile-acids  are  dissolved  by  concentrated  sulphuric  acid  at  the 
ordinary  temperature,  forming  a  reddish-yellow  liquid  which  has  a  beautiful 
green  fluorescence.  According  to  Pregl  an  oxidation  with  a  reduction 
of  the  sulphuric  acid  into  sulphur  dioxide  takes  place.  The  fluorescent 
substance  has  been  called  dehydrocholan  (see  below)  by  Pregl.3  On 
carefully  warming  with  concentrated  sulphuric  acid  and  a  little  cane- 
sugar,  the  bile-acids  give  a  beautiful  cherry-red  or  reddish-violet  liquid. 
Pettenkofer's  reaction  for  bile-acids  is  based  on  this  behavior. 

Pettenkofer's  test  for  bile-acids  is  performed  as  follows:  A  small 
quantity  of  bile  in  substance  is  dissolved  in  a  small  porcelain  dish  in  con- 
centrated sulphuric  acid  and  warmed,  or  some  of  the  liquid  contain- 
ing the  bile-acids  is  mixed  with  concentrated  sulphuric  acid,  taking  special 
care  in  both  cases  that  the  temperature  does  not  rise  higher  than  60- 
70°  C.  Then  a  10-pcr-cent  solution  of  cane-sugar  is  added,  drop  by 
drop,  continually  stirring  with  a  glass  rod.  The  presence  of  bile  is  indi- 
cated by  the  production  of  a  beautiful  red  liquid,  whose  color  does  not 
disappear  at  the  ordinary  temperature,  but  becomes  more  bluish-violet 
in  the  course  of  a  day.  This  red  liquid  shows  a  spectrum  with  two  absorp- 
tion-bands, the  one  at  F  and  the  other  between  D  and  E,  near  E. 

iSee  Chem.  Centralbl.,  1903,  1,  180. 

2  Zeitschr.  f.  physiol.  Chem.,  41. 

3  Zeitschr.  f.  physiol.  Chem.,  45. 


GLYCOCHOLIC  ACID.  419 

This  extremely  delicate  test  fails,  however,  when  the  solution  is 
heated  too  high,  or  if  an  improper  quantity — generally  too  much — of 
the  sugar  is  added.  In  the  last-mentioned  case  the  sugar  easily  car- 
bonizes and  the  test  becomes  brown  or  dark  brown.  The  reaction  fails 
if  the  sulphuric  acid  contains  sulphurous  acid  or  the  lower  oxides  of 
nitrogen.  Many  other  substances,  such  as  proteins,  oleic  acid,  amyl 
alcohol,  and  morphine,  give  a  similar  reaction,  and  therefore  in  doubt- 
ful cases  the  spectroscopic  examination  of  the  red  solution  must  not  be 
forgotten.  '< 

Pettenkofer's  test  for  the  bile-acids  depends  essentially  on  the 
fact  that  furfurol  is  formed  from  the  sugar  by  the  sulphuric  acid  (Mylius). 
According  to  Mylius  and  v.  Udranszky  '  a  1  p.  m.  solution  of  furfurol 
should  be  used.  Dissolve  the  bile,  which  must  first  be  decolorized  by 
animal  charcoal,  in  alcohol.  To  each  cubic  centimeter  of  alcoholic 
solution  of  bile  in  a  test-tube  add  1  drop  of  the  furfurol  solution  and 
1  cc.  concentrated  sulphuric  acid,  and  cool  when  necessary,  so  that  the 
test  does  not  become  too  warm.  This  reaction,  when  performed  as 
described,  will  detect  -fa  to  fa  milligram  cholic  acid  (v.  Udranszky). 
Other  modifications  of  Pettenkofer's  test  have  been  proposed. 

The  reaction  with  furfurol  is  not  identical  with  that  obtained  with 
cane-sugar,  according  to  Ville  and  Derrien,  and  the  absorption-bands 
do  not  occur  in  the  same  place  in  the  two  cases.  The  reaction  with  cane- 
sugar  does  not  depend,  according  to  these  investigators,  upon  a  furfurol 
formation  from  the  sugar.  The  acid  hydrolyzes  the  sugar,  and  from  the 
fructose  produced,  4-methyl-2-oxyfurfurol  is  formed  by  the  further  action 
of  the  acid,  and  this  gives  the  color  reaction  with  the  cholic  acid.  Instead 
of  furfurol  other  aldehydes  such  as  vanillin  and  anisaldehyde  can  be 
used  according  to  Ville  and  Derrien.2 

Glycocholic  Acid.  The  constitution  of  the  glycocholic  acid  occurring 
in  human  and  ox-bile,  and  which  has  been  most  studied,  is  represented 
by  the  formula  C26H43NO6.  Glycocholic  acid  is  absent,  or  nearly  so, 
in  the  bile  of  carnivora.  On  boiling  with  acids  or  alkalies  this  acid, 
which  is  analogous  to  hippuric  acid,  is  converted  into  cholic  acid  and 
glycocoll. 

By  the  action  of  hydrazine  hydrate  upon  the  ethyl  ester  of  cholic  acid 
Bondi  and  Muller  3  prepared  first  cholic-acid  hydrazide,  and  then,  by 
the  action  of  nitrous  acid  upon  this,  they  obtained  the  cholic-acid  azide, 
C23H39O3CO.N3,  and  finally  from  this  last  in  alkaline  solution  with  glyco- 


1  Mylius,  Zeitschr.  f.  physiol.  Chem.,  11;  v.  Udranszky,  ibid.,  12. 

2  Ville  and  Derrien,  Chem.  Centralbl.  1909,  2,  1699  and  Compt.  rend,  eoc,  biol.  64 
and  66. 

3  Zeitschr.  f.  physiol.  Chem.,  47. 


420  THE  LIVER. 

coll  they  synthetically  prepared  the  alkali  salt  of  glycocholic  acid,  at 
the  same  time  splitting  off  nitrogen. 

Glycocholic  acid  crystallizes  in  fine,  colorless  needles  or  prisms.  It 
is  soluble  with  difficulty  in  water  (in  about  300  parts  cold  and  120  parts 
boiling  water),  and  is  easily  precipitated  from  its  alkali-salt  solution 
by  the  addition  of  dilute  mineral  acids.  According  to  Bondi  j  glyco- 
cholic acid  is  a  rather  strong  acid,  about  as  acid  as  lactic  but  much 
stronger  than  acetic  acid.  This  last-mentioned  acid  precipitates  gly- 
cocholic acid  from  the  solution  of  its  alkali  salts  in  water.  It  is  readily 
soluble  in  strong  alcohol,  but  with  great  difficulty  in  ether.  The  solu- 
tions have  a  bitter  but  at  the  same  time  sweetish  taste.  The  acid  melts 
between  132-152°,  depending  upon  the  method  of  preparation.  Accord- 
ing to  Letsche,  the  acid  containing  water  of  crystallization  (1|  mol.) 
deflagrates  on  heating  rapidly  at  126°,  and  at  130°  an  active  frothing  is 
observed.  The  acid  free  from  water  of  crystallization  deflagrates  at 
130-132°,  and  decomposes  at  154-155°  C.  with  frothing.  The  salts  of 
the  alkalies  and  alkaline  earths  are  soluble  in  alcohol  and  water. 

The  solution  of  the  alkali  salt  in  water  can  be  salted  out  by  NaCl, 
but  not  by  KC1.  The  salts  of  the  heavy  metals  are  mostly  insoluble  or 
soluble  with  difficulty  in  water.  The  solution  of  the  alkali  salts  in  water 
is  precipitated  by  sugar  of  lead,  cupric  and  ferric  salts,  and  silver  nitrate. 

On  boiling  with  water  glycocholic  acid  is  probably  transformed  into  its 
physical  isomer  paraglycocholic  acid,  according  to  Letsche,2  and  this 
crystallizes  in  long  leaves  which,  when  containing  water  of  crystalliza- 
tion, show  ready  deflagration  at  186°  and  decompose  with  frothing  at 
198°  C.  On  solution  in  alcohol  or  dilute  alkalies  the  paraglycocholic 
acid  passes  into  the  ordinary  glycocholic  acid. 

Glycocholeic  Acid  is  a  second  glycocholic  acid,  first  isolated  by  Wahl- 
gren  3  from  ox-bile,  and  has  the  formula  C26H43NO5  or  C27H45NO5. 
This  acid,  which  on  hydrolytic  cleavage  yields  glycocoll  and  choleic 
acid,  has  also  been  detected  in  human  bile  and  the  bile  of  the  musk-ox 
(Hammarsten4). 

Glycocholeic  acid  may,  like  glycocholic  acid,  crystallize  in  tufts  of 
fine  needles,  but  is  often  obtained  as  short  thick  prisms.  It  is  much  more 
insoluble  in  water,  even  on  boiling,  than  glycocholic  acid,  and  it  melts 
at  175-176°  C.  The  alkali  salts  are  soluble  in  water,  have  a  pure  bit- 
ter taste,  and  are  more  readily  precipitated  by  neutral  salts  (NaCl)  than 
the  glycocholates.     The  solution  of  the  alkali  salts  is  not  only  precipitated 


1  Zeitschr.  f.  physiol.  Chem.,  53. 

2  Ibid.,  60  and  73. 

3  Ibid.,  36. 
* Ibid.,  43. 


TAUROCHOLIC  ACID.  421 

by  the  salts  of  the  heavy  metals,  but  also  by  the  salts  of  barium,  cal- 
cium and  magnesium. 

The  principle  in  the  preparation  of  the  pure  glycocholic  acids  con- 
sists in  treating  a  2-'.\  per  cent  solution  of  bile  free  from  mucus,  when 
rich  in  glycocholic  acid  (so-called  Hefner's  bile  l),  with  ether,  and  then 
with  2  per  cent  hydrochloric  acid.  If  the  bile  is  not  directly  precipitable 
with  hydrochloric  acid  (bile  relatively  poor  in  glycocholic  acid),  then 
precipitate  the  chief  mass  of  the  glycocholic  acid  with  ferric  chloride, 
or  better  with  lead  acetate,  decompose  the  precipitate  with  soda  and  treat 
the  2  per  cent  solution  as  above  stated  with  ether  and  hydrochloric  acid. 
The  crystalline  and  washed  mass  is  boiled  with  water,  and  on  cooling 
glycocholic  acid  crystallizes  out,  and  then  this  is  recrystallized  from 
water  or  from  alcohol  by  the  addition  of  water.  The  residue  that  remains 
after  boiling  in  water  (paraglycocholic  acid  and  glycocholeic  acid)  is 
converted  into  their  barium  salts,  and  after  a  complicated  method  (see 
Wahlgren)  the  glycocholeic  acid  is  obtained.  The  reader  is  referred  to 
more  exhaustive  works  for  other  methods  of  preparation. 

Hyoglycocholic  Acid,  C27H43NO5,  is  the  crystalline  glycocholic  acid  obtained 
from  the  bile  of  the  pig.  It  is  very  insoluble  in  water.  The  alkali  salts,  whose 
solutions  have  an  intensely  bitter  taste,  without  any  sweetish  after-taste,  are 
precipitated  by  CaCb,  BaCl2,  and  MgCl2,  and  may  be  salted  out  like  a  soap  by 
Na2S04  when  added  in  sufficient  quantity.  According  to  Piettre  it  can  be  salted 
out  entirely,  free  from  sulphur,  by  caustic  alkali  which  is  not  possible  by  other 
methods.  By  precipitation  with  NaCl  in  such  quantity  that  the  precipitate  re- 
dissolves  on  warming,  Hammarsten  2  obtained  the  alkali  salt,  as  macroscopic 
crystals,  on  cooling.  Besides  this  acid  there  occurs  in  the  bile  of  the  pig  still 
another  glycocholic  acid  (Jolin  3). 

The  glycocholate  in  the  bile  of  rodents  is  also  precipitated  by  the  above 
mentioned  earthy  salts,  but  cannot,  like  the  corresponding  salt  in  human  or  ox- 
bile,  be  directly  precipitated  on  saturating  with  a  neutral  salt  (Na2S04).  Guano 
bile-acid  possibly  belongs  to  the  glycocholic-acid  group,  and  is  found  in  Peruvian 
guano,  but  has  not  been  thoroughly  studied. 

Taurocholic  Acid.  This  acid,  which  is  found  in  the  bile  of  man,  car- 
nivora,  oxen,  and  a  few  other  herbivora,  such  as  sheep  and  goats,  has  the 
constitution  C26H45NSO7.  On  boiling  with  acids  and  alkalies  it  splits 
into  cholic  acid  and  taurine.  Taurocholic  acid  has  also  been  prepared 
synthetically  by  Bondi  and  Muller,  using  the  same  method  as  they  used 
for  glycocholic  acid. 

Taurocholic  acid  can  be  readily  obtained,  by  the  method  suggested 
by  Hammarsten,4  as  groups  of  fine  needles  or  as  beautiful  prisms  on 
slow  crystallization.  The  crystals  do  not  change  in  the  air,  but  they 
decompose  above   100°.     They  are  soluble  in  alcohol  but  insoluble  in 


1  Hiifner,  Journ.  f.  prakt.  Chfem.  (N.  F.i,  10,  10,  and  25. 

2  Not  published.     M.  Piettre,  Recherches  sur  la  bile,  Laval,  1910. 

3  Zeitschr.  f.  physiol.  Chem.,  12 and  13. 
1  Ibid.,  43. 


422  THE   LIVER. 

ether,  benzene,  and  acetone.  Taurocholic  acid  is  very  soluble  in  water, 
and  the  solution  has  a  very  sweet  taste,  with  only  a  slight  bitter  taste. 
It  can  hold  the  difficultly  soluble  glycocholic  acid  in  solution.  This  is 
the  reason  why  a  mixture  of  glycocholate  with  a  sufficient  quantity  of 
taurocholate,  which  often  occurs  in  ox-bile,  is  not  precipitated  by  a  dilute 
acid.  Its  salts  are,  as  a  rule,  readily  soluble  in  water,  and  the  solutions 
of  the  alkali  salts  are  not  precipitated  by  copper  sulphate,  silver  nitrate 
or  lead  acetate.  Basic  lead  acetate  gives,  on  the  contrary,  a  precipitate 
which  is  soluble  in  boiling  alcohol.  The  alkali  salts  are  not  only  pre- 
cipitated from  their  solution  by  the  same  neutral  salts  that  precipitate 
glycocholic  acid,  but  also  by  potassium  chloride,  and  by  sodium  and 
potassium  acetates. 

Taurocholeic  Acid  is  a  second  taurocholic  acid,  detected  by  Hammar- 
sten  in  dog-bile  and  isolated  by  Gullbring  1  from  ox-bile,  and  has  the 
formula  C26H45NSO6  or  C27H47NS06-  Thus  far  it  has  been  obtained 
only  in  the  amorphous  form.  It  is  readily  soluble  in  water,  and  has  a 
disagreeably  bitter  taste.  It  is  also  readily  soluble  in  alcohol,  but  insoluble 
in  ether,  acetone,  chloroform,  and  benzene.  The  alkali  salt,  soluble  in 
water,  can  be  salted  out  by  NaCl  as  a  pasty  mass.  The  solutions  of  the 
salts  can  be  precipitated  by  ferric  chloride.  The  cleavage  products  are 
taurine  and  choleic  acid. 

The  taurocholic  acids  are  most  simply  prepared  from  bile,  free  from 
plycocholic  acid  or  poor  therein,  such  as  fish-  or  dog-bile,  easiest  from  the 
latter.  The  aqueous  solution  of  the  mucus-free  bile  is  almost  completely 
precipitated  by  ferric  chloride.  The  precipitate  is  worked  for  tauro- 
choleic acid  and  the  filtrate  for  taurocholic  acid.  The  iron  is  first  removed 
from  the  filtrate  by  Na2COs,  and  then  the  faintly  alkaline  filtrate  satu- 
rated with  Nad.  The  taurocholate  separates  out  and  after  further 
purification  is  decomposed  by  alcohol  containing  hydrochloric  acid.  The 
taurocholic  acid  is  precipitated  from  the  alcoholic  filtrate  by  ether  and 
recrystallized  from  alcohol  containing  water  by  the  addition  of  ether. 
The  taurocholeic  acid  is  obtained  from  the  above  iron  precipitate  by  treat- 
ing it  with  soda,  and  decomposing  the  alkali  salt  of  the  taurocholeic  acid 
with  alcohol,  containing  HC1,  and  precipitating  the  acid  from  the  alcoholic 
solution  with  ether  and  repeating  this  precipitation  from  alcohol  by  ether. 

Cheno-taurocholic  Acid.  This  is  the  most  essential  acid  of  goose-bile  and  has 
the  formula  GaEUNSOe.  This  acid,  but  little  studied,  is  amorphous  and  solu- 
ble in  water  and  alcohol. 

The  taurocholic  acids  differ  from  the  glycocholic  acids  in  being 
readily  soluble  in  water.  In  the  bile  of  the  walrus,  on  the  contrary,  a 
relatively  insoluble,  readily  crystallizable  taurocholic  acid  occurs,  which 


Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  43;  Gullbring,  ibid.,  45. 


CHOLIC  ACID.  423 

can  be  precipitated  from  the  solution  of  the  alkali  salts  by  the  addition 
of  mineral  acids,  like  glycocholic  acid  (Hammarsten  x). 

As  repeatedly  mentioned  above,  the  two  bile-acids  split  on  boiling 
with  acids  or  alkalies  into  non-nitrogenous  cholic  acids  and  into  glycocoll 
or  taurine.  Of  the  various  cholic  acids  the  following  have  been  best 
studied. 

Cholic  Acid  or  Cholalic  Acid.  The  ordinary  cholic  acid  obtained  as 
a  decomposition  product  of  human  and  ox-bile,  which  occurs,  regularly 
in  the  contents  of  the  intestine,  and  also  in  the  urine  in  icterus,  has,  accord- 
int   to  Strecker  and  nearly  all  recent  investigators,   the   constitution 

f  CHOH 
C24H40O5,  =  C2oH3i  j  (CH2OH)2.     According  to  Mylius,2  cholic  acid  is  a 

LCOOH 
monobasic  alcohol-acid  with  one  secondary  and  two  primary  alcohol 
groups.  Curtius  3  has  shown  by  preparing  the  cholamine,  C23H39O3.NH2, 
from  the  above-mentioned  (p.  419)  cholic-acid  azide,  with  cholic-acid 
urethane  as  an  intermediary  step,  that  the  carboxyl  group  is  not  imme- 
diately connected  with  the  CHOH  group,  but  is  combined  with  the  chief 
nucleus  without  the  neighboring  secondary  alcohol  group.  On  oxida- 
tion it  first  yields  dehydrocholic  acid,  C24H34O5  (Hammersten)  from 
which  by  electric  reduction,  Schenck  obtained  the  reducto-dehydro- 
cholic  acid,  C24H36O5.  On  further  oxidation  bilianic  acid,  C24H34OS 
(Cleve),  is  obtained,  or,  more  correctly,  according  to  Latschinoff, 
Lassar-Cohn  and  Pregl,  a  mixture  of  bilianic  and  isobilianic  acids 
discovered  by  Latschinoff.  On  oxidation,  bilianic  acid  yields  cilianic 
acid  (Lassar-Cohn),  whose  formula,  according  to  Pregl,4  is  C20H28O8. 

The  products  formed  on  a  more  active  oxidation  are  of  great  interest. 
If  we  discard  the  still  somewhat  problematic  cholesterinic  acid,  we 
find  in  these  products  in  the  first  place  choloidanic  acid  which  has  also 
been  called  cholecamphoric  acid  and  has  the  formula,  Cis^sOs,  accord- 
ing to  Pregl.  This  acid,  as  well  as  the  acid  obtained  by  Letsche  s 
on  the  oxidation  of  cholic  acid  and  with  the  formula,  C19H28O10,  have 
been  obtained  by  Pregl  6  from  the  three  most  closely  studied  cholic  acids, 


1  Zeitschr.  f.  physiol.  Chem.,  61. 

2  The  important  researches  of  Strecker  on  the  bile-acids  may  be  found  in  Annal.  d. 
Chem.  u.  Pharm.,  65,  67,  and  70;  Mylius,  Ber.  d.  deutsch.  chem.  Gesellsch.,  19. 

•  Ibid.,  39. 

4  Hammarsten,  Ber.  d.  deutsch.  chem.  Gesellsch.,  14;  Schenck,  ibid.,  63  and  69; 
Clevc,  Bull.  Soc.  chim.,  35;  Latschinoff,  Ber.  d.  d.  chem.  Gesellsch.,  15;  Lassar-Cohn, 
Ber.  d.  d.  chem.  Gesellsch.,  32;  Pregl,  Wein.  Sitzungsber.,  Ill,  1902. 

5  Zeitschr.  f.  physiol.  Chem.,  61. 

6  Ibid.,  65. 


424  THE  LIVER 

namely  from  cholic  acid,  choleic  acid  and  desoxycholic  acid,  and  these 
three  acids  are  identically  constructed  in  regard  to  their  19  carbon  atoms. 

The  choloidanic  acid  is  interesting  in  several  respects.  Panzer1  has  obtained 
from  it  by  distillation  with  soda-lime,  a  hydrocarbon,  CuHi6,  a  homologue  of 
benzene,  and  on  the  oxidation  of  the  cholic  acid  he  has  obtained  an  acid  with  the 
formula,  CgO^Os,  which  he  considers  as  an  oxyhexahydro-benzene-l-4-dicarboxylic 
acid  and  from  which  he  obtained  paraoxybenzaldehyde.  Pregl  has  obtained 
from  choloidanic  acid,  by  heating,  pyrocholoidanic  acid,  C15H20O4  which  he  con- 
siders as  parabcnzoic  acid  d-methyl-n-capric  acid,  and  is  produced  from  the 
hexahydrobcnzene  derivative  by  total  dehydrogenation  of  a  benzene  derivative. 

v.  Furth  and  collaborators  have  investigated  the  products  obtained  on  the 
dry  distillation  of  cholic  acid  at  ordinary  pressure,  and  Wieland  and  Weil2  on 
such  distillation  in  vacuum.  In  the  first  case  chiefly  hydrocarbons  with  12  to 
17  carbon  atoms  were  obtained,  and  in  the  second  instance  chiefly  an  unsaturated 
acid,  C24H34O2,  was  obtained,  and  in  both  cases  these  products  and  their  double 
bindings  have  been  carefully  investigated.  We  must  wait  for  further  developments 
in  these  investigations  before  we  attempt  to  draw  any  positive  conclusions  from 
them. 

From  the  investigations  on  the  cholic  acids  carried  out  thus  far  we 
are  not  able  to  draw  any  positive  conclusions  on  their  constitution,  but 
that  they  are  derivatives  of  hexahydrobenzene,  is  very  probable  for  sev- 
eral reasons. 

Cholic  acid  crystallizes  partly  in  rhombic  plates  or  prisms  with  one 
molecule  of  water,  and  partly  in  larger  rhombic  tetrahedra  or  octahedra 
with  one  molecule  of  alcohol  of  crystallization  (Mtlius).  These  crystals 
quickly  become  opaque  and  porcelain-white  in  the  air.  They  are  quite 
insoluble  in  water  (in  4000  parts  cold  and  750  parts  boiling),  rather 
soluble  in  alcohol,  but  soluble  with  difficulty  in  ether.  The  amorphous 
cholic  acid  is  less  insoluble.  The  solutions  have  a  bitter-sweetish  taste. 
The  crystals  lose  their  alcohol  of  crystallization  only  after  a  lengthy 
heating  to  100-120°  C.  The  acid  free  from  water  and  alcohol  melts  at 
195-196°  C.  According  to  Bondi  and  Muller  the  melting-point  of  the 
perfectly  pure  acid  is  198°  C.  It  forms  a  characteristic  blue  compound 
with  iodine  (Mylius).  If  finely  powdered  cholic  acid  is  added  to  25 
per  cent  hydrochloric  acid  at  the  ordinary  temperature,  a  beautiful  violet- 
blue  coloration  gradually  appears,  and  this  color  is  permanent  for  some 
time  and  then  becomes  gradually  green  and  yellow.  The  blue  solution 
shows  an  absorption  band  in  the  neighborhood  of  the  D  line  (Hammar- 
sten  3) . 

The  alkali  salts  are  readily  soluble  in  water,  but  when  treated  with  a 
concentrated   caustic   or   carbonated   alkali   solution,  they  may  then  be 

1  Panzer,  Zeitschr.  f.  physiol.  Chem.,  48  and  60. 

2  v.  Furth  with  Link,  Bioch.  Zeitschr.,  26,  with  Ishihara,  ibid.,  43;  Wieland    and 
Weil,  Zeitschr.  f.  physiol.  Chem.,  80. 

3  Zeitschr.  f.  physiol.  Chem.,  61. 


CHOLEIC  ACID.  425 

separated  as  an  oily  mass  which  becomes  crystalline  on  cooling.     The 

alkali  salts  are  not  readily  soluble  in  alcohol,  and  on  the  evaporation  of 
alcohol  they  may  crystallize.  The  specific  rotatory  power  of  the 
Ljdium  salt  l  is  (a)D= +30.01°  (2.29  per  cent  concentration)  to  +27.40° 
(7..")',)  per  cent  concentration).  The  watery  solution  of  the  alkali  salts, 
when  not  too  dilute,  is  precipitated  immediately  or  after  some  time  by 
Lead  acetate  or  by  barium  chloride.  The  barium  salt  crystallizes  in  fine, 
silky  needles,  and  is  rather  insoluble  in  cold,  but  somewhat  easily  soluble 
in  warm  water.  The  barium  salt,  as  well  as  the  lead  salt,  which  is 
insoluble  in  water,  is  soluble  in  warm  alcohol. 

Choleic  Acid  (C25H42O4,  Latschinoff)  is  another  cholic  acid  which, 
according  to  Lassar-Cohn,2  has  the  formula,  C24H40O4.  This  acid, 
which  occurs  in  varying  but  always  small  quantities  in  ox-bile,  and  also 
in  gall-stones  (H.  Fischer  and  P.  Meyer3)  yields  dehydrocholeic  acid, 
C24H34O4,  and  then  cholanic  acid,  C24H34O7,  and  isocholanic  acid  on 
oxidation. 

Choleic  acid  crystallizes  when  free  from  water  in  hexagonal  vitreous 
prisms  with  pointed  ends,  melting  at  185-187°  C.  The  crystalline  acid 
containing  water  melts  at  135-140°  C.  (Latschinoff).  The  acid 
dissolves  in  water  with  difficulty  and  is  also  relatively  difficultly  soluble 
in  alcohol.  It  has  an  intensely  bitter  taste  and  gives  the  Mylius  iodine 
reaction  for  cholic  acid,  and  also  the  color  reaction  of  cholic  acid  with 
'hydrochloric  acid.  The  specific  rotation  is  (a)D= +48.87°  (Vahlen). 
The  barium  salt  which  crystallizes  from  the  hot  alcoholic  solution  as 
spherical  aggregations  of  radial  needles  is  more  difficultly  soluble  in 
water  than  the  corresponding  cholate. 

Desoxycholic  Acid,  C24H40O4,  is  the  name  given  by  Mylius  4  to  a 
cholic  acid  isolated  by  him  from  putrid  ox-bile,  also  in  gall-stones  (Ktis- 
ter)  and  in  faeces  (Fischer5),  and  which  is  formed  from  the  cholic  acid 
(on  the  putrefaction  of  the  bile)  by  reduction.  This  last  is  still  very 
improbable,  and  the  investigations  of  Ekbom  do  not  support  such  an 
assumption.  On  using  perfectly  pure  cholic  acid  he  was  able  to  regain 
it  almost  quantitatively  after  the  action  of  metallic  sodium  on  the  alcoholic 
solution  of  the  acid,  or  of  zinc  and  alkali.  By  treatment  with  zinc 
and  glacial  acetic  acid  a  reaction  took  place,  but  the  product  was  a 
mixture  of  mono-  and  diacetyl  derivatives.     The  observation  of  Pregl 


1  See  Vahlen,  Zeitschr.  f.  physiol.  Chem.,  21. 

2  Latschinoff,  Ber.  d.  deutsch.  chem.  Gesellsch.,  18  and  20;  Lassar-Cohn,  ibid., 
and  Zeitschr.  f.  physiol.  Chem.,  17.     See  also  Vahlen,  Zeitschr.  f.  physiol.  Chem.,  23. 

3  Zeitschr.  f.  physiol.  Chem.,  76. 

4  Ber  d.  d.  chem.  Gesellsch,  19  and  20. 

5  Kuster,  Zeitschr.  f.  physiol.  Chem.,  69;  H.  Fischer,  ibid.,  73. 


426  THE  LIVER. 

that  desoxycholic  acid,  like  choleic  acid,  yields  dehydrocholeic  acid  and 
cholanic  acid  as  oxidation  products,  makes  the  formation  of  desoxycholic 
acid  from  cholic  acid  by  reduction  very  improbable.  The  conclusion 
of  Latschinoff  that  both  choleic  and  desoxj^cholic  acids  are  identi- 
cal, is  not  to  be  accepted  on  account  of  the  different  properties  of  the 
two  acids,  and  as  shown  by  Langheld  and  also  found  by  Hammarsten,1 
both  acids  can  be  detected  in  the  same  perfectly  fresh  ox-bile.  Pregl2 
has  given  important  proofs  that  we  are  here  dealing  with  two  different, 
probably,  isomeric  acids.  He  found  that  the  two  acids  yielded  dehydro- 
choleic acid  on  oxidation  but  that  the  dehydro-acid  was  not  the  same 
in  both  cases.  The  choleic  acid  yielded  a  dehydro-acid  with  a  lower 
melting-point  and  a  weaker  specific  rotation  than  the  desoxycholic  acid. 
The  desoxycholic  acid  crystallizes  from  glacial  acetic  acid  in  needles 
with  1  molecule  acetic  acid,  having  a  melting-point  of  144-145°.  The 
melting-point  of  the  acid  crystallized  from  alcohol-ether  is  153-155°, 
and  for  the  anhydrous  acid  or  crystallized  from  acetone  it  is  172-173°. 
It  is  soluble  with  difficulty  in  water,  more  readily  soluble  in  alcohol,  but 
somewhat  less  soluble  in  glacial  acetic  acid  than  choleic  acid.  It  has  an 
intensely  bitter  taste.  The  acid  does  not  give  a  blue  iodine  compound, 
and  no  color  reaction  with  hydrochloric  acid.  Its  barium  salt  is  soluble 
with  difficulty  in  cold  water,  but  dissolves  in  boiling  alcohol  and  crys- 
tallizes on  cooling. 

The  cholic  acids  are  best  prepared  from  ox-bile,  which  is  boiled  for 
24  hours  with  5-10  per  cent  caustic  soda.  The  crude  acid  is  precipitated 
by  hydrochloric  acid,  dissolved  in  ammoniacal  water  and  precipitated 
by  BaCb.  The  precipitate  contains  essentially  choleic  and  desoxy- 
cholic acids,  while  the  nitrate  contains  a  part  of  these  and  the  chief  part 
of  the  cholic  acid.  In  regard  to  the  further  rather  complicated  method 
of  separating  the  various  acids,  as  also  in  regard  to  the  many  methods 
suggested  for  the  preparation  of  the  pure  cholic  acids,  we  must  refer  to 
more  extensive  hand-books.3 

Fellic  Acid,  C23H40O4  is  a  cholic  acid,  so  called  by  Schotten,  which  he  obtained 
from  human  bile,  along  with  the  ordinary  acid.  This  acid  is  crystalline,  is  insolu- 
ble in  water,  and  yields  barium  and  magnesium  salts,  which  are  very  insoluble. 
It  does  not  respond  to  Pettenkofer's  reaction  easily  and  gives  a  more  reddish- 
blue  color.     The  existence  of  this  acid  is  still  doubtful. 

The  conjugate  acids  of  human  bile  have  not  been  sufficiently  investi- 
gated.    To  all  appearances  human  bile  contains  under  different  circum- 

1  Ekbom,  Zeitschr.  f.  physiol.  Chem.,  50;  Pregl,  Wien.  Sitz-Ber.  Bd.,  Ill,  Math. 
Naturw.  Kl.,  1902;  Latschinoff,  Ber.  d.  d.  Chem.  Gesellsch.,  20;  Langheld,  ibid., 
41;  Hammarsten  in  Abderhalden's  Handbuch  d.  bioch.  Arbeitsmethoden  Bd.  2,  2. 

2  Zeitschr  f.  physiol.  Chem.,  65. 

Vbderhalden's  Handbuch  d.  bioch.  Arbeitsmethoden  Bd.  II.  2;  also  Pregl  and 
Buchtala,  Zeitsfhr.  f.  physiol.  Chem.,  74  and  Schryver,  Journ.  of  Physiol.,  44. 


SPECIAL  CHOLIC  ACIDS.  427 

stances  various  conjugate1  bile-acids.  In  some  cases  the  bile-Baits  of 
human  bile  arc  precipitated  by  BaCta  and  in  others  not.  According  to 
the  statements  of  Lassar-Cohn  l  three  cholic  acids  may  be  prepared 
from  human  bile,  namely,  ordinary  cholic  acid,  choleic  acid,  and 
fellic  acid. 

Lithofellic  Acid,  CsoHseCb,  is  the  acid  related  to  cholic  acid  which  occurs  in 
the  oriental  bezoar  stones,  which  is  insoluble  in  water,  comparatively  easily  solu- 
ble in  alcohol,  but  only  slightly  soluble  in  ether.2 

Lithocholic  Acid,  C^EUOs,  is  a  cholic  acid  found  by  H.  Fischer  3  in  gall- 
stones.    It  melts  at  184-186°  and  is  tasteless. 

The  hyo-glycocholic  and  cheno-taurocholic  acids,  as  well  as  the 
glycocholic  acid  of  the  bile  of  rodents,  yield  corresponding  cholic  acids. 
This  also  seems  to  be  the  case  with  the  glycocholic  acid  of  the  hippopota- 
mus-bile, which  stands  very  close  to  the  pig-bile  (Hammarsten  4).  In-  the 
polar  bear  a  third  cholic  acid  exists  besides  cholic  and  choleic  acids. 
It  is  called  ursocholeic  acid,  C19H30O4  or  C18H28O4  (Hammarsten5). 
Also  in  the  bile  of  other  animals  (walrus*  seal)  Hammarsten  6  has  found 
special  cholic  acids,  phoccecholic  acids,  of  which  one,  the  cx-acid  crystallizes 
from  benzene  or  petroleum  ether  in  six-sided  thin  plates  which  melt  at 
152-154°  C.  Its  formula  seems  to  be  C22H36O5.  The  other,  tf-phocse- 
cholic  acid  has  the  formula  C24H40O5  and  is  isomeric  writh  cholic  acid. 
The  isocholic  acid  melts  at  220-222°  C. 

On  boiling  with  acids,  on  putrefaction  in  the  intestine,  or  on  heating, 
cholic  acids  lose  water  and  are  converted  into  anhydrides,  the  so-called 
dyslysins.  The  dyslysin,  C24H36O3,  corresponding  to  ordinary  cholic 
acid,  which  occurs  in  faeces,  is  amorphous,  insoluble  in  water  and  alkalies. 
Choloidic  acid,  C24H38O4,  is  called  the  first  anhydride  or  an  intermediary 
product  in  the  formation  of  dyslysin.  On  boiling  dyslysins  with  caustic 
alkali  they  are  reconverted  into  the  corresponding  cholic  acids. 

The  Detection  of  Bile-acids  in  Animal  Fluids.  To  obtain  the 
bile-acids  pure  so  that  Pettenkofer's  test  can  be  applied  to  them,  the 
protein  and  fat  must  first  be  removed.  The  protein  is  removed  by 
making  the  liquid  first  neutral  and  then  adding  a  great  excess  of  alcohol, 
so  that  the  mixture  contains  at  least  85  vols,  per  cent  of  water-free  alcohol. 
Now  filter,  extract  the  precipitated  protein  with  fresh  alcohol,  unite  all 
filtrates,   distil   the  alcohol,  and  evaporate  to  dryness.     The  residue  is 

1  Schotten,  Zeitschr.  f.  physiol.  Chem.,  11;  Lassar-Cohn,  Ber.  d.  deutsch.  chem. 
Gesellsch.,  27. 

2  See  Junger  and  Klages,  Ber.  d.  deutsch.  chem.  Gesellsch.  28  (older  literature). 

3  Zeitschr.  f.  physiol.  Chem.,  73. 

4  Ibid.,  74. 
8  Ibid.,  36. 

6  Ibid.,  61  and  68. 


428  THE  LIVER. 

completely  exhausted  with  strong  alcohol,  filtered,  and  the  alcohol  entirely 
evaporated  from  the  filtrate.  The  residue  is  extracted  with  ether  and 
dissolved  in  water,  and  filtered  if  necessary,  and  the  solution  precipitated 
by  basic  lead  acetate  and  ammonia.  The  washed  precipitate  is  dissolved 
in  boiling  alcohol,  filtered  while  warm,  and  a  few  drops  of  soda  solution 
added.  Then  evaporate  to  dryness,  extract  the  residue  with  absolute 
alcohol,  filter,  and  add  an  excess  of  ether.  The  precipitate  now  formed 
may  be  used  for  Pettenkofer's  test.  It  is  not  necessary  to  wait  for 
cystallization;  but  one  must  not  consider  the  crystals  which  form  in  the 
liquid  as  being  positively  crystallized  bile.  It  is  also  possible  for  needles 
of  alkali  acetate  to  be  formed.  In  this  connection  it  must  be  remarked 
that  a  confusion  with  phosphatides,  which  also  give  Pettenkofer's 
reaction,  is  not  excluded,  and  a  further  testing  and  separation  are  advisable. 

Bile-pigments.  The  bile-coloring  matters  known  thus  far  are  rela- 
tively numerous,  and  in  all  probability  there  are  still  more  of  them.  Most 
of  the  known  bile-pigments  are  not  found  in  the  normal  bile,  but  occur 
either  in  post-mortem  bile  or  principally  in  the  bile  concrements.  The 
pigments  which  occur  under  physiological  conditions  in  human  bile  are 
the  reddish-yellow  bilirubin,  the  green  biliverdin,  and  sometimes  also 
urobilin  (and  urobilinogen)  or  a  closely  related  pigment.  The  pigments 
found  in  gall-stones  are  (besides  the  bilirubin  and  biliverdin)  choleyrasin, 
bilifuscin,  biliprasin,  bilihumin,  bilicyanin  and  (choletelinf).  Besides 
these,  others  have  been  noticed  in  human  and  animal  bile  by  various 
observers.  The  two  above-mentioned  physiological  pigments,  bilirubin 
and  biliverdin,  are  those  which  serve  to  give  the  golden-yellow  or  orange- 
yellow  or  sometimes  greenish  color  to  the  bile;  or  when,  as  is  most  fre- 
quently the  case  in  ox-bile,  the  two  pigments  are  present  in  the  bile  at 
the  same  time,  they  produce  the  different  shades  between  reddish-brown 
and  green. 

Bilirubin.  This  pigment  has  the  formula,  C1GH18N2O3,  or  according 
to  Orndorff  and  Teeple  and  Kuster,1  more  correctly  C32H36N4O6, 
and  is  designated  by  the  names  cholepyrrhin,  biliph^ein,  bilifulvin, 
and  h^ematoidin.  It  occurs  chiefly  in  the  gall-stones  as  calcium  bilirubin. 
Bilirubin  is  present  in  the  liver-bile  of  all  vertebrates,  and  in  the  bladder- 
bile  especially  in  man  and  carnivora;  sometimes,  however,  the  latter 
may  have  a  green  bile  when  fasting  or  in  a  starving  condition.  It  also 
occurs  in  the  contents  of  the  small  intestine,  in  the  blood  serum  of  the 
horse,  in  old  blood  extravasations  (as  haematoidin) ,  and  in  the  urine  and 
the  yellow-colored  tissue  in  icterus. 

On  reduction  with  sodium  amalgam  Maly  obtained  a  reduction 
product,  which  he  called  hydrobilirubin,  with  the  formula,  C32H40N4O7, 

1  Orndorff  and  Teeple,  Salkowski's  Festschrift,  Berlin,  1904;  Kuster,  Zeitschr.  f. 
Physiol.  Chem.,  59. 


BILIRUBIN.  429 

and  which  shows  great  similarity  to  tho  urinary  pigment,  urobilin,  as  well 
as  to  siercobilin  found  in  the  contents  of  the  intestine  (Masius  and 
Vanlaib  j).  The  reduction  products  have  been  carefully  investigated 
by  H.  Fischer  and  then  by  Paul  Meter  and  F.  Meyek-Betz.  They 
have  found  that  hy<  In  (bilirubin  is  a  mixture  of  bodies,  among  which  there 
is  one  which  forms  at  least  one-half  and  therefore,  called  henribilirvbin, 
gives  colorless  crystals,  and  according  to  Fischer  and  Meyer-Betz 
is  identical  with  the  urobilinogen  of  the  urine.  The  formula  of  this 
body  is,  C32H44N4O6  or  C33H44N4OG.  The  other  body  is  amorphous 
but  in  properties  and  composition  shows  great  similarity  to  the  hemi- 
bilirubin.  The  analyses  correspond  closely  to  the  formula,  C32H40X4O6. 
This  body  as  well  as  the  hemibilirubin  yields  hsematinic  acid  and  met  Inl- 
et hylmaleic  imide  on  oxidation.  As  Kuster  2  first  showed,  bilirubin 
yields  hsematinic  acid  as  oxidation  product.  It  does  not  on  the  con- 
trary yield  methylethyl  maleic  imide. 

Piloty  and  Thannhauser3  obtained  bilinic  acid,  C17H2GN2O3 
from  bilirubin  on  reduction  with  hydriodic  acid  and  iodophosphonium. 
Tliis  acid  corresponded  to  the  hsematopyrrolidine  carboxylic  acid  obtained 
from  haematoporphyrin.  This  bilinic  acid  is  identical  with  the  bilirubinic 
acid  described  below  and  hence  has  this  name.4  They  also  obtained  an 
isomeric  acid  to  phonopyrrolic  acid,  the  isophonopyrrol  carboxylic  acid 
and  in  the  potash  fusion  they  found  partly  a  dimethyl-  and  partly  a 
trimethylpyrrol.  From  bilinic  acid  they  later  obtained  on  mild  oxida- 
tion an  intensely  yellow  colored  acid,  the  dehydrobilinic  acid. 

From  bilirubin  and  hemibilirubin,  on  heating  with  sodium  methylate, 
H.  Fischer  and  Rose5  have  obtained  2,  4,  5-  tri  methyl  pyrrol-S-propionic 
acid  which  was  previously  obtained  by  H.  Fischer  and  Bartholomai s 
from  phonopyrrolcarboxylic  acid.  From  bilirubinic  acid  on  the  con- 
trary, with  the  same  procedure  they  did  not  obtain  this  acid  but  another, 
xanthobilirubinic  acid,  C17H22X2O3,  which  is  probably  identical  with 
dehydrobilinic  acid,  and  which  contains  two  atoms  of  hydrogen  less  than 
bilirubinic  acid,  and  which  can  be  retransformed  into  the  latter  by  glacial 
acetic  acid  and  hydriodic  acid.  As  bilirubin,  as  well  as  hemibilirubin, 
yields  xanthobilirubinic  acid  as  a  side  product  with  sodium  methylate, 
these  experimenters  consider  this  as  a  proof  that  the  bilirubinic  acid  con- 

1  Maly,  Wien.  Sitzungsber.,  57,  and  Annal.  d.  Chem.,  163;  Masius  and  Vanlair, 
Centralbl.  f.  d.  med.  Wissenseh.,  1871,  369. 

2  Hans  Fischer,  Zeitschr.  f.  physiol.  Chem.,  73,  with  Paul  Meyer,  ibid.,  75,  with. 
Meyer-Betz,  ibid.,  75;  Kuster,  ibid.,  26  and  Ber.  d.  d.  chem.  Gesellsch,  32  and  35. 

3  Piloty  and  Thannhauser,  Annal.  d.  chem.  u.  Pharm.,  390  and  Ber  d.  d.  chem. 
Gesellsch.,  45. 

4  Piloty,  Ber.  d.  d.  chem.  Gesellsch.,  46,  1000;  H.  Fischer,  ibid.,  46,  1574. 
s  Ber.  d.  d.  chem.  Gesellsch.,  46,  439. 


430  THE  LIVER. 

figuration  exists  already  formed  in  these  two  bodies,  and  that  the  above- 
mentioned  tetrasubstituted  acid,  which  is  not  obtained  from  bilirubinic 
acid,  must  come  from  a  special  third  pyrrol  nucleus  in  the  bilirubin  and 
hemibilirubin.  Hsematinic  acid  (Kuster  from  bilirubin)  and  methyl- 
ethylmaleic  imide  (H.  Fischer  and  Meyer  from  hemibilirubin)  have 
been  obtained  from  the  two  other  pyrrol  nuclei.  Hsematinic  acid  as 
well  as  methylethylmaleic  imide  have  also  been  obtained  from  biliru- 
binic acid. 

Fischer  and  Rose  *  have  earlier  obtained,  from  hemibilirubin  as 
well  as  from  the  above-mentioned  bodies  and  from  bilirubin,  by  reduc- 
tion with  hydricdic  acid,  glacial  acetic  acid,  a  new  crystalline  acid, 
the  bilirubinic  acid,  C1-H24N2O3.  This  acid,  to  which  Pilot y  and 
Thannhauser's  bilinic  acid  stands  in  close  relation,  yields  hsematinic 
as  well  as  methylethylmaleic  imide  on  oxidation.  By  changing  the 
method  of  reduction  Fischer  and  Rose  2  have  obtained  cryptopyrrol 
and  isophonopyrrolcarboxylic  acid  from  bilirubin.  The  bilirubinic  acid 
also  yielded  the  same  products. 

The  close  relation  of  the  blood  pigments  to  the  bile  pigments  was 
first  shown  by  Kuster  when  he  obtained  the  two  hsematinic  acids  (as 
imide)  as  oxidation  products  of  these.  This  close  relation  is  further 
shown  by  the  investigations  given  above  although  it  is  perhaps  too 
early  to  draw  positive  conclusions  in  regard  to  the  structure  of  the  two 
groups  of  pigments  and  the  differences  existing  between  them. 

Bilirubin  is  sometimes  amorphous  and  sometimes  crystalline.  The 
amorphous  bilirubin  is  a  reddish-yellow  or  reddish-brown  powder;  the 
crystals  have  a  reddish-yellow,  reddish-brown,  or  more  reddish  color, 
and  sometimes  they  have  nearly  the  color  of  crystalline  chromic  acid. 
The  crystals,  which  can  easily  be  obtained  by  allowing  a  solution  of  bili- 
rubin in  chloroform  to  evaporate  spontaneously,  are  reddish-yellow, 
rhombic  plates,  whose  obtuse  angles  are  often  rounded.  On  crystalliz- 
ing from  hot  dimethylaniline  it  forms,  on  cooling,  broad  columns  with 
both  ends  sharply  cut  (Kuster3).  On  dissolving  in  chloroform  both 
kinds  of  crystals  are  converted  into  long  needles  or  whetstones. 

Bilirubin  is  insoluble  in  water,  behaves  like  an  acid,  and  occurs  in 
animal  fluids  as  soluble  alkali  bilirubin.  It  is  very  slightly  soluble  in 
ether,  benzene,  carbon  disulphide,  amyl  alcohol,  fatty  oils,  and  glyc- 
erin. It  is  somewhat  more  soluble  in  alcohol.  In  cold  chloroform  it 
dissolves  with  difficulty,  and  is  much  more  readily  soluble  in  warm  chloro- 
form. Its  solubility  varies,  and  supersaturated  solutions  are  readily 
formed   (Orndorpf  and  Teeple).     The  varying  solubility  of  bilirubin 

1  Zeitschr.  f.  physio].  Choin.,  82. 

2  Ber.  d.  d.  chem.  Gesellsch,  45. 

'  Ibid.,  30  and  35,  and  Zeitschr.  f.  physiol.  Chem.,  47. 


BILIRUBIN.  431 

in  chloroform  depends,  according  to  Kuster,  on  the  fact  that  in  its 
preparation,  derivatives  which  are  readily  soluble  and  contain  chlorine 
or  other  transformation  products  are  formed,  or  perhaps  the  bilirubin 
goes  over  into  polymeric  modifications  having  different  solubilities.  In 
cold  dimethylaniline  it  dissolves  in  the  proportion  of  1:100,  and  in  hot 
dimethylaniline  much  more  readily.  Its  solutions  show  no  absorption- 
Viands,  but  only  a  continuous  absorption  from  the  red  to  the  violet  end 
of  the  spectrum,  and  they  have  a  decided  yellow  color,  even  on  diluting 
greatly  (1:500000),  in  .a  layer  1.5  cm.  thick.  The  combinations  of 
bilirubin  with  alkali  are  insoluble  in  chloroform,  and  the  bilirubin  in  solu- 
tion in  chloroform  can  be  removed  from  this  solution  by  shaking  with 
dilute  alkali  (differing  from  lutein).  Solutions  of  bilirubin-alkali  in 
water  are  precipitated  by  the  soluble  salts  of  the  alkaline  earths  and  also 
by  metallic  salts.  If  a  dilute  solution  of  alkali  bilirubin  in  water  is 
treated  with  an  excess  of  ammonia  and  then  with  a  zinc-chloride  solution, 
the  liquid  is  first  colored  deep  orange  and  then  gradually  olive-brown 
and  then  green.  This  solution  first  gives  a  darkening  of  the  violet  and 
blue  part  of  the  spectrum,  and  then  the  bands  of  alkaline  cholecyanin 
(see  below),  or  at  least  the  bands  of  this  pigment  in  the  red  between 
C  and  D,  close  to  C.  This  is  a  good  reaction  for  bilirubin.  The  fol- 
lowing reaction  has  been  suggested  by  Auche  l.  Treat  5  cc.  of  an  alcoholic 
solution  of  bilirubin  (1:20000)  which  contains  1  drop  of  ammonia  in 
100  cc,  with  5  to  6  drops  of  an  alcoholic  zinc  acetate  solution  (1:1000) 
and  then  1  drop  alcoholic  iodine  solution  (1:100)  when  a  beautiful  bluish- 
green  coloration  with  a  beautiful  garnet-red  fluorescence  is  obtained  on 
shaking.  The  spectrum  shows  a  dark  band  between  B  and  C,  and  a 
pale  band  at  D.  If  a  few  drops  of  hydrochloric  acid  are  added  to  the 
solution  the  color  becomes  violet,  the  fluorescence  disappears  and  the 
two  Jaffa's  cholecyanin  bands  appear.  This  reaction  is  extremely 
delicate. 

As  Ehrlich  first  showed,  bilirubin  forms  combinations  with  diazo 
compounds,  which  have  been  closely  studied  by  Proscher,  Orndorff 
and  Teeple.2  A  test  suggested  by  Ehrlich  for  bilirubin  is  based  upon 
this  behavior  with  sulphodiazobenzene. 

If  an  alkaline  solution  of  bilirubin  be  allowed  to  stand  in  contact 
with  the  air,  it  gradually  absorbs  oxygen,  and  green  biliverdin  is  formed. 
This  process  is  accelerated  by  warming.  According  to  Kuster,  in  this 
case  the  alkali  also  has  a  splitting  action  upon  the  pigment,  and  among 
the  products  formed  we  find  hsematinic  acid.     Biliverdin  is  formed  only 


1  Compt.  rend.  soc.  biol.,  64. 

2  Ehrlich,  Zeitschr.  f.  anal.  Chem.,  23;    Proscher,  Zeitschr.  f.  physiol.  Chem.,  39; 
Orndorff  and  Teeple,  1.  c. 


432  THE  LIVER. 

from  bilirubin  by  oxidation  under  special  conditions  (Kuster).  A  green 
coloring-matter  similar  in  appearance  is  formed  by  the  action  of  other 
reagents  such  as  CI,  Br,  and  I.  According  to  Jolles,1,  biliverdin  is 
produced  by  the  use  of  Hubl's  iodine  solution,  while  according  to  others 
(Thudichum,  Malt  2)  substitution  products  of  bilirubin  are  formed. 

Gmelin's  Reaction  for  Bile-pigments.  If  one  carefully  pours  nitric 
acid,  containing  some  nitrous  acid,  under  an  aqueous  solution  of  alkali 
bilirubin,  there  is  obtained  a  series  of  colored  layers  at  the  juncture  of  the 
two  liquids  in  the  following  order  from  above  downward:  Green,  blue, 
violet,  red,  and  reddish-yellow.  This  color  reaction,  Gmelin's  test, 
is  very  delicate,  and  serves  to  detect  the  presence  of  one  part  bilirubin 
in  80,000  parts  liquid.  The  green  ring  must  never  be  absent;  and  also  the 
reddish-violet  must  be  present  at  the  same  time,  otherwise  the  reaction 
may  be  confused  with  that  for  lutein,  which  gives  a  blue  or  greenish  ring. 
The  nitric  acid  must  not  contain  too  much  nitrous  acid,  for  then  the  reac- 
tion takes  place  too  quickly  and  it  does  not  become  typical.  Alcohol 
must  not  be  present  in  the  liquid,  because,  as  is  well  known,  it  gives  a 
play  of  colors,  in  green  or  blue,  with  the  acid. 

Hammarsten's  Reaction.  An  acid  is  first  prepared  consisting  of  1 
vol.  nitric  acid  and  19  vols,  hydrochloric  acid  (each  acid  being  about 
25  per  cent).  One  volume  of  this  acid  mixture,  which  can  be  kept  for 
at  least  a  year,  is,  when  it  has  become  yellow  by  standing,  mixed  with 
4  vols,  alcohol.  If  a  drop  of  bilirubin  solution  is  added  to  a  few  cubic 
centimeters  of  this  colorless  mixture  a  permanent  beautiful  green  color 
is  obtained  immediately.  On  the  further  addition  of  the  acid  mixture 
to  the  green  liquid  all  the  colors  of  Gmelin's  scale,  as  far  as  choletelin, 
can  be  produced  consecutively. 

Huppert's  Reaction.  If  a  solution  of  alkali  bilirubin  is  treated  with 
milk  of  lime  or  with  calcium  chloride  and  ammonia,  a  precipitate  is 
produced  consisting  of  calcium  bilirubin.  If  this  moist  precipitate,  which 
has  been  washed  with  water,  is  placed  in  a  test-tube  and  the  tube  half 
filled  with  alcohol  which  has  been  acidified  with  hydrochloric  acid,  and 
heated  to  boiling  for  some  time,  the  liquid  becomes  emerald-green  or 
bluish-green  in  color. 

In  regard  to  the  modifications  of  Gmelin's  test  and  certain  other 
reactions  for  bile-pigments,  see  Chapter  XIV  (Urine). 

That  the  characteristic  play  of  colors  in  Gmelin's  test  is  the  result 
of  an  oxidation  is  generally  admitted.     The  first  oxidation  step  is  the 


1  Kuster,  Ber.  d.  d.  chem.  Gesellsch.,  35  and  59;  Jolles,  Journ.  f  .prakt.  Chem.  (N.F.), 
59,  and  Pfluger's  Arch.,  75. 

2  Thudichum,  Journ.  of    Chem.  Soc.  (2),'  13,  and  Journ.  f.  prakt.  Chem.  (N.F.), 
53;  Maly,  Wien.  Sitzungsber.,  72. 


BILIRUBIN,   BILIVEKDIN.  433 

green  hiliverdin.  Then  follows  a  blue  coloring-matter  which  Heinsm  B 
and  Campbell  call  bilicyanin,  and  Stokvis  calls  cholecyanin,  and  which 
shows  a  characteristic  absorption-spectrum.  The  neutral  solutions  of 
this  coloring-matter  are,  according  to  Stokvis,  bluish-green  or  steel-blue 
with  a  beautiful  blue  fluorescence.  The  alkaline  solutions  are  green 
and  have  no  marked  fluorescence,  and  show  three  absorption-bands: 
one,  sharp  and  dark,  in  the  red  between  C  and  D,  nearer  to  C;  a  second, 
less  well  defined,  covering  D;  and  a  third  between  E  and  F,  near  E. 
The  strongly  acid  solutions  are  violet-blue  and  show  two  bands,  described 
by  Jaffe  between  the  lines  C  and  E,  separated  from  each  other  by  a 
narrow  space  near  D.  A  third  band  between  b  and  F  is  seen  with  dif- 
ficulty. The  next  oxidation  step  after  these  blue  coloring-matters  is 
a  red  pigment,  and  lastly  a  yellowish-brown  pigment,  called  choletelin, 
by  Malt,  which  in  neutral  alcoholic  solutions  does  not  give  any  absorp- 
tion-spectrum, but  in  acid  solution  gives  a  band  between  b  and  F.  On 
oxidizing  cholecyanin  with  lead  peroxide,  Stokvis1  obtained  a  "product 
which  he  calls  choletelin,  which  is  quite  similar  to  urinary  urobilin,  to 
be  discussed  later. 

Bilirubin  is  best  prepared  from  gall-stones  of  oxen,  these  concretions 
being  very  rich  in  calcium  bilirubin.  The  finely  powdered  concrement 
is  first  exhausted  with  ether  and  then  with  boiling  water,  so  as  to  remove 
the  cholesterin  and  bile-acids.  In  order  to  .remove  the  mineral  con- 
stituents it  is  better  to  use  10  per  cent  acetic  acid  instead  of  hydrochloric 
acid  (Kuster2).  A  green  pigment  is  now  removed  by  extraction  with 
alcohol,  and  the  choleprasin  is  extracted  with  hot  glacial  acetic  acid. 
After  washing  with  water  it  is  dried,  and  extracted  repeatedly  with  boil- 
ing chloroform.  The  bilirubin  separates  from  the  chloroform  as  crusts, 
which  are  treated  once  or  twice  in  the  above  manner.  It  is  then  extracted 
with  alcohol  and  precipitated  from  its  chloroform  solution  by  alcohol,  or 
crystallized  from  boiling  dimethylaniline.  Further  details  are  given  by 
Kuster.3 

The  quantitative  estimation  of  bilirubin  may  be  made  by  the  spectro- 
photometric  method,  according  to  the  steps  suggested  for  the  blood- 
coloring  matters.4 

Biliverdin,  Ci6Hi8N204  or  C32H36N4O8.  This  body,  which  is  formed 
by  the  oxidation  of  bilirubin,  occurs  in  the  bile  of  many  animals,  in 
vomited  matter,  in  the  placenta  of  the  bitch  (?),  in  the  shells  of  birds' 


1  Heinsius  and  Campbell,  Pfluger's  Arch.,  4;  Stokvis,  Centralbl.  f.  med.  Wis- 
sensch.,  1872,  785;  ibid.,  1873,  211  and  449;  Jaffe\  ibid.,  1868;  Maly,  Wien.  Sitzungs- 
ber.,  59. 

2  Zeitschr.  f.  physiol.  Chem.,  47. 

3  Ibid.,  59. 

4  See  also  Herzfeld,  Zeitschr.  f.  physiol.  Chem.,  77  and  78. 


434  THE  LIVER. 

eggs,  in  the  urine  in  icterus,  and  sometimes  in  gall-stones,  although  in 
very  small  quantities. 

Biliverdin  is  amorphous;  at  least  it  has  not  been  obtained  in  well- 
defined  crystals.  It  is  insoluble  in  water,  ether,  and  chloroform  (this  is 
true  at  least  for  the  artificially  prepared  biliverdin)  but  is  soluble  in 
alcohol  or  glacial  acetic  acid,  showing  a  beautiful  green  color.  It  is  dis- 
solved by  alkalies,  giving  a  brownish-green  color,  and  this  solution  is 
precipitated  by  acids,  as  well  as  by  calcium,  barium,  and  lead  salts. 
Biliverdin  gives  Huppert's,  Gmelin's,  and  Hammarsten's  reactions, 
commencing  with  the  blue  color.  It  is  converted  into  hydrobilirubin 
by  nascent  hydrogen.  On  allowing  the  green  bile  to  stand,  also  by  the 
action  of  ammonium  sulphide,  the  biliverdin  may  be  reduced  to  bilirubin 
(Haycraft  and  Scofield  *). 

Biliverdin  is  most  simply  prepared  by  allowing  a  thin  layer  of  an 
alkaline  solution  of  bilirubin  to  stand  exposed  to  the  air  in  a  dish  until 
the  color  is  brownish-green.  The  solution  is  then  precipitated  by  hydro- 
chloric acid,  the  precipitate  washed  with  water  until  no  HC1  reaction 
is  obtained,  then  dissolved  in  alcohol  and  the  pigment  again  separated  by 
the  addition  of  water.  Any  contaminating  bilirubin  may  be  removed 
by  means  of  chloroform.  Kuster  has  shown  that  the  biliverdin  is  only 
formed  by  the  oxygen  of  the  air  from  bilirubin  under  certain  conditions: 
The  presence  of  2  molecules  caustic  alkali  with  the  addition  of  water  so 
that  the  solution  contains  0.2  per  cent  and,  a  temperature  not  above  5°  C. 
Hugounenq  and  Doyon  2  prepared  biliverdin  from  bilirubin  by  the 
action  of  sodium  peroxide  and  a  little  hydrochloric  acid. 

Choleprasin  is  a  green  pigment  isolated  by  Kuster  3  from  gall-stones,,  which 
is  soluble  in  glacial  acetic  acid  but  insoluble  in  alcohol.  It  differs  from  the  other 
bile-pigments  by  containing  sulphur.  On  distillation  with  zinc  powder  it  gives, 
the  pyrrol  reaction,  and  on  oxidation  with  chromic  acid,  Kuster  could  not 
observe  any  formation  of  hsematinic  acid. 

Bilifuscin,  so  named  by  Stadeler,4  is  an  amorphous  brown  pigment  soluble 
in  alcohol  and  alkalies,  almost  insoluble  in  water  and  ether,  and  soluble  with  great 
difficulty  in  chloroform  (when  bilirubin  is  not  present  at  the  same  time).  Pure 
bilifuscin  does  not  give  Gmelin's  reaction.  This  is  also  true  for  the  bilifuscin 
prepared  by  v.  Zumbusch,5  which  is  more  like  a  humin  substance,  and  the  formula 
of  which  is,  Cc^H^NyOu.  Bilifuscin  has  been  found  in  gall-stones.  Biliprasin 
is  a  green  pigment  prepared  by  Stadeler  from  gall-stones,  and  is  generally  con- 
sidered as  a  mixture  of  biliverdin  and  bilirubin.     Dastre  and  Floresco,6  on  the 


1  Centralbl.  f.  Physiol.,  3,  222,  and  Zeitschr.  f.  physiol.  Chem.,  14. 

2  Hugounenq  et  Doyon,  Arch,  de  Phyisol.  (5),  8;  Kuster,  Zeitschr.  f.  physiol.  Chem.,, 
59. 

'Zeitschr.  f.  physiol.  Chem.,  47. 

4  Cited  from  Hoppe-Seyler,  Physiol,  u.  Path.  chem.  Analyse,  6.  Aufl.,  p.  225. 

6  Zeitschr.  f.  physiol.  Chem.,  31. 

•Arch,  de  Physiol.  (5),  9. 


SPECIAL   BILE  PIGMENTS.  435 

contrary,  consider  biliprasin  as  an  intermediate  step  between  bilirubin  and  bili- 
verdin.  According  to  them  it  occurs  as  a  physiologica]  pigmenl  in  the  bladder- 
bile  of  several  animals,  and  is  derived  from  bilirubin  by  oxidation.  Thia  oxida- 
tion is  brought  about  by  an  oxidative  ferment  existing  in  the  bile.  Bilihumin 
is  the  name  given  by  Stadeler  to  that  brownish  amorphous  residue  which  is  left 
after  extracting  gall-stones,  with  chloroform  alcohol,  and  ether.  It  does  not 
give  Gmelin's  test.  Bilicyani/i  is  also  found  in  human  gall-stones  (Heinsics  and 
Campbell).  Cholohcematin,  so-called  by  MacMunn,  is  a  pigment  often  occurring 
in  sheep-  and  ox-bile  and  characterized  by  four  absorption-bands,  which  is 
formed  from  hsematin  by  the  action  of  sodium  amalgam.  In  the  dried  condition, 
as  when  obtained  by  the  evaporation  of  the  chloroform  solution,  it  is  green,  and  in 
alcoholic  solution  olive-brown.  This  pigment,  which  has  also  been  found  by 
Hammarsten  in  the  bile  from  the  musk-ox  and  hippopotamus,  is,  according  to 
Marchlewski,  identical  with  the  crystalline  bilipurvurin  isolated  by  Loebisch 
and  Fischler  from  ox-bile.  This  latter  pigment,  according  to  Marchlewski, 
is  not  a  bile-pigment,  but  phjlloerythrin,  a  transformation  product  of  chlorophyll. 
1  hvlloervthrin  has  been  detected  by  Marchlewski  l  in  the  excrement  of  cows 
fed  on  green  grass. 

Gmelin's  and  Huppert's  reactions  are  generally  used  to  detect  the 
presence  of  bile-pigments  in  animal  fluids  or  tissues.  The  first,  as  a  rule, 
can  be  performed  directly,  and  the  presence  of  proteins  does  not  interfere 
with  it,  but,  on  the  contrary,  it  brings  out  the  play  of  colors  more  strik- 
ingly. If  blood-coloring  matters  are  present  at  the  same  time,  the  bile- 
coloring  matters  are  first  precipitated  by  the  addition  of  sodium  phos- 
phate and  milk  of  lime.  This  precipitate  containing  the  bile-pigments 
may  be  used  directly  in  Huppert's  reaction,  or  a  little  of  the  precipitate 
may  be  dissolved  in  Hammarsten's  reagent.  Bilirubin  is  detected  in 
blood,  according  to  Hedenius,  by  precipitating  the  proteins  with  alcohol, 
filtering  and  acidifying  the  filtrate  with  hydrochloric  or  sulphuric  acid, 
and  boiling.  The  liquid  becomes  of  a  greenish  color.  Serum  and  serous 
fluids  may  be  boiled  directly  with  a  little  acid  after  the  addition  of  alcohol. 
According  to  Obermeyer  and  Popper  2  the  alcoholic  filtrate  from  the 
protein  precipitation  can  be  tested  with  an  alcoholic  solution  of  iodine 
or  ferric  chloride. 

Besides  the  bile-acids  and  the  bile-pigments,  there  occur  in  the  bile 
also  cholesterin,  lecithin,  jecorin  or  other  phosphatides  (Hammarstex), 
palmitin,  stearin,  olein,  myristic  acid  (Lassar-Cohn3),  soaps,  ethereal 
sidphuric  acids,  conjugated  glucuronates,  diastatic  and  proteolytic  enzymes, 
oxidases  and  catalases.  Choline,  and  glycerophosphoric  acid,  when  they 
are  present,  may  be  considered  as  decomposition  products  of  lecithin. 
Urea  occurs,  though  only  in  traces,  as  a  physiological  constituent  of  human, 


1  MacMunn,  Journ.  of  Physiol.,  6;  Loebisch  and  Fischler,  Wien.  Sitzungsber.,  112 
(1903);  Marchlewski,  Zeitschr.  f.  physiol.  Chem.,  41,  43,  and  45;  Hammarsten,  ibid., 
43,  and  investigations  not  published. 

2  Hedenius  Upsala  Lakaref.  Forh.,  29  and  Maly's  Jahresber.,  24;  Obermeyer  and 
Popper,  Wien.  med.  Wochenschr.,  60. 

3  Zeitschr.  f.  physiol.  Chem.,  17;  Hammarsten,  ibid.,  32,  36  and  43. 


436  THE  LIVER. 

ox-,  and  dog-bile.  Urea  occurs  in  the  bile  of  the  shark  and  ray  in  such 
large  quantities  that  it  forms  one  of  the  chief  constituents  of  the  bile.1 
The  mineral  constituents  of  the  bile  are,  besides  the  alkalies,  to  which  the 
bile-acids  are  united,  sodium  and  potassium  chloride,  calcium  and 
magnesium  phosphate,  and  iron — 0.04-0.115  p.  m.  in  human  bile, 
chiefly  combined  with  phosphoric  acid  (Young2).  Traces  of  copper 
are  habitually  present,  and  traces  of  zinc  are  often  found.  Sulphates  are 
entirely  absent,  or  occur  only  in  very  small  amounts. 

The  quantity  of  iron  in  the  bile  varies  greatly.  According  to  Novi 
it  is  dependent  upon  the  kind  of  food,  and  in  dogs  it  is  lowest  with  a  bread 
diet  and  highest  with  a  meat  diet.  According  to  Dastre  this  is  not  the 
case.  The  quantity  of  iron  in  the  bile  varies  even  though  a  constant 
diet  is  maintained,  and  the  variation  is  dependent  upon  the  forma- 
tion and  destruction  of  blood.  According  to  Beccari3  the  iron  does 
not  disappear  from  the  bile  in  inanition,  and  the  percentage  shows  no 
constant  diminution.  The  question  as  to  the  extent  of  elimination  by 
the  bile  of  the  iron  introduced  into  the  body  has  received  various  answers. 
There  is  no  doubt  that  the  liver  has  the  property  of  collecting  and  retain- 
ing iron,  as  well  as  other  metals,  from  the  blood.  Certain  investigators, 
such  as  Novi  and  Kunkel,  are  of  the  opinion  that  the  iron  introduced 
and  transitorily  retained  in  the  liver  is  eliminated  by  the  bile,  while 
others,  such  as  Hamburger,  Gottlieb,  and  Anselm,4  deny  any  such 
elimination  of  iron  by  the  bile. 

Quantitative  Composition  of  the  Bile.  Complete  analyses  of  human 
bile  have  been  made  by  Hoppe-Seyler  and  his  pupils.  The  bile  was 
removed  from  the  gall-bladder  of  cadavers,  hence  these  analyses  can 
be  of  little  interest.  Older  and  less  complete  analyses  of  perfectly  fresh 
human  bile  have  been  made  bjr  Frerichs  and  v.  Gorup-Besanez.5 
The  bile  analyzed  by  them  was  from  perfectly  healthy  persons  who 
had  been  executed  or  accidentally  killed.  The  two  analyses  of 
Frerichs  are,  respectively,  of  (I)  an  18-year-old  and  (II)  a  22-year- 
old  male.  The  analyses  of  v.  Gorup-Besanez  are  of  (I)  a  man  of  49 
and  (II)  a  woman  of  29.     The  results  are,  as  usual,  in  parts  per  1000. 


1  Hammarsten,  ibid.,  24. 

2  Journ.  of  Anat.  and  Physiol.,  5,  158. 

3  Novi,  see  Maly's  Jahresber.,  20;  Dastre,  Arch,  de  Physiol.  (5),  3;  Beccari,  Arch, 
ital.  de  Biol.,  28. 

4  Kunkel,  Pfliiger's  Arch.,  14;  Hamburger,  Zeitschr.  f.  physiol.  Chem.,  2  and  4; 
Gottlieb,  ibid.,  15;  Anselm,  "  Ueber  die  Eisenausscheidung  der  Galle,"  Inaug.-Diss. 
Dorpat,  1891 .     See  also  the  works  cited  in  footnote  3,  p.  339. 

5,See  Hoppe-Seyler  Physiol.  Chem.,  301;  Socoloff,  Pfliiger's  Arch.,  12;  Trifanow- 
Bki,  ibid.,  9;  Frerichs  in  Hoppe-Seyler's  Physiol.  Chem.,  299;  v.  Gorup-Besanez,  ibid. 


v.  Gone 

p-Besanez. 

I. 

II. 

822 . 7 

898.1 

177.3 

101.9 

107.9 

56.5 

22.1 

14.5 

47.3 

30.9 

10.8 

6.2 

COMPOSITION  OF  THE  BILE.  437 

Frerichb. 

I.  II. 

Water 800. 0  850  2 

Solids 140.0  140.8 

Biliary  salts 72.2  91.4 

Mucus  and  pigments 26.6  29.8 

Cholesterin 1.6  2.6 

Fat 3.2  9.2 

Inorganic  substances 6.5  7.7 

Human  liver-bile  is  poorer  in  solids  than  the  bladder-bile.  In 
several  cases  it  contained  only  12-18  p.  m.  solids,  but  the  bile  in  these 
cases  is  hardly  to  be  considered  as  normal.  Jacobsen  found  22.4-22.8 
p.  m.  solids  in  a  specimen  of  bile.  Hammarsten,  who  had  occasion  to 
analyze  the  liver-bile  in  seven  cases  of  biliary  fistula,  has  often 
found  25-28  p.  m.  solids.  In  a  case  of  a  corpulent  woman  the  quantity 
of  solids  in  the  liver-bile  varied  between  30.10-38.6  p.  m.  in  ten  days. 
Brand  '  observed  still  higher  figures,  more  than  40  p.  m.,  in  two 
cases.  This  investigator  suggests  that  the  bile  from  an  imperfect 
fistula,  when  it  is  partly  absorbed,  is  richer  in  solids  than  when  it  comes 
from  a  perfect  fistula. 

The  molecular  concentration  of  human  bile,  according  to  Brand, 
Bonanni,  and  Strauss,2  is  generally  identical  with  that  of  the 
blood,  although  the  amount  of  water  and  solids  varies.  The  freezing- 
point  varies  only  between  —0.54°  and  —0.58°.  This  constancy  of  the 
osmotic  pressure  is  explained  by  the  fact  that  in  concentrated  biles  with 
larger  amounts  of  organic  substances  (with  larger  molecules)  the  amount 
of  inorganic  salts  is  lower.3 

Human  bile,  sometimes,  but  not  always,  contains  sulphur  in  an  ethereal 
sulphuric-acid-like  combination  (Hammarsten,  Oerum,  Brand).  The 
quantity  of  such  sulphur  may  even  amount  to  |-|  of  the  total  sulphur. 
We  do  not  know  the  nature  of  these  ethereal  sulphuric  acids.  According 
to  Oerum  4  they  are  not  precipitated  by  lead  acetate,  but  are  precipitated 
by  basic  lead  acetate,  especially  with  ammonia.  Human  bile  is  habitually 
richer  in  glycocholic  than  in  taurocholic  acid.  In  six  cases  of  liver-bile 
analyzed  by  Hammarsten  the  relation  of  taurocholic  to  glycocholic  acid 
varied  between  1 : 2.07  and  1 :  14.36.  The  bile  analyzed  by  Jacobsen 
contained  no  taurocholic  acid. 

As  an  example  of  the  composition  of  human  liver-bile  the  following 


1  Jacobsen,  Ber.  d.  deutsch.  chem.  Gesellsch.,  6;  Hammarsten,  Nova  Acta    Reg. 
Soc.  Scient.  Upsala.  16;  Brand,  Pfluger's  Arch.,  90. 

2  Brand,  1.  c,  Bonanni,  Biochem.  Centralbl.,  1;  Strauss,  Berl.  klin.    Wochenschr., 
1903. 

s  See  Brand,  1.  c;  Hammarsten,  1.  c. 
4  Skand.  Archiv.  f.  Physiol.,  16. 


438  THE  LIVER. 

results  of  three  analyses  made  by  Hammarsten  are  given.     The  results 
are  calculated  in  parts  per  1000 : l 

Solids 25.200  35.260  25.400 

Water    974.800  964.740  974.600 

Mucin  and  pigments 5 .  290  4 .  290  5 .  150 

Bile-salts 9.310  18.240  9.040 

Taurocholate 3.034  2.079  2.180 

Glycocholate 6.276  16.161  6.860 

Fattv  acids  from  soaps 1 .  230  1 .  360  1 .  010 

Cholesterin 0.630  1.60J  1.500 

Lecithin \    n  990  0574  °-650 

Fat          J  0.956  0.610 

Soluble  salts 8.070  6.760  7.250 

Insoluble  salts 0.250  0.490  0.210 

Among  the  mineral  constituents  the  chlorine  and  sodium  occur  to 
the  greatest  extent.  The  relation  between  potassium  and  sodium 
varies  considerably  in  different  samples,  Sulphuric  acid  and  phosphoric 
acid  occur  only  in  very  small  quantities. 

Baginskt  and  Sommerfeld  2  found  true  mucin,  mixed  with  some 
nucleoalbumin,  in  the  bladder-bile  of  children.  The  bile  contained 
on  an  average  896.5  p.  m.  water;  103.5  p.  m.  solids;  20  p.  m.  mucin; 
9.1  p.  m.  mineral  substances;  25.2  p.  m.  bile-salts  (of  which  16.3  p.  m. 
were  glycocholate  and  8.9  p.  m.  taurocholate);  3.4  p.  m.  cholesterin; 
6  p.  m.  lecithin;  6.7  p.  m.  fat,  and  2.8  p.  m.  leucine.3 

The  quantity  of  pigment  in  human  bile  is,  according  to  Noel-Paton, 
0.4-1.3  p.  m.  (in  a  case  of  biliary  fistula).  The  method  used  in  deter- 
mining the  pigments  in  this  case  was  not  quite  trustworthy.  Accurate 
results  for  dog-bile  obtained  by  spectrophotometric  methods  are  on 
record.  According  to  Stadelmann  4  dog-bile  contains  on  an  average 
0.6-0.7  p.  m.  bilirubin.  At  the  most  only  7  milligrams  of  pigment  are 
secreted  per  kilo  of  body  in  the  twenty-four  hours. 

In  animals  the.  relative  proportion  of  the  two  acids  varies  con- 
siderably. It  has  been  found,  on  determining  the  amount  of  sulphur, 
that,  so  far  as  the  experiments  have  gone,  taurocholic  acid  is  the  pre- 
vailing acid  in  carnivorous  mammals,  birds,  snakes,  and  fishes.  Among 
the  herbivora,  sheep  and  goats  have  a  predominance  of  taurocholic 
acid  in  the  bile.  Ox-bile  sometimes  contains  taurocholic  acid  in  excess, 
in  other  cases  glycocholic  acid  predominates,  and  in  a  few  cases  the 
latter  occurs   almost   alone.     The   bile   of   the   rabbit,    hare,    kangaroo, 

decent  quantitative  analyses  may  be  found  in  Brand,  1.  c;  v.  Zeynek,  Wien. 
klin.  Wochensehr.,  1899;  Bonanni,  1.  c. 

2  Verhandl.  d.  physiol.  Gesellsch.  zu  Berlin,  189-95. 

3  Analyses  of  bile  from  children  may  be  found  in  Heptner,  Maly's  Jahresber.,  30. 

4  Noel-Paton,  Rep.  Lab.  Roy.  Soc.  Coll.  Phys.  Edinburgh,  3;  Stadelmann,.  Der 
Icterus. 


CONSTITUTION  OF  THE   BILE.  439 

hippopotamus,  and  orang-utang  (Hammarsten  !)  contains,  like  the 
bile  of  the  pig,  almost  exclusively  glycocholic  acid.  A  distinct  influence 
on  the  relative  amounts  of  the  two  bile-acids  exerted  by  differences  in 
diet  has  not  been  detected.  Hitter2  claims  to  have  found  a*  decrease 
in  the  quantity  of  taurocholic  acid  in  calves  when  they  pass  from  the 
milk  to  the  vegetable  diet. 

In  the  above-mentioned  calculation  of  the  taurocholic  acid  from  the 
quantity  of  sulphur  in  the  bile-salt,  it  must  be  remarked  that  no  definite 
conclusion  can  be  drawn  from  such  a  determination,  since  it  is  known 
that  other  kinds  of  bile  (e.  g.,  human  and  shark  bile)  contain  sulphur  in 
compounds  other  than  taurocholic  acid.3 

The  phosphorized  constituents  of  bile  are  not  well  known;  never- 
theless, there  is  no  doubt  that  bile  contains  other  phosphatides  besides 
lecithin  (Hammarsten).  These  phosphatides  are  in  part  precipitated  in 
the  precipitation  of  the  bile-salts  and  they  in  part  keep  the  bile-salts  in 
solution,  preventing  their  complete  precipitation,  and  hence  they  have  a 
double  disturbing  action  in  the  quantitative  analysis  of  bile.  Those  biles 
richest  in  phosphatides,  so  far  as  known,  are  the  following,  in  the  order  of 
their  amount:  Polar  bear,  man  (in  special  cases),  dog,  black  bear,  orang- 
utang.  The  bile  of  certain  fishes  contains  but  little  phosphatides 
(Hammarsten4). 

The  cholesterin,  which,  according  to  several  investigators,  originates 
not  only  from  the  liver  but  also  from  the  biliary  passages,  occurs  in 
larger  quantities  in  the  bladder-bile  than  in  the  liver-bile,  and  is  present 
to  a  greater  extent  in  the  non-filtered  than  in  the  filtered  bile  (Doyon 
and  Dufourt).  The  quantity  seems  to  be  very  variable  and  in  patients 
with  bile  fistulas  Bacmeister  5  found  0.24-0.59  p.  m.  The  gases 
of  the  bile  consist  of  a  large  quantity  of  carbon  dioxide,  which  increases 
with  the  amount  of  alkalies,  only  traces  of  oxygen,  and  a  very  small 
quantity  of  nitrogen. 

Little  is  known  in  regard  to  the  composition  of  the  bile  in  disease.  The  quantity 
of  urea  is  found  to  be  considerably  increased  in  uraemia.  Leucine  and  tyrosine  are 
observed  in  acute  yellow  atrophy  of  the  liver  and  in  typhoid.  Traces  of  albumin 
(without  regard  to  nucleoalbumin)  have  several  times  been  found  in  the  human 
"bile.  The  so-called  pigmentary  acholia,  or  the  secretion  of  a  bile  containing 
bile-acids  but  no  bile-pigments,  has  also  been  repeated!}'  noticed.  In  all  such 
eases  observed  by  Ritter  he  found  a  fatty  degeneration  of  the  liver-cells,  in  return 
for  which,  even  in  excessive  fatty  infiltration,  a  normal  bile  containing  pigments 
was   secreted.    The  secretion  of  a  bile  nearly  free  from   bile-acids  has   been 

1  See  Ergebnisse  der  Physiol.,  4. 

1  Cited  from  Maly's  Jahresber.,  6,  195. 

3  Hammarsten,  Zeitschr.  f.  physiol.  Chem.,  32,  and  Ergebnisse,  der  Physiol.,  4. 

4  Zeitschr.  f.  physiol.  Chem.,  36,  and  Ergebnisse  der  Physiol.,  4. 

6  Doyon  and  Dufort,  Arch,  de  Physiol.  (5),  8;  Bacmeister,  Bioch.  Zeitschr.,  26. 


440  THE  LIVER. 

observed  by  Hoppe-Seyler  :  in  amyloid  degeneration  of  the  liver.  In  animals,, 
dogs,  and  especially  rabbits,  it  has  been  observed  that  the  blood-pigments  pass 
into  the  bile  in  poisoning  and  in  other  conditions,  causing  a  destruction  of  the 
blood-corpuscles,  as  also  after  intravenous  haemoglobin  injection  (Wertheimer 
and  Meyer,  Filehne,  Stern  2).  Albumin  can  pass  into  the  bile  after  the  intra- 
venous injection  of  a  foreign  protein  (casein)  (Gurber  and  Hallauer),  as  well 
as  after  poisoning  with  phosphorus  or  arsenic  (Pilzecker),  or  after  the  irrita- 
tion of  the  liver  by  the  introduction  of  ethyl  alcohol  or  amyl  alcohol  (Brauer). 
Sugar  occurs  in  bile  only  in  exceptional  cases.3 

The  physiological  secretion  of  the  gall-bladder  in  man  is,  according 
to  Wahlgren4  a  viscous,  alkaline  fluid  with  11.24-19.63  p.  m.  solids. 
The  mucilaginous  properties  are  not  due  to  mucin,  but  to  a  phosphorized 
protein  substance  (nucleoalbumin  or  nucleoprotein) . 

Instead  of  bile  there  is  sometimes  found  in  the  gall-bladder  under  pathological 
conditions  a  more  or  less  viscous,  thready,  colorless  fluid  which  contains  pseudo- 
mucins  or  other  peculiar  protein  substances.5 

Chemical  Formation  of  the  Bile.  The  first  question  to  be  answered 
is  the  following:  Do  the  specific  constituents  of  the  bile,  the  bile-acids 
and  bile-pigments  originate  in  the  liver;  and  if  this  is  the  case,  do  they 
come  from  this  organ  alone,  or  are  they  also  formed  elsewhere? 

The  investigations  of  the  blood,  and  especially  the  comparative 
investigations  of  the  blood  of  the  portal  and  hepatic  veins  under  normal 
conditions,  have  not  given  any  answer  to  this  question.  To  decide  this, 
therefore,  it  is  necessary  to  extirpate  the  liver  of  animals  or  to  isolate 
it  from  the  circulation.  If  the  bile  constituents  are  not  formed  in  the 
liver,  or  at  least  not  alone  in  this  organ,  but  are  eliminated  only  from 
the  blood,  then,  after  the  extirpation  or  removal  of  the  liver  from  the 
circulation,  an  accumulation  of  the  bile  constituents  is  to  be  expected 
in  the  blood  and  tissues.  If  the  bile  constituents,  on  the  contrary,  are 
formed  exclusively  in  the  liver,  then  the  above  operation  naturally  would 
give  no  such  result.  If  the  ductus  choledochus  is  tied,  then  the  bile 
constituents  will  be  collected  in  the  blood  or  tissues  whether  they  are 
formed  in  the  liver  or  elsewhere. 

From  these  principles  Kobner  has  tried  to  demonstrate  by  exper- 
iments on  frogs  that  the  bile-acids  are  produced  exclusively  in  the  liver. 
While  he  was  unable  to  detect  any  bile-acids  in  the  blood  and  tissues  of 

fitter,  Compt.  Rend.,  74,  and  Journ.  de  l'anat.  et  de  la  physiol.  (Robin),  1872; 
Hoppe-Seyler,  Physiol.  Chem.,  317. 

2  Wertheimer  and  Meyer,  Compt.  Rend.,  108;  Filehne,  Virchow's  Arch.,  121;  Stern, 
ibid,  123. 

3  Gurber,  and  Hallauer,  Zeitschr.  f.  Biologie,  45;  Pilzecker,  Zeitschr.  f.  physiol. 
Chem.,  41;  Brauer,  ibid.,  40. 

1  Sec  Maly'.s  Jahresber.,  32. 

'  Winternitz,  Zeitschr.  f.  physiol.  Chem.,  21;  Sollmann,  Amer.  Medicine,  5  (1903). 


FORMATION  OF  BILE  PIGMENTS.  441 

these  animals  after  extirpation  of  the  liver,  he  was  able  to  discover  them 
on  tying  the  ductus  choledochus.  The  investigations  of  Ludwig  and 
Fleischl  '  show  that  in  the  dog  the  bile-acids  originate  in  the  liver  alone. 
After  tying  the  ductus  choledochus,  they  observed  that  the  bile  constituents 
were  absorbed  by  the  lymphatic  vessels  of  the  liver  and  passed  into  the 
blood  through  the  thoracic  duct.  Bile-acids  could  be  detected  in  the 
blood  after  such  an  operation,  while  they  could  not  be  detected  in  the 
normal  blood.  But  when  the  common  bile  and  thoracic  ducts  were  both 
tied  at  the  same  time,  then  not  the  least  trace  of  bile-acids  could  be 
detected  in  the  blood,  while  if  they  are  also  formed  in  other  organs  and 
tissues  they  should  have  been  present. 

From  earlier  reports  of  Cloez  and  Vulpian,  as  well  as  Virchow,  the  bile- 
acids  also  occur  in  the  suprarenal  capsule.  These  claims  have  not  been  confirmed 
by  later  investigations  of  Stadelmann  and  Beier.2  At  the  present  time  there 
is  no  ground  for  supposing  that  the  bile-acids  are  formed  elsewhere  than  in 
the  liver. 

It  has  been  undoubtedly  proved  that  the  bile-pigments  may  be  formed 
in  other  organs  besides  the  liver,  for,  as  is  generally  admitted,  the  color- 
ing-matter haematoidin,  which  occurs  in  old  blood  extravasations,  is 
identical  with  the  bile-pigment  bilirubin  (see  page  301).  Latschen- 
berger3  also  observed  in  horses,  under  pathological  conditions,  a 
formation  of  bile-pigments  from  the  blood-coloring  matters  in  the  tissues. 
The  occurrence  of  bile-pigments  in  the  placenta  also  seems  to  depend 
on  their  formation  in  that  organ,  while  the  occurrence  of  small  quantities 
of  bile-pigments  in  the  blood-serum  of  certain  animals  probably  depends 
on  an  absorption  of  these  substances. 

Although  the  bile-pigments  may  be  formed  in  other  organs  besides 
the  liver,  still  it  is  of  first  importance  to  know  what  bearing  this  organ 
has  on  the  elimination  and  formation  of  bile-pigments.  In  this  regard 
it  must  be  recalled  that  the  liver  is  an  excretory  organ  for  the  bile-pig- 
ments circulating  in  the  blood.  Tarchanoff  observed  in  a  dog  with 
biliary  fistula,  that  intravenous  injection  of  bilirubin  causes  a  very 
considerable  increase  in  the  bile-pigments  eliminated.  This  statement 
has  been  later  confirmed  by  the  investigations  of  Vossius.4 

Numerous  experiments  have  been  made  to  decide  the  question  whether 
the  bile-pigments  are  only  eliminated  by  the  liver,  or  whether  they  are 
also   formed   therein.     By   experimenting   on   pigeons,    Stern   was   able 


1  Kobner,  see  Heidenhain,  Physiologie  der  Absonderungsvorgange,  in  Hermann's 
Handbuch,  5;  Fleischl,  Arbeiten  aus  der  physiol.  Anstalt  zu  Leipzig,  Jahrgang,  9. 

2  Zeitschr.  f.  physiol.  Chem.,  18,  in  which  the  older  literature  may  be  found. 

•  See  Maly's  Jahresber.,  16,  and  Monatshefte  f.  Chem.,  9. 

*  Tarchanoff,  Pfliiger's  Arch.,  9;  Vossius,  cited  from  Stadelmann,  Der  Icterus. 


442  THE  LIVER. 

to  detect  bile-pigments  in  the  blood-serum  five  hours  after  tying  the 
biliary  passages  alone,  while  after  tying  all  the  vessels  of  the  liver  and  also 
the  biliary  passages,  no  bile-pigments  could  be  detected  either  in  the 
blood  or  the  tissues  of  the  animal,  which  was  killed  10-24  hours  after 
the  operation.  Minkowski  and  Naunyn  1  also  found  that  poisoning 
with  arseniureted  hydrogen  produces  a  liberal  formation  of  bile-pig- 
ments, and  the  secretion,  after  a  short  time,  of  a  urine  rich  in  biliverdin 
in  previously  healthy  geese.  In  geese  with  extirpated  livers  this  does 
not  occur. 

With  experiments  on  dogs,  Whipple  and  Hooper  2  found  after  intra- 
venous injection  of  blood-corpuscles  of  the  same  animal  hsemolyzed 
with  water,  that  a  transformation  of  the  haemoglobin  into  bile-pigments 
occurred  with  the  same  rapidity  in  normal  animals  as  with  animals  with 
Eck  fistulas,  or  with  such  a  fistula  and  the  hepatic  artery  ligatured.  The 
formation  of  bile-pigments  also  occurred  on  removing  the  liver,  spleen 
and  abdomen  from  the  circulation,  as  well  as  by  circulation  through  the 
head  and  thorax.  A  transformation  of  haemoglobin  into  bile-pigments, 
at  least  in  dogs,  can  take  place  easily  without  the  medium  of  the  liver 
and  these  experimenters  suggest  the  possibility  that  the  endothelial 
cells  are  here  active. 

No  such  experiments  can  be  carried  out  on  mammalia,  as  they  do 
not  live  long  enough  after  the  operation;  still  there  is  no  doubt  that  this 
organ  is  the  chief  seat  of  the  formation  of  bile-pigments  under  physiolog- 
ical conditions. 

In  regard  to  the  materials  from  which  the  bile-acids  are  produced, 
it  may  be  said  with  certainty  that  the  two  components,  glycocoll  and 
taurine,  which  are  both  nitrogenized,  are  formed  from  the  protein  bodies. 
The  close  relation  of  taurine  to  the  cystine  group  of  the  protein  mole- 
cule has  been  especially  shown  by  the  investigations  of  Freidmann, 
(see  Chapter  III),  and  recently  v.  Bergmann  3  has  shown  by  feeding 
dogs  with  sodium  cholate  and  cystine  that  the  animal  body  can  trans- 
form cystine  into  taurine,  and  that  the  taurine  of  the  bile  originates 
from  the  proteins  of  the  food.  In  regard  to  the  origin  of  the  non-nitro- 
genized  cholic  acid,  which  was  formally  considered  as  originating  from 
the  fats,  nothing  is  positively  known;  to  all  appearances  it  is  from 
proteins. 

The  blood-coloring  matters  are  considered  as  the  mother-substances 
of  the  bile-pigments.     If  the  identity  of  haematoidin  and  bilirubin  was 


1  Stern,  Arch.  f.  exp.  Path.  u.  Pharm.,  19;  Minkowski  and  Naunyn,  ibid.,  21. 

2  Journ.  of  exp.  Med.,  17. 

1  Hofmeister's  Beitrage,  4.     See  also  Wohlgemuth,  Zeitschr.  f.  physiol.  Chem.,  40. 


FORMATION  OF  BILE  PIGMENTS.  443 

settled  beyond  a  doubt,  then  this  view  might  be  considered  as  proved: 
Independently,  however,  of  this  identity,  which  is  not  admitted  by 
all  investigators,  the  view  that  the  bile-pigments  are  derived  from  the 
blood-coloring  matters  has  strong  arguments  in  its  favor.  It  has  been 
shown  by  several  experimenters  that  a  yellow  or  yellowish-red  pigment 
can  be  formed  from  the  blood-coloring  matters,  which  gives  Gmelin's 
test,  and  which,  though  it  may  not  form  a  complete  bile-pigment,  is 
at  least  a  step  in  its  formation  (Latschenberger).  The  previously 
mentioned  relationship  between  the  blood  and  bile-pigments  must  be 
recalled,  and  the  formation  of  bilirubin  from  the  blood-pigments  is 
shown,  according  to  the  unanimous  observations  of  several  investi- 
gators,1 bv  the  fact  that  the  appearance  of  free  haemoglobin  in  the  plasma, 
produced  by  the  destruction  of  the  red  corpuscles  by  widely  differing 
influences  (see  below)  or  by  the  injection  of  haemoglobin  solution,  causes 
an  increased  formation  of  bile-pigments.  The  amount  of  pigments  in 
the  bile  is  not  only  considerably  increased,  but  the  bile-pigments  may 
even  pass  into  the  urine  under  certain  circumstances  (icterus).  After 
the  injection  of  haemoglobin  solution  into  a  dog  either  subcutaneously 
or  in  the  peritoneal  cavity,  Stadelmann  and  Gorodecki  2  observed  an 
increase  of  61  per  cent  in  the  secretion  of  pigments  by  the  bile,  which 
lasted  for  more  than  twenty-four  hours.  Recently  Brusch  and  Yosh- 
imoto,3  by  quantitative  estimations  of  the  bile-pigments  and  urobilin 
in  animals  with  bile  fistulas  with  ligated  ductus  choledochus,  have 
shown  the  increased  formation  of  bile-pigments  after  the  injection  of 
known  amounts  of  haematin,  and  in  this  manner  further  proved  the 
genetic  relationship  between  the  bile-pigments  and  haematin. 

If  bilirubin,  which  contains  no  iron,  is  derived  from  haematin,  which 
contains  iron,  then  iron  must  be  split  off.  The  question  in  what  form  or 
combination  the  iron  is  split  off  is  of  special  interest,  and  also  whether 
it  is  eliminated  by  the  bile.  This  latter  does  not  seem  to  be  the  case, 
at  least  to  any  great  extent.  In  100  parts  of  bilirubin  which  are  eliminated 
by  the  bile  there  are  only  1.4-1.5  parts  iron,  according  to  Kunkel,  while 
100  parts  haematin  contain  about  9  parts  iron.  Minkowski  and  Base- 
rin4  also  found  that  the  abundant  formation  of  bile-pigments  occurring 
in  poisoning  by  arseniureted  hydrogen  does  not  increase  the  quantity 
of  iron  in  the  bile.  The  quantity  apparently  does  not  seem  to  correspond 
with  that  in  the  decomposed  blood-coloring  matters.     It  follows  from  the 


1  See  Stadelmann,  Der  Icterus,  etc.,  Stuttgart,  1891. 
1  See  Stadelmann,  ibid. 

3  Zeitschr.  f.  exp.  Path.  u.  Therap.,  8. 

4  Kunkel,   Pfluger's  Arch.,   14;    Minkowski  and  Baserin,  Arch.  f.  exp.   Path.  u. 
Pharm.,  23. 


444  THE  LIVER. 

researches  of  several  investigators  *  that  the  iron  is,  at  least  chiefly, 
retained  by  the  liver  as  a  ferruginous  pigment  or  protein  substance. 

What  relation  does  the  formation  of  bile-acids  bear  to  the  forma- 
tion of  bile-pigments?  Are  these  two  chief  constituents  of  the  bile  derived 
simultaneously  from  the  same  material,  and  can  we  detect  a  certain 
connection  between  the  formation  of  bilirubin  and  bile-acids  in  the  liver? 
The  investigations  of  Stadelmann  teach  us  that  this  is  not  the  case. 
With  increased  formation  of  bile-pigments  the  amount  of  bile-acids 
is  decreased,  and  the  introduction  of  haemoglobin  into  the  liver  strongly 
increases  the  formation  of  bilirubin,  but  simultaneously  strongly  decreases 
the  production  of  bile-acids.  According  to  Stadelmann  the  formation 
of  bile-pigments  and  bile-acids  is  due  to  a  special  activity  of  the  cells. 

An  absorption  of  bile  from  the  liver,  and  the  passage  of  the  bile  con- 
stituents into  the  blood  and  urine  occurs  in  retarded  discharge  of  the 
bile,  and  usually  in  different  forms  of  hepatogenic  icterus.  But  bile- 
pigments  may  also  pass  into  the  urine  under  other  circumstances,  espe- 
cially when  a  solution  or  destruction  of  the  red  blood-corpuscles  takes 
place  in  animals  through  injection  of  water  or  a  solution  of  biliary  salts, 
through  poisoning  by  ether,  chloroform,  ars3niureted  hydrogen,  phos- 
phorus, or  toluylenediamine,  and  in  other  cases.  This  also  occurs  in  man 
in  severe  infectious  diseases  where  the  red  blood-corpuscles  are  dissolved 
or  destroyed.  It  has  also  been  claimed  many  times  that  a  transformation 
of  blood-pigments  into  bile-pigments  occurs  elsewhere  than  in  the  liver, 
namely,  in  the  blood.  Such  a  belief  has  been  made  very  improbable 
and  in  some  of  the  above-mentioned  cases,  as  after  poisoning  with  phos- 
phorus, toluylenediamine,  and  arseniureted  hydrogen,  it  has  been  dis- 
proved by  direct  experiment.2  In  these  cases  we  are  also  dealing  with 
an  abundant  working  up  of  the  blood-pigments  in  the  liver. 

Bile  Concretions. 

The  concrements  which  occur  in  the  gall-bladder  vary  considerably 
in  size,  form,  and  number,  and  are  of  three  kinds,  depending  upon  the 
kind  and  nature  of  the  bodies  forming  their  principal  mass.  One  group  of 
gall-stones  contains  lime-pigment  as  chief  constituent,  another  cholesterin, 
and  the  third  calcium  carbonate  and  phosphate.  The  concrements 
of  the  last-mentioned  group  occur  very  seldom  in  man.  The  so-called 
cholesterin-stones  are  those  which  occur  most  frequently  in  man,  while 

<    Naunyn  and  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  21;    Latschenberger, 
\.c;  Neumann,  Virchow's  Arch.,  Ill,  and  the  literature  in  footnote  2,  p.  383. 

2  The  literature  belonging  to  this  subject  is  found  in  Stadelmann,  Der  Icterus,  etc., 
Stuttgart,  1891. 


BILE  CONCRETIONS.     CHOLESTERIN.  445 

the  lime-pigment  stones  are  not  found  very  often  in  man,  hut  often  in 
oxen. 

The  pigment-stones  are  generally  not  large  in  man,  but  in  oxen  and 
pigs  they  are  sometimes  found  the  size  of  a  walnut  or  even  larger.  In 
most  cases  they  consist  principally  of  calcium-bilirubin  with  little  or  no 
biliverdin,  and  they  also  often  contain  very  small  amounts  of  cholic  acids. 
Sometimes  also  small  black  or  greenish-black,  metallic-looking  stones 
are  found,  which  consist  chiefly  of  bilifuscin  along  with  biliverdin.  Iron 
and  copper  seem  to  be  regular  constituents  of  pigment-stones.  Man- 
ganese and  zinc  have  also  been  found  in  a  few  cases.  The  pigment-stones 
are  generally  heavier  than  water. 

The  cholesterin-stones,  whose  size,  form,  color,  and  structure  may  vary 
greatly,  are  often  lighter  than  water.  The  fractured  surface  is  radiated, 
crystalline,  and  frequently  shows  crystalline,  concentric  layers.  The 
cleavage  fracture  is  waxy  in  appearance,  and  the  fractured  surface  when 
rubbed  by  the  finger-nail  also  becomes  like  wax.  By  rubbing  against 
each  other  in  the  gall-bladder  they  often  become  faceted  or  take  other 
remarkable  shapes.  Their  surface  is  sometimes  nearly  white  and  wax- 
like, but  generally  their  color  is  variable.  They  are  sometimes  smooth, 
in  other  cases  they  are  rough  or  uneven.  The  quantity  of  cholesterin 
in  the  stones  varies  from  G42  to  981  p.  m.  (Ritter  l).  The  cholesterin- 
stones  sometimes  contain  variable  amounts  of  lime-pigments,  which  may 
give  them  a  very  changeable  appearance. 

Cholesterin.  The  formula  for  this  body,  although  not  positively 
determined,  is  generally  given  as  C27H46O  (Obermuller)  or  C27H44O 
(Mauthner  and  Suida). 

Because  of  the  fact  that  from  cholesterin,  hydrocarbons  which 
have  been  called  cholesteriline,  cholesterone  and  cholesterilene,  can  be 
prepared  in  different  ways,  it  was  believed  that  a  certain  analogy  exists 
between  the  cholesterin  and  the  terpenes.  The  color  reactions  as 
well  as  the  recent  investigations  on  the  constitution  of  cholesterin  indicate 
that  this  body  belongs  to  the  terpenes. 

The  constitution  of  cholesterin  has  not  been  completely  determined, 
although  we  have  the  very  laborious  and  thorough  investigations  of 
many  workers  of  whom  we  especially  mention  Mauthner  and  Suida, 
Windaus,  Stein,  Diels  and  Abderhalden.2  From  these  investigations 
we  conclude  that  cholesterin  is  a  monoatomic,  unsaturated,  secondary 
alcohol   whose   hydroxyl  group   exists   in   a   hydrogenized  ring,  between 


1  Journ.  de  l'anat.  et  de  la  physiol.  (Robin),  1872. 

2  The  literature  on  cholesterin  can  be  found  in  Windaus,  Arch.  d.  Pharm.,  246, 
Hft.  2,  and  in  Abderhalden's  Bioch.  Handlexikon,  Bd.,  3,  and  also  in  Glikin,  Bioch. 
Centralbl.,  7,  372-377. 


446  THE  LIVER. 

two  methyl  groups,  and  which  also  contains  an  isopropyl  group. 
It  is  also  generally  admitted  that  cholesterin  contains  only  one  double 
bond,  which  occurs  in  a  vinyl  group,  CH:CH2,  at  the  end.  No  constitu- 
tional formula  for  cholesterin  can  be  given;  still  there  is  no  doubt  but 
that  it  is  a  complex  terpene  which  stands  in  close  relation  to  retene  as 
well  as  to  the  cholic  acids. 

By  the  reduction  of  cholesterin  by  metallic  sodium  and  amyl  alcohol,  Diels 
and  Abderhalden  as  well  as  Neuberg  and  Rauchwerger  obtained  a  dihydro- 
cholesterin,  the  a-cholestanol,  C27H480.  On  treating  cholestenon,  the  ketone  of 
cholesterin,  Diels  and  Abderhalden  obtained  a  second  dihydrocholesterin, 
the  @<holesta?wl,  which  Willstatter  and  E.  W.  Mayer  obtained  directly  from 
cholesterin  in  ethereal  solution  by  reduction  with  hydrogen  and  platinum-black. 
According  to  Diels  and  Linn  *  /3-cholestanol  is  obtained  from  cholestenon  by 
the  action  of  sodium  and  amyl  alcohol,  and  a-cholestanol  with  sodium  amy  late. 
The  relation  of  these  bodies  to  each  other  is  still  not  understood.  These  dihydro- 
cholesterins  have  a  physiological  interest  in  regard  to  the  question  whether  they 
are  identical  or  not  with  koprosterin,  which  will  be  discussed  below. 

On  heating  cholesterin,  when  contaminated  with  iron,  to  300-320°,  according 
to  Diels  and  Linn,2  it  in  part  yields  cholestenon  and  partly  an  isomeric  cholesterin, 
the  fl-cholesterin.  This  last  body  can  be  retransformed  into  cholesterin  by  the 
saponification  of  the  cholesteryl  benzoate. 

Cholesterin  occurs  in  small  amounts  in  nearly  all  animal  fluids  and 
juices.  It  occurs  only  rarely  in  the  urine,  and  then  in  very  small  quanti- 
ties. It  is  also  found  in  the  different  tissues  and  organs,  especially 
abundant  in  the  brain  and  the  nervous  system;  further,  in  the  yolk  of 
the  egg,  in  semen,  in  wool-fat  (together  with  isocholesterin),  and  in 
sebum.  It  also  appears  in  the  contents  of  the  intestine,  in  excrements, 
and  in  the  meconium.  It  especially  occurs  pathologically  in  gall-stones 
as  well  as  in  atheromatous  cysts,  in  pus,  in  tuberculous  masses,  old 
transudates,  cystic  fluids,  sputum,  and  tumors.  It  does  not  exist  free 
in  all  cases;  for  example,  it  exists  in  part  as  fatty-acid  esters  in  wool- 
fat,  blood,  lymph,  brain,  vernix  caseosa  and  epidermis  formations.  Sev- 
eral kinds  of  cholesterin,  called  phytosterines,  have  been  found  in  the 
vegetable  kingdom. 

Cholesterin  which  has  been  crystallized  from  warm  alcohol  on  cooling, 
and  also  that  which  is  present  in  old  transudates,  contains  one  molecule 
of  water  of  crystallization,  and  melts  at  148.5°  C.  when  dried  in  a  vacuum, 
and  forms  colorless,  transparent  plates  whose  sides  and  angles  frequently 
appear  broken,  and  whose  acute  angle  is  often  76°  30'  or  87°  30'.  In 
large  quantities  it  appears  as  a  mass  of  white  plates  which  shine  like 
mother-of-pearl  and  have  a  greasy  touch. 

Cholesterin  is  insoluble  in  water,  dilute  acids,  and  alkalies.  It  is 
neither   dissolved   nor   changed   by   boiline;   caustic   alkali.     It   is   easily 

1  Willstatter  and  Mayer,  Ber  d.  d.  chem.  Gesellsch.,  41;  Diels  and  Linn,  ibid.,  41. 

2  Ibid.,  41. 


CHOLESTERIN.  447 

soluble  in  boiling  alcohol,  and  crystallizes  on  cooling.  It  dissolves  readily 
in'ether,  chloroform,  and  benzene,  and  also  in  the  volatile  or  fatty  oils. 
It  is  dissolved  to  a  slight  extent  by  alkali  salts  of  the  bile-acids,  better 
in  the  presence  of  oleic  soap  (Gerard  l).  The  solutions  in  ether  and 
chloroform  are  levorotatory,  (a)D= —31.12°  (2  per  cent  ethereal  solu- 
tion). 

Among  the  many  compounds  of  cholesterin  the  propionic  ester 
C2H5.CO.O.C27H45  is  of  special  interest  because  of  the  behavior  of  the 
fused  compound  on  cooling,  and  it  is  used  in  the  detection  of  choles- 
terin. For  the  detection  of  cholesterin  use  is  made  of  its  reaction  with 
concentrated  sulphuric  acid,  which  gives  colored  products. 

If  a  mixture  of  five  parts  sulphuric  acid  and  one  part  water  acts  on 
cholesterin  crystals,  they  show  colored  rings,  first  a  bright  carmine-red 
and  then  violet.  This  test  is  employed  in  the  microscopic  detection 
of  cholesterin.  Another  test,  and  one  very  good  for  the  microscopical 
detection  of  cholesterin,  consists  in  treating  the  crystals  first  with  the 
above  dilute  acid  and  then  with  some  iodine  solution.  The  crystals 
will  be  gradually  colored  violet,  bluish-green,  and  a  beautiful  blue. 

Salkowski's  2  Reaction.  The  cholesterin  is  dissolved  in  chloroform 
and  then  treated  with  an  equal  volume  of  concentrated  sulphuric  acid. 
The  cholesterin  solution  becomes  first  bluish-red,  then  gradually  more 
violet-red,  while  the  sulphuric  acid  appears  dark  red  with  a  greenish 
fluorescence.  If  the  chloroform  solution  is  poured  into  a  porcelain  dish 
it  becomes  violet,  then  green,  and  finally  yellow. 

Liebermann-Burchard's  3  Reaction.  Dissolve  the  cholesterin  in 
about  2  cc.  chloroform  and  add  first  10  drops  of  acetic  anhydride  and  then 
concentrated  sulphuric  acid  drop  by  drop.  The  color  of  the  mixture 
will  first  be  a  beautiful  red,  then  blue,  and  finally,  if  not  too  much 
cholesterin  or  sulphuric  acid  is  present,  a  permanent  green.  In  the  pres- 
ence of  very  little  cholesterin  the  green  color  may  appear  immediately. 

Neuberg-Rauchwerger's  4  Reaction.  With  rhamnose,  or  better  still 
with  6-methylfurfurol  and  concentrated  sulphuric  acid,  an  alcoholic 
solution  of  cholesterin  gives  a  pink  ring,  or  after  mixing  the  liquids  and 
cooling,  a  pink  solution.  On  proper  dilution  an  absorption-band  can 
be  seen  just  beginning  before  E  and  whose  other  side  coincides  with  b. 
This  reaction  is  of  interest  because  it  is  also  given  by  bile-acids,  some 
camphor  derivatives,  abietinic  acid,  and  a  hydride  of  retene. 


1  Compt.  rend.  soc.  biol.,  58. 

2  Pfluger's  Arch.,  6. 

3  C.  Liebermann,  Ber.  d.  deutsch.  chem.  Gesellsch.,   18;  1804,  H.  Burchard,  Bei- 
trage  zur  Kenntnis  der  Cholesterine,  Rostock,  1899. 

4  Salkowski's  Festschrift,  1904. 


448  THE  LIVER. 

LiFSCHtJTz's 1  Reaction.  Dissolve  a  few  milligrams  of  cholesterin 
in  2-3  cc.  glacial  acetic  acid,  add  a  little  benzoylsuperoxide  thereto,  and 
boil  once  or  twice.  On  adding  4  drops  concentrated  sulphuric  acid  to 
the  cooled  solution  and  shaking,  a  pure  green  coloration  is  obtained, 
which  changes  immediately  into  blue  or  with  violet-red  as  an  intermediary 
color.  An  absorption-band  is  formed  between  C  and  d,  and  a  broad  band 
at  D.  In  this  reaction  an  oxidation  of  the  cholesterin  occurs,  and  Lif- 
schutz  2  therefore  uses  the  glacial  acetic  acid-sulphuric  acid  reaction 
(color  and  spectrum)  for  the  detection  of  oxidation  products  of  choles- 
terin in  blood  and  tissues. 

Pure,  dry  cholesterin  when  fused  in  a  test-tube  over  a  low  flame  with  two  or 
three  drops  of  propionic  anhydride  yields  a  mass  which  on  cooling  is  first  violet, 
then  blue,  green,  orange,  carmine-red,  and  finally  copper-red.  It  is  best  to  re-fuse 
the  mass  on  a  glass  rod  and  then  to  observe  the  rod  on  cooling,  holding  it  against 
a  dark  background  (Obermuller).3 

Schiff's  Reaction.  If  a  little  cholesterin  is  placed  in  a  porcelain  dish  with 
the  addition  of  a  few  drops  of  a  mixture  of  2  or  3  vols,  of  concentrated  hydrochloric 
acid  or  sulphuric  acid  and  1  vol.  of  a  rather  dilute  solution  of  ferric  chloride,  and 
carefully  evaporated  to  dryness  over  a  small  flame,  a  reddish-violet  residue  is. 
first  obtained  and  then  a  bluish- violet. 

If  a  small  quantity  of  cholesterin  is  evaporated  to  dryness  with  a  drop  of 
concentrated  nitric  acid,  one  obtains  a  yellow  spot  which  becomes  deep  orange-red 
with  ammonia  or  caustic  soda  (not  a  characteristic  reaction).         ' 

Cholesterin  combines  with  saponin  (Windaus,  Yagi)  and  when  a  solution 
of  cholesterin  in  boiling  95  per  cent  alcohol  is  treated  with  a  warm  1  per  cent 
solution  of  digitonin  in  90  per  cent  alcohol,  a  precipitate  of  digitonin-cholesteride 
is  obtained.  If  the  amount  of  the  washed  and  dried  digitonin-cholesteride  is 
multiplied  by  0.25  the  quantity  of  cholesterin  is  obtained  (Windaus  4).  The 
cholesterin  esters  are  not  precipitated  by  digitonin. 

Koprosterin  is  the  name  given  by  Bondzynski  to  the  cholesterin  which  was 
isolated  by  him  from  human  feces,  although  it  was  prepared  earlier  by  Flint 
and  designated  as  stercorin.  It  dissolves  in  cold,  absolute  alcohol  and  very  readily 
in  ether,  chloroform,  and  benzene.  It  crystallizes  in  fine  needles  which  melt  at 
95-96°  C.  (89-90°  according  to  Hausmann),  and  is  dextrorotatory  (of)D  =  +24°. 
It  gives  the  same  color  reactions  as  cholesterin,  with  the  exception  that  it  does 
not  give  a  reaction  with  propionic  anhydride.  According  to  Bondzynski  and 
Humnicki  it  is  a  dihydrocholesterin,  with  the  formula  C27H48O,  which  is  formed 
in  the  human  intestine  by  the  reduction  of  ordinary  cholesterin.  According  to 
Kusumoto  as  well  as  Dorbe  and  Gardner,  koprosterin  also  occurs  in  the  intes- 
tine of  dogs.  The  koprosterin  prepared  by  H.  Fischer  from  human  feees  seems 
to  be  identical  with  that  prepared  by  Bondzynski.     It  is  remarkable  that  Boehm  6 


1  Ber.  d.  d.  chem.  Gesellsch.,  41. 

2  Zeitschr.  f.  physiol.  Chem.,  50,  53,  58,  and  Ber.  d.  d.  chem.  Gesellsch.,  41. 

3  Zeitschr.  f.  physiol.  Chem.,  15. 

*  Windaus,  Zeitschr.  f.  physiol.  Chem.,  65;   Yagi,  Arch.  f.  exp.  Path.  u.  Pharm.,  64. 

6  Bondzynski,  Ber.  d.  deutsch.  chem.  Gesellsch.,  20;  Bondzynski  and  Humnicki, 
Zeitschr.  f.  physiol.  Chem.,  22;  Flint,  ibid.,  23,  and  Amer.  Journ.  Med.  Sciences, 
1862;  Muller,  Zeitschr.  f.  physiol.  Chem.,  20;  Hausmann,  Hofmeister's  Beitrage,  6; 
Kusumoto,  Bioch.  Zeitschr.,  14;  Dor6e  and  Gardner,  Proc.  Roy.  Soc.  London,  80, 
Bet    I'  :   II.  Fischer,  Zeitschr.  f.  physiol.  Chem.,  73;  Boehm,  Bioch.  Zeitschr.,  33. 


CH0LESTERIN8.  449 

found  a  dihydrocholesterin  in  the  contents  in  a  part  of  the  ileum  which  had  been 
disconnected  from  the  other  part  of  the  intestine  for  14  years.  This  had  the 
same  optical  rotation  and  the  same  melting-point,  142-143°  C,  as  the  dihydro- 
cholesterin  (j8-cholestanol)  prepared  by  Diels  and  Abderhalden,  Willstatter 
and  Mayer. 

Hippokoprosterin  is  another  cholesterin  richer  in  hydrogen,  which  Bondzynbkj 
and  Hi  M\i(  Ki  found  in  the  feces  of  the  horse.  Its  formula  is  C^Ho.O.  According 
to  I>i>kki:  and  Gardner  it  is  not  an  animal  cleavage  product,  but  a  constituent 
of  the  grass  used  as  fodder.     It  melts  at  78.5-79.5°  C. 

Isocholesterin  is  a  cholesterin,  so  called  by  Schulze,1  with  the  formula 
ChHuO,  which  occurs  in  wool-fat,  and  is  therefore  found  to  a  great  extent  in 
so-called  lanolin.  It  gives  the  Liebermann-Burchard  reaction,  but  .does  not 
give  Salkowski's  reaction.  It  melts  at  138-138.5°  C.  The  specific  rotation  in 
7  per  ceni  ethereal  solution  is  (a)D  =  4-59.1°. 

Spongosterin,  C^ILsO  is  themame  given  by  Henze2  to  a  cholesterin  isolated  by 
him  from  a  silicious  sponge.  It  is  very  similar  to  cholesterin,  but  is  not  identical 
with  it  or  with  phytocholesterins.  It  gives  the  Liebermann-Burchard  reaction  as 
well  as  Salkowski's  reaction,  but  the  last  test  is  not  quite  so  beautiful  a  red. 
Obermuller's  reaction  is  negative.     Melting-point  123-124°. 

Bombicesterin  is  the  name  given  by  Menozzi  and  Moreschi  3  to  a  cholesterin 
isolated  by  them  from  the  chrysalis  of  the  silkworm,  which  has  a  melting-point 
of  148°  and  a  specific  rotation  of  (<*)d  =  —34°. 

The  cholesterin  occurring  in  the  intestine  is  derived  in  part  from  the 
food,  in  part  from  the  bile  and  part,  as  shown  from  the  contents  of  a 
ligatured  portion  of  the  intestine  (see  Chapter  VIII),  from  the  epithelium 
or  the  secretion  of  the  intestinal  mucosa.  That  a  part  of  the  cholesterin 
of  the  intestine  disappears  has  been  shown  by  Kusumoto,  although 
it  remains  undecided  whether  this  takes  place  by  bacterial  decomposi- 
tion or  by  absorption.  Levites  4  on  the  contrary,  recovered  the  cho- 
lesterin introduced  into  dogs  almost  quantitatively.  The  behavior  of 
cholesterin  in  metabolism  is  not  well  known;  Lifschutz  believes  that  he 
has  detected  by  his  color-reaction  the  oxidation  products  of  cholesterin 
in  the  blood  and  in  bone-fat. 

The  cholesterins  belong  to  the  so-called  lipoids,  which  have  been 
mentioned  in  previous  chapters  (I  and  VI),  and  are  of  the  greatest 
importance  as  constituents  of  the  outer  envelope  of  erythrocytes  and 
the  cells  in  general.  Cholesterin  is  also  of  great  interest  because  it  inhibits 
or  prevents  the  haemolysis  produced  by  certain  bodies,  and  therefore 
acts  as  a  certain  protective  power  in  the  animal  bod}'.  This  action  of 
the  cholesterins  in  regard  to  inhibiting  the  haemolytic  action  of  saponin, 

1  Ber.  d.  deutsch.  chem.  Gesellsch.,  6;  Journal  f.  prakt.  Chem.  (N.  F.),  25;  and 
Zeitschr.  f.  physiol.  Chem.,  14,  522.  See  also  E.  Schulze  and  J.  Barbieri,  Journal  f. 
prakt.  Chem.  (N.  F.)(  25,  159.  In  regard  to  the  formula  for  isocholesterin,  see 
Darmstiidter  and  Lifschutz,  Ber.  d.  deutsch.  chem.  Gesellsch.,  31,  and  E.  Schulze, 
ibid.,  1200. 

2  Zeitschr.  f.  physiol.  Chem.,  41  and  55. 

s  Cited  from  Chem.  Centralbl.,  1908,  1377  and  1910,  872. 
4  Zeitschr.  f.  physiol.  Chem.,  57. 


450  THE  LIVER. 

as  first  discovered  by  Ransom,  is  destroyed,  as  shown  by  Hausmann,  by 
replacing  the  hydroxyl  groups.  These  combinations  between  cholesterin 
and  saponins  have  been  studied  by  Madsen  and  Noguchi  and  by 
Windaus.1 

The  so-called  cholesterin-stones  are  best  employed  in  the  preparation 
of  cholesterin.  The  powder  is  first  boiled  with  water  and  then  repeatedly 
boiled  with  alcohol.  The  cholesterin  which  on  cooling  separates  from  the 
warm  filtered  solution  is  boiled  with  a  solution  of  caustic  potash  in  alcohol 
so  as  to  saponify  any  fat.  After  the  evaporation  of  the  alcohol  the  choles- 
terin is  extracted  from  the  residue  with  ether,  by  which  the  soaps  are 
not  dissolved;  filter,  evaporate  the  ether,  and  purify  the  cholesterin  by 
recrystallization  from  alcohol-ether.  The  cholesterin  may  be  extracted 
with  fat  from  tissues  and  fluids  by  first  extracting  with  ether  and  then 
proceeding  as  suggested  by  Ritter.2  The  essential  points  in  his  method 
consist  in  saponifying  the  fat  with  sodium  alcoholate,  removing  the  alcohol 
by  evaporating  to  dryness  with  NaCl,  and  finally  extracting  the  dried 
pulverized  mass  with  ether.  After  evaporating  the  ether  the  residue  is 
dissolved  in  as  little  alcohol  as  possible  and  the  cholesterin  precipitated 
by  the  addition  of  water.  It  is  ordinarily  easily  detected  in  transudates 
and  pathological  formations  by  means  of  the  microscope.  In  regard  to 
the  methods  of  preparation,  detection  and  quantitative  estimation  of 
cholesterin  we  refer  to  the  larger  text-books. 

Ransom,  Deutsch.  med.  Wochenschr.,  1901;  Hausmann,  Hofmeister's  Beitrage, 
6;  Madsen  and  Noguchi,  Kgl.  Dansk.  Vidensk.  Selskabs.  Forh.,  1904;  Windaus,  Ber. 
d.  d.  chem.  Gesellsch.,  42. 

2  Zeitschr.  f.  physiol.  Chem.,  34.    See  also  Corper,  Journ.  of  biol.  Chem.,  11. 


CHAPTER  VIII. 
DIGESTION. 

The  purpose  of  digestion  is  to  separate  those  constituents  of  the 
food  which  serve  as  nutriment  for  the  body  from  those  which  are  use- 
less, and  to  separate  each  in  such  a  form  that  it  may  be  taken  up  by  the 
blood  from  the  alimentary  canal  and  employed  for  various  purposes  in 
the  organism.  This  demands  not  only  mechanical,  but  also  chemical, 
action.  The  first  action,  which  is  essentially  dependent  upon  the  physical 
properties  of  the  food,  consists  in  a  tearing,  cutting,  crushing,  or  grinding 
of  the  food,  while  the  second  serves  chiefly  in  converting  the  nutritive 
bodies  into  a  soluble  and  easily  absorbable  form,  or  in  splitting  them  into 
simpler  compounds  for  use  in  the  animal  syntheses.  The  solution  of  the 
nutritive  bodies  may  take  place  in  certain  cases  by  the  aid  of  water  alone, 
but  in  most  cases  a  chemical  metamorphosis  or  cleavage  is  necessary; 
this  is  effected  by  means  of  the  acid  or  alkaline  fluids  secreted  by  the 
glands.  The  study  of  the  processes  of  digestion  from  a  chemical  stand- 
point must  therefore  begin  with  the  digestive  fluids,  their  qualitative 
and  quantitative  composition,  as  well  as  their  action  on  the  nutriments 
and  foods. 

I.     THE   SALIVARY   GLANDS  AND   THE   SALIVA. 

The  salivary  glands  are  partly  albuminous  glands  (as  the  parotid 
in  man  and  mammals,  and  the  submaxillary  in  rabbits),  partly  mucous 
glands  (as  some  of  the  small  glands  in  the  buccal  cavity  and  the  sub- 
lingual and  submaxillary  glands  of  many  animals),  and  partly  mixed 
glands  (as  the  submaxillary  gland  in  man).  The  alveoli  of  the  albuminous 
glands  contain  cells  which  are  rich  in  protein  but  which  contain  no  mucin. 
The  alveoli  of  the  mucin-glands  contain  cells  rich  in  mucin  but  poor  in 
protein.  Cells  arranged  in  different  ways,  but  rich  in  proteins,  also  occur 
in  the  submaxillary  and  sublingual  glands.  According  to  the  analyses 
of  Magnus-Levy  1  the  human  salivary  glands  contain  274  p.  m.  solids, 
of  which  114  p.  m.  was  fat  and  154  p.  m.  was  protein.  Among  the 
solids  we  find  mucin,  proteins,  nucleoproteins,  nuclein,  enzymes  and  theix 

1  Bioch.  Zeitschr.,  24. 

451 


452  DIGESTION. 

zymogens,    besides   extractive   bodies,    leucine,    purine   bases,    and   mineral 
substances. 

The  occurrence  of  a  mucinogen  has  not  been  proved.  On  the  complete  removal 
of  all  mucin  E.  Holmgren  1  found  no  mucinogen  in  the  submaxillary  gland  of 
the  ox,  but  a  mucin-like  gluconucleoproteid. 

1  he  saliva  is  a  mixture  of  the  secretion  of  the  above-mentioned  groups 
of  glands;  therefore  it  is  proper  that  a  study  be  made  of  each  of  the  dif- 
ferent secretions  by  itself  and  then  of  the  mixed  saliva. 

The  submaxillary  saliva  in  man  may  be  easily  collected  by  intro- 
ducing a  canula  through  the  papillary  opening  into  Wharton's  duct. 

The  submaxillary  saliva  has  not  always  the  same  composition  or 
properties;  this  depends  essentially,  as  shown  by  experiments  on  animals, 
upon  the  conditions  under  which  the  secretion  takes  place.  That  is  to 
say,  the  secretion  is  partly  dependent  on  the  cerebral  system,  through 
the  facial  fibers  in  the  chorda  tympani,  and  partly  on  the  sympathetic 
nervous  system,  through  the  fibers  entering  the  vessels  in  the  gland.  In 
consequence  of  this  dependence  the  two  distinct  varieties  of  submaxillary 
secretion  are  distinguished  as  chorda-  and  sympathetic  saliva.  A  third 
kind  of  saliva,  the  so-called  paralytic  saliva,  is  secreted  after  poisoning 
with  curare  or  after  the  severing  of  the  glandular  nerves. 

The  difference  between  chorda-  and  sympathetic  saliva  (in  dogs) 
consists  chiefly  in  their  quantitative  constitution;  the  less  abundant 
sympathetic  saliva  is  more  viscous  and  richer  in  solids,  especially  in 
mucin,  than  the  more  abundant  chorda-saliva.  The  specific  gravity  of 
the  chorda-saliva  of  the  dog  is  1.0039-1.0056,  and  contains  12-14  p.  m. 
solids  (Eckhard  2) .  The  sympathetic  has  a  specific  gravity  of  1 .0075-1 .018, 
with  16-28  p.  m.  solids.  The  freezing-point  of  the  chorda-saliva  obtained 
from  dogs  on  electric  stimulation  varies,  according  to  Nolf,3  between 
A  =  -0.193°  and  -0.396°,  with  a  content  of  3.3-6.5  p.  m.  salts  and  4.1- 
11.5  p.  m.  organic  substances.  The  osmotic  pressure  is  on  am  average  a 
little  higher  than  one-half  the  osmotic  pressure  of  the  blood-serum.  The 
spontaneously  secreted  submaxillary  saliva  is  ordinarily  somewhat  diluted. 
On  changing  the  osmotic  pressure  of  the  blood  the  osmotic  pressure 
of  the  saliva,  according  to  Jappelli,4  changes  in  the  same  direction. 
According  to  Demoor,  Locke's  solution  with  some  dog  serum  is  well 
suited  by  transfusion  to  keep  the  submaxillary  gland  of  the  dog  in 
activity,  while  ox  serum  is  unsuited.5    The  gases  of  the  chorda-saliva 

1  Upsala  Lakaref.  F6rb.  (N.  F.),  2;  also  Maly's  Jahresber.,  27. 

2  Cited  from  Kiihne'a  Lebrb.  d.  physiol.  Chem.,  7. 
<<■<-.  Maly's  Jahresber.,  81,  494. 

4  .Jappelli,  ibid.,  48  and  51. 

5  Arch,  intern,  de  Physiol.,  10  (1911). 


SALIVA.  453 

have  been  investigated  by  PflUgeb.1  He  found  0.5-0.8  per  cent  oxygen, 
0.9-1  per  cent  nitrogen,  and  64.73-85.13  per  cent  carbon  dioxide — 
all  results  calculated  at  0°  C.  and  760  mm.  pressure.  The  greater  part 
of  the  carbon  dioxide  was  chemically  combined. 

The  two  kinds  of  submaxillary  secretion  just  named  have  not  thus 
far  been  separately  studied  in  man.  The  secretion  may  be  excited  by  an 
emotion,  by  mastication,  and  by  irritating  the  mucous  membrane  of  the 
mouth,  especially  with  acid-tasting  substances.  The  submaxillary  saliva 
in  man  is  ordinarily  clear,  rather  thin,  a  little  ropy,  and  froths  easily. 
Its  reaction  is  alkaline  toward  litmus.  The  specific  gravity  is  1.002- 
1.003,  and  it  contains  3.6-4.5  p.  m.  solids.2  As  organic  constituents 
are  found  mucin,  traces  of  protein  and  diastatic  enzyme,  which  latter  is 
absent  in  several  species  of  animals.  The  inorganic  bodies  are  alkali 
chlorides,  sodium  and  magnesium  phosphates,  and  bicarbonates  of  the 
alkalies  and   calcium.     Potassium   sulphocyanide   occurs   in   this  saliva. 

The  Sublingual  Saliva.  The  secretion  of  this  saliva  is  also  influenced 
by  the  cerebral  and  the  sympathetic  nervous  system.  The  chorda-saliva, 
which  is  secreted  only  to  a  small  extent,  contains  numerous  salivary 
corpuscles,  but  is  otherwise  transparent  and  very  ropy.  Its  reaction 
is  alkaline,  and  it  contains,  according  to  Heidenhain,3  27.5  p.  m.  solids 
(in  dogs). 

The  sublingual  secretion  in  man  is  clear,  mucilaginous,  more  alka- 
line than  the  submaxillary  saliva,  and  contains  mucin,  diastatic  enzyme, 
and  potassium  sulphocyanide. 

Buccal  mucus  can  be  obtained  pure,  from  animals  only,  by  the  method 
suggested  by  Bidder  and  Schmidt,  which  consists  in  tying  the  exit  to 
all  the  large  salivary  glands  and  cutting  off  their  secretion  from  the  mouth. 
The  quantity  of  liquid  secreted  under  these  circumstances  (in  dogs)  was 
so  very  small  that  the  investigators  named  were  able  to  collect  only  2 
grams  of  buccal  mucus  in  the  course  of  one  hour.  It  is  a  thick,  ropy, 
sticky  liquid  containing  mucin;  it  is  rich  in  form-elements,  above  all 
in  flat  epithelium  cells,  mucous  cells,  and  salivary  corpuscles.  The 
quantity  of  solids  in  the  buccal  mucus  of  the  dog  is,  according  to 
Bidder  and  Schmidt,4  9.98  p.  m. 

Parotid  Saliva.  The  secretion  of  this  saliva  is  also  partly  dependent 
on  the  cerebral  nervous  system  (n.  glossopharyngeus)  and  partly  on  the 
sympathetic.     The  secretion  may  be  excited  by  emotions  and  by  irri- 


1  Pfluger's  Arch.,  1. 

2  See  Maly's  "Chemie  der  Verdauungssafte  und  der  Verdauung,"  in  Hermann's 
Handb.,  5,  part  II,  18.     This  article  contains  also  the  pertinent  literature. 

3  Studien.  d.  physiol.  Instituts  zu  Breslau,  Heft  4. 

*  Die  Verdauungssafte  und  der  Stoffwechsel  (Mitau  and  Leipzig,  1852),  p.  5. 


454  DIGESTION. 

tation  of  the  glandular  nerves,  either  directly  (in  animals),  or  reflexly, 
by  mechanical  or  chemical  irritation  of  the  muccus  membrane  of  the 
mouth.  Among  the  chemical  irritants  the  acids  take  first  place.  Mas- 
tication also  exercises  a  strong  influence  upon  the  secretion  of  parotid 
saliva,  which  is  specially  marked  in  certain  herbivora. 

Human  parotid  saliva  may  be  readily  collected  by  the  introduction 
of  a  canula  into  Stenson's  duct.  This  saliva  is  thin,  less  alkaline  than 
the  submaxillary  saliva  (the  first  drops  are  sometimes  neutral  or  acid), 
without  special  odor  or  taste.  It  contains  a  little  protein  but  no  mucin, 
which  is  to  be  expected  from  the  construction  of  the  gland.  It  also  con- 
tains a  diastatic  enzyme,  which,  however,  is  absent  in  many  animals. 
The  quantity  of  solids  varies  between  5  and  16  p.  m.  The  specific  gravity 
is  1.003-1.012.  Potassium  sulphocyanide  seems  to  be  present,  though 
it  is  not  a  constant  constituent.  Kulz  *  found  a  maximum  of  1.46  per 
cent  oxygen,  3.8  per  cent  nitrogen,  and  in  all  66.7  per  cent  carbon  dioxide 
in  human  parotid  saliva.  The  quantity  of  firmly  combined  carbon  dioxide 
was  62  per  cent. 

The  quantity  and  composition  of  the  saliva,  from  the  mucin  glands 
as  well  as  from  the  albuminous  glands,  show  differences  in  the  various 
classes  of  animals  but  these  cannot  be  entered  into  here.  According 
to  Pawlow  2  and  his  pupils  the  quantity  as  well  as  the  composition  of 
the  saliva  of  the  various  glands  and  the  mixed  saliva  in  dogs  is  to  a  great 
degree  dependent  upon  the  psychical  stimulation,  but  also  upon  the 
kind  of  substances  introduced  into  the  mouth,  and  an  adaptation  of 
the  glands  for  various  mechanical  and  chemical  irritants  is  found  to  occur. 

Popielski  3  disputes  the  existence  of  such  an  accommodation  (in 
dogs)  to  the  kind  of  food  and  to  the  kind  of  stimulation.  In  man  an 
accommodation  of  the  salivary  glands,  to  the  needs,  has  also  been  sug- 
gested but  the  statements  are  still  not  unanimous.1  See  also  Chapter 
I  (page  53). 

The  mixed  buccal  saliva  in  man  is  a  colorless,  faintly  opalescent, 
slightly  ropy,  easily  frothing  liquid  without  special  odor  or  taste.  It 
is  made  turbid  by  epithelium  cells,  mucous  and  salivary  corpuscles, 
and  often  by  food  residues.  Like  the  submaxillary  and  parotid  saliva, 
on  exposure  to  the  air  it  becomes  covered  with  an  incrustation  consist- 
ing of  calcium  carbonate  and  a  small  quantity  of  an  organic  substance, 


1  Zeitschr.  f .  Biologie,  23. 

2  Arch,  internat.  de  Physiol.,  1,  1904.  See  also  Boos,  Maly's  Jahresber.,  36.  390, 
and  Neilson  and  Terry,  Amer.  Journ.  of  Physiol.,  15,  as  well  as  the  work  of  Mendel 
and  Underbill,  Journ.  of  biol.  Chem.,  3. 

'Popielski,  Pfluger's  Arch.,  127;  Zebrowski,  Pfluger's  Arch.,  110;  Neilson  and 
Lewis,  Journ.  of  biol.  Chem.,  4,  with  Scheele,  ibid.,  5;  Carlson  and  Chittenden,  Amer.. 
Journ.  of  Physiol.,  2(J. 


MIXED  SALIVA.  455 

or  it  gradually  becomes  cloudy.  Its  reaction  is  generally  alkaline  to 
litmus.  The  degree  of  alkalinity  varies  considerably  not  only  in  dif- 
ferent individuals  but  also  in  the  same  individual  during  different  parts 
of  the  day,  so  that  it  is  difficult  to  state  the  average  alkalinity.  Accord- 
ing to  Chittenden  and  Ely  it  corresponds  to  the  alkalinity  of  0.8  p.  m. 
Na2C03  solution,  or  to  0.2  p.  m.  solution  according  to  Cohn.  According 
to  Foa  the  actual  alkalinity  (OH-ion  concentration)  is  always  consider- 
ably less  than  that  found  by  titration,  and  the  reaction  determined 
electrometrically  is  very  nearly  neutral.  The  reaction  may  also  be  acid, 
as  found  to  be  the  case  by  Sticker  some  time  after  a  meal,  but  this  is 
not  true,  at  least  for  all  individuals.  The  specific  gravity  varies  between 
1.002  and  1.008,  and  the  quantity  of  solids  between  5  and  10  p.  m. 
According  to  Cohn,1  A=  —0.20°  on  an  average,  and  the  amount  of  NaCl 
is  1.6  p.  m.  The  solids,  irrespective  of  the  form-constituents  men- 
tioned, consist  of  protein,  mucin,  oxidases,2  two  enzymes,  ptyalin  and 
maltase,  as  well  as  a  dipeptid  and  a  tripeptid  splitting  enzyme3  and 
mineral  bodies.  It  is  also  claimed  that  urea  is  a  normal  constituent  of 
the  saliva.  The  mineral  bodies  are  alkali  chlorides,  bicarbonates  of 
the  alkalies  and  calcium,  phosphates,  and  traces  of  sulphates,  nitrites, 
ammonia,  and  sulphocyanides,  which  latter  average  about  0.1  p.  in. 
(Munk  and  others).  Smaller  quantities,  0.03-0.04  p.  m.,  are  found  in 
the  saliva  of  non-smokers  (Schneider  and  Kruger),  while  from  ordin- 
ary smokers  the  quantity  of  sulphocyanides  may  rise  to  0.2  p.  m. 
(Fleckseder4). 

Sulphocyanides,  which,  although  not  constant,  occur  in  the  saliva  of 
man  and  certain  animals,  may  be  easily  detected  by  acidifying  the  saliva 
with  hydrochloric  acid  and  treating  with  a  very  dilute  solution  of  ferric 
chloride.  As  control,  especially  in  the  presence  of  very  small  quantities, 
it  is  best  to  compare  the  test  with  another  test-tube  containing  an  equal 
amount  of  acidulated  water  and  ferric  chloride.  Other  methods  have 
been  suggested  by  Gscheidlen,  Solera,  and  Ganassini.  The  quantita- 
tive estimation  can  be  done  according  to  Munk's  5  method. 

Chittenden  and  Ely,  Amer.  Chem.  Journ.,  4,  1883;  Chittenden  and  Richards, 
Amer.  Journ.  of  Physiol.,  1;  Foa,  Compt.  rend.  soc.  biol.,  58;  Sticker,  cited  from 
Centralbl.  f.  Physiol.,  3,  237;  Cohn,  Deutsch.  med.  Wochenschr.,  1900. 

2  Bogdanow-Beresowski,  cited  from  Biochem.  Centralbl.,  2,  653;  Herlitzka,  Maly's 
Jahresber.,  40;  Spanjer-Herford,  Virchow's  Arch.,  205. 

MVarfield,  Johns  Hopkins  Hosp.  Bull.  22  (1911);  Koelker,  Zeitschr.  f.  physiol. 
Chem.,  76,  (1911). 

4  Munk,  Virchow's  Arch.,  69;  Schneider,  Amer.  Journ.  of  Physiol.,  5;  Kruger, 
Zeitschr.  f.  Biologie,  37;  Fleckseder,  Centralbl.  f.  innere  Med.,  1905.  In  regard  to 
the  variation  in  the  amount  of  various  constituents  in  saliva  see  Fleckseder,  1.  c,  and 
Tezner,  Arch,  internat.  de  Physiol.,  2. 

5  Gscheidlen,  Maly's  Jahresber.,  4;  Solera,  see  ibid.,  7  and  8;  Munk,  Virchow's 
Arch.,  69;  Ganassini,  Biochem.  Centralbl.,  2,  p.  361. 


456  DIGESTION. 

Ptyalin,  or  salivary  diastase,  is  the  amylolytic  enzyme  of  the  saliva. 
This  enzyme  is  found  in  human  saliva,1  but  not  in  that  of  all  animals, 
especially  not  in  the  typical  carnivora.  It  occurs  not  only  in  adults, 
but  also  in  new-born  infants.  In  opposition  to  Zweifel's  views,  Ber- 
ger  2  claims  that  it  is  present  not  only  in  the  parotid  gland  of  children, 
but  also  in  the  mucin  glands. 

According  to  H.  Goldschmidt  3  the  saliva  (parotid  saliva)  of  the  horse  does 
not  contain  ptyalin,  but  a  zymogen  of  the  same,  while  in  other  animals  and  man 
the  ptyalin  is  formed  from  the  zymogen  during  secretion.  In  horses  the  zymogen 
is  transformed  into  ptyalin  during  mastication,  and  bacteria  seem  to  give  the 
impulse  to  this  change.  During  precipitation  with  alcohol  the  zymogen  is  changed 
into  ptyalin. 

Ptyalin  has  not  been  isolated  in  a  pure  form  up  to  the  present  time. 
It  can  be  obtained  purest  by  Cohnheim's  4  method,  which  consists  in 
carrying  the  enzyme  down  mechanically  with  a  calcium-phosphate 
precipitate,  and  washing  the  precipitate  with  water,  which  dissolves  the 
ptyalin,  and  from  which  it  can  be  obtained  by  precipitating  with  alcohol. 
For  the  study  or  demonstration  of  the  action  of  ptyalin  one  employs  a 
watery  or  glycerin  extract  of  the  salivary  glands,  or  simply  the  saliva 
itself. 

Ptyalin,  like  other  enzymes,  is  characterized  by  its  action.  This 
consists  in  converting  starch  into  dextrins  and  sugar.  Our  knowledge 
as  to  the  process  going  on  here  is  just  as  uncertain  as  our  knowledge 
on  the  formation  of  sugar  from  starch  (see  page  229).  The  nature  of 
the  sugar  thus  produced  is  known  with  certainty.  For  a  long  time  it 
was  considered  that  glucose  was  the  sugar  formed  from  starch  and 
glycogen,  but  Seegen  and  O.  Nasse  have  shown  that  this  is  not  true. 
Muculus  and  v.  Mering  have  shown  that  the  sugar  formed  by  the 
action  of  saliva,  amylopsin,  and  diastase  upon  starch  and  glycogen  is 
for  the  most  part  maltose.  This  has  been  substantiated  by  Brown 
and  Heron.  E.  Kulz  and  J.  Vogel  5  have  also  demonstrated  that 
in  the  saccharification  of  starch  and  glycogen,  isomaltose,  maltose,  and 
some  glucose  are  formed,  the  varying  quantities  depending  upon  the 
amount  of   ferment   and    the  length  of  its  action.     The  formation  of 


1  In  regard  to  the  variation  in  the  quantity  of  ptyalin  in  human  saliva  see  Hof- 
bauer,  Centralbl.  f.  Physiol.,  10,  and  Chittenden  and  Richards,  Amer.  Journ.  of  Physiol., 
1;  Schiile,  Maly's  Jahresber.,  29;  Tezner,  1.  c. 

2  Zweifel,  Untersuchungen  iiber  den  Verdauungsapparat  der  Neugeborenen  (Berlin, 
1874);  Berger,  see  Maly's  Jahresber.,  30,  399. 

1  Zeitschr.  f.  physiol.  Chem.,  10. 

* Virchow's  Arch.,  28. 

6  Seegen,  Centralbl.  f.  d.  med.  Wissensch.,  1876,  and  Pfliiger's  Arch.,  19;  Nasse, 
ibid.,  14;  Musculus  and  v.  Mering,  Zeitschr.  f.  physiol.  Chem.,  2;  Brown  and  Heron, 
Liebig's  Annal.,  199  and  204;  Kulz  and  Vogel,  Zeitschr.  f.  Biologie,  31. 


PTYALIN.  457 

glucose  is  claimed  by  Tebb,  Rohmann,  and  Hamburger  1  to  be  only  a 
product  of  the  inversion  of  the  maltose  by  the  maltase. 

The  action  of  ptyalin  in  various  reactions  has  been  the  subject  of 
1  umerous  investigations.2  Natural  alkaline  saliva  is  very  active,  but 
it  is  not  so  active  as  when  made  neutral.  It  may  be  still  more  active 
under  certain  circumstances  in  faintly  acid  reaction,  and  according  to 
Chittenden  and  Smith  it  acts  better  when  enough  hydrochloric  acid 
is  added  to  saturate  the  proteins  present  than  when  only  neutralized. 
When  the  acid-combined  protein  exceeds  a  certain  amount,  then  the 
diastatic  action  is  diminished.  The  addition  of  alkali  to  the  saliva 
decreases  its  diastatic  action;  on  neutralizing  the  alkali  with  acid  or 
carbon  dioxide  the  retarding  or  preventive  action  of  the  alkali  is  arrested. 
According  to  Schierbeck,  carbon  dioxide  has  an  accelerating  action  in 
neutral  liquids,  while  Ebstein  claims  that  it  has,  as  a  rule,  a  retarding 
action.  Organic  as  well  as  inorganic  acids,  when  added  in  sufficient 
quantity,  may  stop  the  diastatic  action  entirely.  The  degree  of  acidity 
necessary  in  this  case  is  not  always  the  same  for  a  certain  acid,  but  is 
dependent  upon  the  quantity  of  ferment.  The  same  degree  of  acidity 
in  the  presence  of  large  amounts  of  ferment  has  a  weaker  action  than  in 
the  presence  of  smaller  quantities.  Hydrochloric  acid  is  of  special 
physiological  interest  in  this  regard,  for  it  prevents  the  formation  of 
sugar  even  in  very  small  amounts  (0.03  p.  m.).  Hydrochloric  acid  has 
not  only  the  property  of  preventing  the  formation  of  sugar,  but,  as 
shown  by  Langley,  Nylen,  and  others,  may  entirely  destroy  the 
enzyme.  This  is  important  in  regard  to  the  physiological  significance 
of  the  saliva. 

Foreign  substances,  such  as  metallic  salts,3  have  different  effects. 
Certain  salts,  even  in  small  quantities,  completely  arrest  the  action; 
for  example,  HgCl2  accomplishes  this  result  completely  in  the  presence 
of  only  0.05  p.  m.  Others  have  an  accelerating  action,  and  this  seems 
to  apply  to  the  salts  of  the  saliva.  According  to  Guyenot  the  saliva 
has  a  weaker  action  the  more  it  is  freed  from  salts  by  dialysis.     On  the 


1  Tebb,  Journ.  of  Physiol.,  15;  Rohmann,  Ber.  d.  deutsch.  chem.  Gesellsch.,  27; 
Hamburger,  Pfluger's  Arch.,  60. 

2  See  Hammarsten,  Maly's  Jahresber.,  1;  Chittenden  and  Griswold,  Amer.  Chem. 
Journ.,  3;  Langley,  Journal  of  Physiol.,  3;  Nylen,  Maly's  Jahresber.,  12,  241;  Chit- 
tenden and  Ely,  Amer.  Chem.  Journ.,  4;  Langley  and  Eves,  Journal  of  Physiol.,  4: 
Chittenden  and  Smith,  Yale  College  Studies,  1,  1885,  1;  Schlesinger,  Virchow's  Arch. 
125;  Schierbeck,  Skand.  Arch.  f.  Physiol.,  3;  Ebstein  and  C.  Schulze,  Virchow's 
Arch.,  134;  Kubel,  Pfluger's  Arch.,  56. 

3  See  O.  Nasse,  Pfluger's  Arch.,  11,  and  Chittenden  and  Painter,  Yale  College 
Studies,  1,  1885,  52;  Kubel,  Pfluger's  Arch.,  76;  Patten  and  Stiles,  Amer.  Journ.  of 
Physiol.,  17. 


458 


DIGESTION. 


addition  of  salts  the  dialyzed  saliva  becomes  active  again,  especially 
on  the  addition  of  calcium  or  potassium  chloride  (see  also  page  71). 
Roger1  believes  that  the  presence  of  phosphates  is  a  necessity  for  the 
action  of  saliva.  The  amount  of  salts  added  is  of  special  importance 
for  the  action  of  the  saliva,  and  one  salt,  which  in  small  quantities  has 
an  accelerating  action,  may  in  large  quantities  have  a  retarding  action. 
The  presence  of  peptone  has  an  accelerating  action  on  the  sugar  forma- 
tion (Chittenden  and  Smith  and  others). 

To  show  the  action  of  saliva  or  ptyalin  on  starch  the  three  ordinary 
tests  for  glucose  may  be  used,  namely,  Moore's  or  Trommer's  test  or 
the  bismuth  test  (see  Chapter  III).  It  is  also  necessary,  as  a  control, 
to  first  test  the  starch-paste  and  the  saliva  for  the  presence  of  glucose. 
The  steps  in  the  transformation  of  starch  into  amidulin,  erythrodextrin, 
and  achroodextrin  may  be  shown  by  testing  with  iodine. 

Maltase  occurs  in  saliva  to  only  a  slight  extent.  It  converts  maltose 
into  glucose.  According  to  Sticker,2  saliva  also  has  the  power  of  split- 
ting sulphureted  hydrogen  from  the  sulphur  oils  of  radishes,  onions, 
and  certain  other  vegetables. 

The  quantitative  composition  of  the  mixed  saliva  must  vary  consider- 
ably, not  only  because  of  individual  differences,  but  also  because  under 
varying  conditions  there  may  be  an  unequal  division  of  the  secretion 
from  the  different  glands.  We  give  herewith  a  few  analyses  of  human 
saliva  as  examples  of  its  composition.     The  results  are  in  parts  per  1000. 


as 
D 

B 
M 
PS 

W 

pa 

a 

m 

O 

m 
P 
o 
< 

m 
W 

o 

5 
s 
a 

13 

a 
a 

z  . 

s  J 
"a 
sO 
H 

a 

H 

« 
B 
W 

55 
Z 
<! 

a 
« 

H 

i  « 

K  a 
S  W 

<  « 

Water 

992.9 
7.1 

1.4 
3.8 

995 . 16 
4.84 

1.62 
1.34 

994.1 
5.9 

2.13 
1.42 

988.3 
11.7 

994.7 
5.3 

3.5-8.4 

in 
filtered 
saliva. 

994.2 

Solids.              

5.8 

Mucus  and  epithelium .... 
Soluble  organic  substances. 

2.2 

3.27 

1.4 

(Ptyalin  of  early  investigators.) 

0.06 

0.10 

0.064 

0.04 

Salts 

1.9 

1.82 

2.19 

1.30 

to 
0.090 

2.2 

1  Guyenot,  Compt.  rend.  soc.  biol.,  63;    Roger,  ibid.,  65;    see  also  Bang,  Bioch. 
Zeitechr.,  32  (1911). 

2  Miinch.  med.  Wochenschr.,  43. 

»  Zeitechr.  f.  physiol.  Chem.,  5.     The  other  analyses  are  cited  from  Maly,  Chemie 
der  Verflauvmgssafte,  Hermann's  Handbuch  d.  Physiol.,  5,  Part  II,  14. 


SECRETION  OF  SALIVA.  459 

Hammerbacher  found  in  1000  parts  of  the  ash  from  human  saliva:  potash 
457.2,  soda  95.9,  iron  oxide  50.11,  magnesia  1.55,  sulphuric  anhydride  (S03)  G3.8, 
phosphoric  anhydride  (PiO»)  188.48,  and  chlorine  183.52. 

The  quantity  of  saliva  secreted  during  twenty-four  hours  cannot 
be  exactly  determined,  but  has  been  calculated  by  Bidder  and  Schmidt 
to  be  1400-1500  grams.  The  most  abundant  secretion  occurs  during 
meal-times.  According  to  the  calculations  and  determinations  of 
Tuczek,1  in  man  1  gram  of  gland  yields  13  grams  of  secretion  in  the  course 
of  one  hour  during  mastication.  These  figures  correspond  fairly  well 
with  those  representing  the  average  secretion  from  1  gram  of  gland  in 
animals,  namely,  14.2  grams  in  the  horse  and  8  grams  in  oxen.  The 
quantity  of  secretion  per  hour  may  be  8  to  14  times  greater  than  the  entire 
mass  of  glands,  and  there  is  probably  no  gland  in  the  entire  body,  so  far 
as  is  known  at  present — the  kidneys  not  excepted — whose  ability  of 
secretion  under  physiological  conditions  equals  that  of  the  salivary  glands. 
But  as  the  secretion  of  saliva  is  so  very  variable  under  different  con- 
ditions no  positive  results  can  be  given  as  to  the  extent.  A  remark- 
ably abundant  secretion  of  saliva  is  induced  by  pilocarpine,  while 
atropine,  on  the  contrary,  inhibits  it. 

That  the  secretion  of  saliva,  even  if  we  do  not  consider  such  sub- 
stances as  ptyalin,  mucin,  and  the  like,  is  not  a  process  of  filtration, 
follows  for  many  reasons,  especially  the  following:  The  salivary  glands 
have  a  specific  property  of  eliminating  certain  substances,  such  as 
potassium  salts  (Salkowpki  2),  iodine,  and  bromine  compounds,  but 
not  others,  for  example,  iron  compounds  and  glucose.  It  is  also  notice- 
able that  the  saliva  is  richer  in  solids  when  it  is  eliminated  quickly  by 
gradually  increased  stimulation,  and  in  larger  quantities  than  when  the 
secretion  is  slower  and  less  abundant  (Heidenhain).  The  amount  of 
salts  increases  also  to  a  certain  degree  by  an  increasing  rapidity  of 
elimination  (Heidenhain,  Werther,  Langley  and  Fletcher,  Novi3). 

Like  the  secretion  processes  in  general,  the  secretion  of  saliva  is 
closely  connected  with  the  processes  in  the  cells.  The  chemical  processes 
going  on  in  these  cells  during  secretion  are  still  unknown. 

The  Physiological  Importance  of  the  Saliva. — The  quantity  of  water 
in  the  saliva  renders  possible  the  action  of  certain  bodies  on  the  organs 
of  taste,  and  it  also  serves  as  a  solvent  for  a  part  of  the  nutritive  sub- 
stances. The  importance  of  the  saliva  in  mastication  is  especially 
marked  in  herbivora,  and  there  is  no  question  as  to  its  importance  in 

1  Bidder  and  Schmidt,  1.  c,  13;  Tuczek,  Zeitschr.  f.  Biologie,  12. 

2  Virchow's  Arch.,  53. 

1  Heidenhain,  Pfluger's  Arch.,  17;  Werther,  ibid.,  38;  Langley  and  Fletcher, 
Proc.  Roy.  Soc,  45,  and  especially  Phil.  Trans.  Roy.  Soc.  London,  180;  Novi,  Arch, 
f.  (Anat.  u.)  Physiol.,  1888. 


460  DIGESTION. 

facilitating  the  act  of  swallowing.  The  saliva  containing  mucin  is  espe- 
cially important  in  this  regard,  and  Pawlow's  school  has  shown  that  the 
secretion  also  regulates  itself  in  this  regard.  The  saliva  is  also  of  import- 
ance, as  it  serves  in  washing  out  the  mouth  and  thereby  acts  as  a  pro- 
tection against  destructive  substances  or  bodies  foreign  to  the  mouth. 
The  power  of  converting  starch  into  sugar  is  not  inherent  in  the  saliva 
of  all  animals,  and  even  when  it  possesses  this  property  the  intensity 
varies  in  different  animals.  In  man,  whose  saliva  forms  sugar  rapidly, 
a  production  of  sugar  from  (boiled)  starch  undoubtedly  takes  place  in  the 
mouth,  but  how  far  this  action  proceeds  after  the  morsel  has  entered  the 
stomach  depends  upon  the  rapidity  with  which  the  acid  gastric  juice  mixes 
with  the  swallowed  food,  and  also  upon  the  relative  amounts  of  the 
gastric  juice  and  food  in  the  stomach.  The  large  quantity  of  water  which 
is  swallowed  with  the  saliva  must  be  absorbed  and  pass  into  the  blood, 
and  it  must  in  this  way  go  through  an  intermediate  circulation  in  the 
organism.  Thus  the  organism  possesses  in  the  saliva  an  active  medium 
by  which  a  constant  stream,  conveying  the  dissolved  and  finely  divided 
bodies,  passes  into  the  blood  from  the  intestinal  canal  during  digestion. 
The  relation  of  the  saliva  or  the  salivary  glands  to  the  secretion  of  gastric 
juice  will  be  mentioned  in  the  next  section. 

Salivary  Concrements.  The  so-called  tartar  is  yellow,  gray,  yellowish-gray, 
brown  or  black,  and  has  a  stratified  structure.  It  may  contain  more  than  200- 
p.  m.  organic  substances,  which  consist  of  mucin,  epithelium,  and  leptothrix- 
chains.  The  chief  part  of  the  inorganic  constituents  consists  of  calcium  car- 
bonate and  phosphate.  The  salivary  calculi  may  vary  in  size  from  that  of  a 
small  grain  to  that  of  a  pea  or  still  larger  (a  salivary  calculus  has  been  found 
weighing  18.6  grams),  and  they  contain  variable  quantities  of  organic  substances 
(50-380  p.  m.),  which  remain  on  extracting  the  calculus  with  hydrochloric  acid. 
The  chief  inorganic  constituent  is  calcium  carbonate. 

H.    THE  GLANDS  OF  THE  MUCOUS   MEMBRANE  OF  THE  STOMACH,  AND 

THE   GASTRIC  JUICE. 

The  glands  of  the  mucous  coat  of  the  stomach  have  long  been 
divided  into  two  distinct  classes.  Those  which  occur  in  the  greatest 
abundance  and  which  have  the  greatest  size  in  the  fundus  are  called 
fundus,  rennin  or  pepsin  glands,  and  the  others,  which  occur  onl}r  in 
the  neighborhood  of  the  pylorus,  have  received  the  name  of  pyloric 
glands,  sometimes  also,  though  incorrectly,  called  mucous  glands.  The 
division  of  these  two  forms  of  glands  in  the  mucous  membrane  of  the 
stomach  is  essentially  different  in  various  animals.  The  mucous  coat- 
ing of  the  stomach  is  covered  throughout  with  a  layer  of  columnar 
epithelium,  which  is  generally  considered  as  consisting  of  goblet  cells  that 
produce  mucus  by  a  metamorphosis  of  the  protoplasm. 


GASTRIC  JUICE.  461 

The  fundus  glands  contain  two  kinds  of  cells:  adelomorphic  or  chief 
cells,  and  delomorphic  or  cover  cells,  the  latter  formerly  called  rennin 
or  pepsin  cells.  Both  kinds  consist  of  protoplasm  rich  in  proteins; 
but  their  relation  to  coloring-matters  seems  to  show  that  the  protein 
substances  of  both  are  not  identical.  The  nucleus  must  consist  princi- 
pally of  nuclein.  Besides  the  above-mentioned  constituents,  the  fundus 
glands  contain  as  more  specific  constituents  several  enzymes  or  their 
zymogens,  besides  a  little  fat  and  cholesterin. 

The  pyloric  glands  contain  cells  which  are  generally  considered  as 
related  to  the  above-mentioned  chief  cells  of  the  fundus  glands.  As 
these  glands  were  formerly  thought  to  contain  a  larger  quantity  of 
mucin,  they  were  also  called  mucous  glands.  According  to  Heiden- 
hain,  independent  of  the  columnar  epithelium  of  the  excretory  ducts, 
they  take  no  part  worthy  of  mention  in  the  formation  of  mucus,  which 
according  to  his  views  is  effected  by  the  epithelium  covering  the  mucous 
membrane.  The  pyloric  glands  also  seem  to  contain  zymogens.  Alkali 
chlorides,  alkali  phosphates,  and  calcium  phosphates  are  found  in  the 
mucous  coating  of  the  stomach. 

The  Gastric  Juice.  The  observations  of  Helm  and  Beaumont  on 
persons  with  gastric  fistula  led  to  the  suggestion  that  gastric  fistulas 
be  made  on  animals,  and  this  operation  was  first  performed  by  Basso w  l 
in  1842  on  a  dog.  Verneuil  performed  the  same  on  a  man  in  1876  with 
successful  results.  Pawlow  2  has  recently  improved  the  surgery  of 
gastric  fistula  and  has  added  much  to  the  study  of  gastric  secretion. 

As  most  investigations  upon  gastric  digestion,  and  also  upon  diges- 
tion as  a  whole,  are  based  on  observations  upon  dogs  and  then  upon  man, 
and  for  this  reason,  when  not  otherwise  stated,  in  this  chapter  on  the 
study  of  digestion  we  give  the  conditions  in  dogs  and  man. 

The  secretion  of  gastric  juice  is  not  continuous,  at  least  in  man  and 
in  the  mammals  experimented  upon.  It  only  occurs  under  psychic 
influence,  and  also  by  stimulation  of  the  mucous  membrane  of  the  stomach 
or  the  intestine.  The  most  exhaustive  researches  on  the  secretion  of 
gastric  juice  (in  dogs)  have  been  made  by  Pawlow  and  his  pupils. 

In  order  to  obtain  gastric  juice  free  from  saliva  and  food  residues,  they  arranged 
besides  a  gastric  fistula  also  an  oesophageal  fistula  from  which  the  swallowed  food 
could  be  withdrawn  with  the  saliva  without  entering  the  stomach,  and  in  this 
manner  an  apparent  or  sham  feeding  was  possible.    In  this  way  it  was  possible  to 

1  Helm,  Zwei  Krankengeschichten,  Wien,  1803,  cited  from  Hermann's  Handbuch, 
5,  part  II,  39;  Beaumont,  "The  Physiology  of  Digestion,"  1833;  Bassow,  Bull,  de 
la  eoc.  des  natur.  de  Moscou,  16,  cited  from  Maly  in  Hermann's  Handbuch,  5,  38; 
Verneuil,  see  Ch.  Richet,  "Du  sue  gastrique  chez  l'homme,"  etc.  (Paris,  1878),  158. 

2  Pawlow,  The  Work  of  the  Digestive  Glands,  (translated  by  Thompson,  Phila- 
delphia, 1910),  where  the  works  of  his  pupils  are  also  mentioned.  See  also  Ergebnisse 
der  Physiologie,  1,  Abt.  1. 


462  DIGESTION. 

study  the  influence  of  psychical  moments  on  one  side  and  the  direct  action  of  food 
on  the  mucous  membrane  on  the  other.  After  a  method  suggested  by  Heidenhain 
and  later  improved  by  Pawlow  and  Chigin,  they  have  succeeded  in  preparing 
a  blind  sac  by  partial  dissection  of  the  fundus  part  of  the  stomach,  and  the  secre- 
tion processes  could  be  studied  in  this  sac  while  the  digestion  in  the  other  parts 
of  the  stomach  was  going  on.  In  this  way  they  were  able  to  study  the  action  of 
different  foods  on  the  secretion. 

The  most  essential  results  of  the  investigations  of  Pawlow  and  his 
pupils  are  as  follows:  Mechanical  stimulation  of  the  mucosa  does  not 
produce  any  secretion.  Mechanical  irritation  of  the  mucous  membrane 
of  the  mouth  causes  no  reflex  excitation  of  the  secretory  nerves  of  the 
stomach.  There  are  two  moments  which  cause  a  secretion,  namely, 
the  psychical  moment — the  passionate  desire  for  food  and  the  sensa- 
tion of  satisfaction  and  pleasure  on  partaking  it — and  the  chemical 
moment,  the  action  of  certain  chemical  substances  on  the  mucous  mem- 
brane of  the  stomach.  The  first  moment  is  the  most  important.  The 
secretion  occurring  under  its  influence  by  the  vagus  fibers  appears  earlier 
than  that  produced  by  chemical  irritants,  but  only  after  an  interval  of 
at  least  4^  minutes.  This  secretion  is  more  abundant  but  less  contin- 
uous than  the  "  chemical."  It  yields  a  more  acid  and  active  juice  than 
the  latter.  As  chemical  excitants  which  cause  a  secretion  reflexively 
through  the  stomach  mucosa  we  include  water  (slight  action)  and  cer- 
tain unknown  extractive  substances  contained  in  meat  and  meat  extracts, 
in  impure  peptone,  and  also,  it  seems,  in  milk.  According  to  Herzen 
and  Radzikowski  1  and  others,  alcohol  is  also  a  strong  agent  in  produc- 
ing a  flow  of  juice.  The  claims  in  regard  to  the  action  of  sodium  chloride 
and  alkali  carbonates  are  somewhat  disputed.  That  the  alkali  carbonates 
retard  or  inhibit  secretion  is  the  opinion  of  many,  but  from  more 
recent  determinations  2  it  would  seem  as  if  the  concentration  of  the  car- 
bonate as  well  as  of  sodium  chloride  exercises  a  certain  influence,  so  that 
a  weaker  concentration  is  indifferent  or  retarding,  while  somewhat  stronger 
concentration  has  an  accelerating  action  upon  secretion,  though  inves- 
tigators are  not  agreed  as  to  results.  Bitter  substances  partaken  of  in 
small  amounts  a  certain  time  before  a  meal  increase  the  secretion,  while 
larger  amounts  have  a  retarding  action  (Bomssow,  Strashesko3). 
Fats  have  a  retarding  action  on  the  appearance  of  secretion  and  diminish 
the  quantity  of  juice  secreted  as  well  as  the  amount  of  enzyme.  The 
substances,  such  as  egg-albumin,  which  do  not  act  as  chemical  stimulants, 


1  Pfluger's  Arch.,  84,  513. 

2  See  Rozenblatt,  Bioch.  Zeitschr,  4;  Mayeda,  ibid.,  2;  Pimenow,  Bioch,  Centralbl., 
6;  Lonnquist,  Maly's  Jahresb.,  36. 

'  Borissow,  Arch.  f.  exp.  Path.  u.  Pharm.,  51;  Strashesko,  see  Biochem.  Centralbl., 
4,  148. 


GASTRIC  JUICE.  463 

may  be  digested  by  the  "  psychical  "  secretion,  and  then  perhaps  cause 
a  chemical  secretion  by  their  decomposition  products. 

The  secretion  in  the  stomach  may  also  be  influenced  by  the  small 
intestine,  and  in  this  way,  as  shown  by  the  investigations  of  Pawlow 
and  his  pupils,  the  fats  have  a  retarding  action  upon  the  secretion  of 
juice  and  upon  digestion  by  acting  reflexly  upon  the  duodenal  mucosa. 
In  dogs  on  feeding  fat  (oil)  with  food  containing  starch,  the  secretion  of 
gastric  juice  remains  reduced  during  the  entire  period  of  feeding,  and 
Cat  in  connection  with  protein  food  has  a  similar  action,  with  the  excep- 
tion that  in  this  case  the  retarding  action  is  observed  only  in  the  first 
hours  of  digestion.  According  to  Piontkowski  x  the  oil-soaps  differ  from 
the  neutral  fats  by  having  a  strong  action  on  the  flow  of  juice,  and  this 
is  the  reason  why  about  five  to  six  hours  after  a  meal  with  fat  the  secre- 
tion of  juice  is  stopped,  as  just  at  this  time  the  soaps  are  being  formed. 
According  to  Frouin  the  food  in  the  intestine  produces  a  secretion  of 
gastric  juice  which  continues  after  the  action  of  the  psychic  moment 
has  ceased.  Lecoxte  -  arrived  at  similar  results,  and  he  ascribes  a  less 
subordinate  importance  to  the  chemical  secretion  as  compared  with  the 
psychic  secretion,  than  Pawlow  does. 

The  behavior  of  the  different  parts  of  the  stomach  in  secretion  is 
also  of  interest.  The  work  of  Pawlow  and  his  pupils  Gross  and 
Krshyschkowsky,  has  shown  that  meat  and  its  extractives  as  well  as 
the  digestion  products  and  milk  especially  act  upon  the  pyloric  partr 
although  not  entirely,  while  they  are  inactive  upon  the  fundus.  Alcohol 
also  acts  upon  the  fundus  part.  Popielski  3  found  that  meat  extracts 
had  an  exciting  action  upon  the  secretion  of  gastric  juice,  even  when  intro- 
duced subcutaneously.  In  close  relation  to  what  has  been  said  above 
stands  the  observation  of  Edkins  that  the  pylorus  part  of  the  stomach 
contains  a  substance,  a  prosecreti?i,  which  by  acids  and  certain  other 
substances  is  transformed  into  a  secretin,  which  when  introduced  into 
the  blood  circulation  causes  a  secretion  of  gastric  juice.  Hemmeter, 
claims  that  a  secretin  for  the  secretion  of  gastric  juice  is  also  produced 
in  the  salivary  glands.  The  extirpation  of  all  the  salivary  glands  in 
dogs  causes  a  marked  diminution  in  the  secretion  of  gastric  juice,  while 
the  intravenous  or  peritioneal  injection  of  an  extract  of  the  salivary 
glands  of  dogs  produces  a  secretion  of  gastric  juice.  Emsmann4  has 
also  obtained  bodies  having  a  similar  action,  from   the  mucosa  of  the 

1  See  Biochem.  Centralbl.,  3,  660. 

2  Frouin,  Compt.  rend.  soe.  biol.,  53;  Leconte,  La  Cellule,  17. 

3  Gross,  Bioch.  Centralbl.,  5,  669;    Krshyschkowsky,  Maly's    Jahresb.,  36,  403; 
Popielski,  ibid..  39. 

*  Edkins,   Journ.   of  Physiol.,   34;    Hemmeter,   Bioch.   Zeitschr.,    11;    Emsmannr 
Intern.  Beitr.  zu.  Path.  u.  Ther.  d.  Ernahrungs  storungen  3  (1911). 


464  DIGESTION. 

duodenum,  jejunum,  and  ileum  as  well   as  from  the  liver  and  pancreas 
by  hydrochloric  acid. 

We  know  very  little,  positively,  in  regard  to  the  gastric  secretion  in 
man.  According  to  the  earlier  authorities  the  irritants  may  be  mechan- 
ical, thermic,  and  chemical.  Among  the  chemical  excitants  we  include 
alcohol  and  ether,  which  in  too  great  a  concentration  bring  about  no 
physiological  secretion,  but  rather  the  transudation  of  a  neutral  or 
faintly  alkaline  fluid.  Certain  acids,  such  as  carbonic  acid,  neutral 
salts,  meat  extracts,  spices,  and  other  bodies  also  belong  to  this  group. 
The  reports  on  this  subject  are  unfortunately  very  uncertain  and  con- 
tradictory. 

The  question  as  to  how  far  the  observations  made  by  PAWLOwand 
his  school  can  be  applied  to  man  is  of  special  interest.  Many  observa- 
tions on  this  question  have  been  collected  1  and  they  compare  favor- 
ably with  the  observations  made  upon  dogs.  Thus  in  man  a  psychic 
secretion  of  gastric  juice  can  be  brought  about,  and  it  has  also  fceen 
observed  that  it  can  be  stopped  by  emotions.  As  in  dogs,  so  also  in  man, 
after  sham  feeding,  a  secretion  takes  place  after  a  pause,  the  duration 
of  which  varies  in  different  cases.  In  some  cases,  as  in  dogs  after  meat 
feeding,  the  pause  was  about  five  minutes.  The  chewing  of  indifferent 
bodies  did  not  affect  the  glands,  while  bodies  acting  upon  the  organs  of 
smell  and  taste  had  an  exciting  action.  Umber  observed  besides  this,  that 
after  the  introduction  of  a  nutritive  enema  into  the  rectum,  a  secretion 
of  gastric  juice  was  produced  by  reflex  action. 

From  these  observations  of  Hornborg  and  Umber,  as  well  as  from 
some  earlier  observations  of  Schule,  Troller,  Riegel,  and  Scheuer,2 
we  conclude  that  in  man  the  psychic  secretion  is  much  less  than  that 
produced  by  the  introduction  of  food  or  bodies  having  a  pleasant  taste. 
That  the  preparation  of  the  food  in  the  mouth  has  an  essential  influence 
upon  the  secretion  is  proved  without  doubt,  but  we  do  not  agree  as  to 
how  this  action  takes  place.  Certain  experimenters  consider  the  secreted 
and  swallowed  saliva  as  the  most  essential  factor  in  this  action,  while 
others  believe  that  the  act  of  chewing,  and  still  others  that  the  chemical 
action  and  the  sense  of  taste,  are  the  most  important. 

In  regard  to  the  action  of  saliva,  Hemmeter  finds  that  after  the 
extirpation  of  the  salivary  glands,  the  introduction  into  the  stomach 
of  chewed  food  soaked  with  dog-saliva,  has  no  special  action  upon  the 


1  Hornborg,  Maly's  Jahresb.,  33,  547;  Umber,  Berl.  klin.  Wochenschr.,  1905; 
Cade  and  Latarjet,  Compt.  rend.  soe.  biol.,  57;  Kaznelson,  Pfliiger's  Arch.,  118; 
Bogen,  ibid.,  117;  Bickel,  Deutsch.  med.  Wochenschr.,  32,  and  Maly's  Jahresb.,  36, 
411.     See  also  Maly's  Jahresb.  39,  40,  and  Bioch,  Centralbl.  12. 

'The  literature  may  be  found  in  Umber's  v.ork,  1.  c. 


COMPOSITION  OF  THE  GAS1RIC  JUICE.  465 

secretion  of  juice.  On  the  other  hand  Frouin  1  observed  that  the  intro- 
duction of  saliva  into  the  large  stomach  of  dogs,  acts  favorably  upon 
the  secretion  in  the  small  stomach  (see  page  462),  and  the  acidity  as  well 
as  the  digestive  activity  of  the  juice  is  increased.  This  action  does  not 
depend,  according  to  Frouin,  upon  the  alkali  of  the  saliva. 

The  Qualitative  and  Quantitative  Composition  of  the  Gastric  Juice. 
The  human  gastric  juice,  which  can  seldom  be  obtained  pure  and  free 
from  residues  of  the  food  or  from  mucus  and  saliva,  is  a  clear,  or  only 
very  faintly  cloudy,  and  nearly  colorless  fluid  of  an  insipifcl,  acid  taste 
and  strong  acid  reaction.  It  contains,  as  form-elements,  glandular  cells 
or  their  nuclei,  and  more  or  less  changed  columnar  epithelium. 

The  acid  reaction  of  the  gastric  juice  depends  on  the  presence  of  free 
acid,  whith,  as  has  been  learned  from  the  investigations  of  C.  Schmidt, 
Richet,  and  others,  consists,  when  the  gastric  juice  is  pure  and  free 
from  particles  of  food,  chiefly  or  in  large  part  of  hydrochloric  acid.  Con- 
tejean 2  regularly  found  traces  of  lactic  acid  in  the  pure  gastric  juice 
of  fasting  dogs.  After  partaking  of  food,  especially  after  a  meal  rich  in 
carbohydrates,  lactic  acid  occurs  abundantly,  and  sometimes  acetic 
and  butyric  acids.  In  new-born  dogs  the  acid  of  the  stomach  is  lactic 
acid,  according  to  Gmelin.3  The  quantity  of  free  hydrochloric  acid  in 
the  gastric  juice  is,  according  to  Pawlow  and  his  pupils,  in  dogs  5-6 
p.  m.,  and  in  cats  an  average  cf  5.20  p.  m.  HO.  In  man  the  results 
obtained  are  variable  but  regularly  much  lower.  Since  it  has  been 
possible  to  obtain  pure  human  gastiic  juice  for  investigation  it  has  been 
found  (Umber,  Hornborg,  Bickel,  Sommerfeld  4)  that  the  amount 
of  hydrochloric  acid  is  about  4-5  p.  m.  There  is  hardly  any  doubt  that 
at  least  a  part  of  the  hydrochloric  acid  of  the  gastric  juice  does  not 
exist  free  in  the  ordinary  sense,  but  combined  Avith  organic  substances. 
The  results  obtained  in  testing  for  the  acidity  of  gastric  juice  by  phys- 
ical methods  are  almost  identical  with  those  obtained  by  titration  (P. 
Franc  kel5). 

The  specific  gravity  of  gastric  juice  is  low,  1.001-1.010.  It  is  corre- 
spondingly poor  in  solids.  Earlier  analyses  of  gastric  juice  from  man, 
the  dog,  and  the  sheep  were  made  by  C.  Schmidt.6     As  these  analyses 

1  Compt.  rend.  soc.  biol.,  62. 

2  Bidder  and  Schmidt,  Die  Verdauungssafte,  etc.,  44;  Richet,  1.  c;  Contejean,  Con- 
tributions a  l'etude  de  la  physiol.  de  l'estomac,  Theses,  Paris,  1892. 

3  Pfliiger's  Arch.,  90  and  103. 

4  See  Richet,  1.  c;  Contejean,  1.  c;  Verhaegen,  "La  Cellule,"  1896  and  1897; 
Sommerfeld,  Bioch,  Zeitschr,  9,  and  also  footnote  1,  page  464,  and  the  literature  on 
the  estimation  of  hydrochloric  acid  in  the  gastric  juice  contents  (p.  489)  ;  see  also 
Cohnheim  and  Dreyfus,  Zeitschr.  f.  physiol.  Chem.  58  (1908). 

5  Zeitschr,  f.  exp.  Path.  u.  Therap.,  1. 
6i.  c. 


466  DIGESTION.      . 

refer  only  to  impure  gastric  juice  they  are  of  little  value.  Rosemann,1 
who  has  investigated  the  gastric  juice  secreted  by  a  dog  after  sham 
feeding,  found  an  average  of  4.22  p.  m.  solids,  among  which  1.32  p.  m. 
were  mineral  bodies  and  about  2.90  p.  m.  organic  substance.  The 
amount  of  nitrogen  in  one  case  was  0.36  p.  m.,  in  another  0.54  p.  m.  and 
the  quantity  of  HC1  was  about  5.6  p.  m.  The  ash  consisted  chiefly  of 
potassium  chloride,  namely  980-990  p.  m.  of  the  inorganic  part.  Nencki 
and  Sieber2  found  3.06  p.  m.  solids  in  the  pure  gastric  juice  of  a  dog. 
Nencki  3  found  5  milligrams  sulphocyanic  acid  per  liter  of  gastric  juice 
of  a  dog. 

In  the  ash  of  human  gastric  juice  after  sham-feeding  Albu4  found 
356.2  p.  m.  K20;  226.5  p.  m.  Na20,  and  497.3  p.  m.  CI.  The  amount 
of  salts  insoluble  in  water  was  23.9  p.  m.  In  hyperacidity  he  found 
almost  the  same  composition. 

Besides  the  free  hydrochloric  acid,  pepsin,  rennin,  and  a  lipase  are 
the  other  physiologically  important  constituents  of  gastric  juice. 

Pepsin.  This  enzyme  is  found,  with  the  exception  of  certain  fishes, 
in  all  vertebrates  thus  far  investigated. 

Pepsin  occurs  in  adults  and  in  new-born  infants.  This  condition 
is  different  in  new-born  animals.  While  in  a  few  herbivora,  such  as  the 
rabbit,  pepsin  occurs  in  the  mucous  coat  before  birth,  this  enzyme  is 
entirely  absent  at  the  birth  of  those  carnivora  which  have  thus  far  been 
examined,  such  as  the  dog  and  cat. 

In  various  invertebrates  enzymes  have  also  been  found  which  have 
a  proteolytic  action  in  acid  solutions.  It  has  been  shown  that  these 
enzymes,  nevertheless,  are  not  in  all  animals  identical  with  ordinary 
pepsin.  According  to  Klug  and  Wr6blewski5  the  pepsins  found 
in  man  and  various  higher  animals  are  somewhat  different,  an  observa- 
tion which  according  to  the  experience  of  Hammarsten  is  very  prob- 
able. Enzymes  also  occur  in  various  plants  and  animal  organs,  although 
not  identical  with  pepsin,  but  which  act  in  acid  reaction.  The  enzyme 
obtained  from  the  Nepenthes,  which  dissolves  proteins  only  in  acid 
reaction,  stands  very  close  to  pepsin.  An  enzyme  more  closely  related 
to 'trypsin  or  erepsin  (see  sections  III  and  IV)  is,  on  the  contrary, 
Glaessner's  pseudopepsin,  which  according  to  him  is  the  only  peptic 
enzyme  in  the  pyloric  end.  Pseudopepsin,  whose  existence  is  disputed 
by  Klug,  while  others  (Reach,  Pekelharing)  affirm  its  occurrence  in 


1  Pfliiger's  Arch.,  118. 

2  Zeitschr.  f.  physiol.  Chem.,  32. 

3  Ber.  d.  d.  Chem.  Gesellsch.,  28. 

4  Zeitschr.  f.  Path.  u.  Therap.,  5. 

6  Klug.  Pfliiger's  Arch.  60;  Wr6blewski,  Zeitschr.  f.  physiol.  Chem.,  21. 


PEPSIN.  467 

the  mucous  membrane,  cannot,  according  to  Hammarsten,  either  be  the 
only  or  the  most  prominent  peptic  enzyme  of  the  pyloric  part.  According 
to  Glaessner,  it  also  acts  in  neutral  and  alkaline  reaction  and  yields 
tryptophane  among  other  cleavage  products.  According  to  Bergmann  l 
it  is  identical  with  erepsin  (see  below).  Among  the  enzymes  of  the 
mucosa  of  the  stomach  belongs  the  so-called  antipepsin  discovered  by 
Weinland,2  which  has  a  retarding  action  upon  pepsin  digestion  and, 
as  some  claim,  prevents  the  self-digestion  of  the  mucous  membrane. 

Pepsin  is  as  difficult  to  isolate  in  a  pure  condition  as  are  other 
enzymes.  The  pepsin  prepared  by  Brucke  and  Sundberg  gave  negative 
results  with  most  reagents  for  proteins,  and  showed  nevertheless  a 
powerful  action,  which  seems  to  indicate  that  it  was  very  pure.  Schou- 
mow-Simanowski,  Nencki  and  Sieber,  have  designated  as  the  true 
enzyme  the  substance  containing  chlorine,  which  they  obtain  by  strongly 
cooling  the  gastric  juice.  That  this  precipitate  is  not  a  chemical  indi- 
vidual, and  hence  cannot  be  pepsin,  follows  from  the  investigations  of 
Pekelharing.  While  pepsin,  according  to  Nencki  and  Sieber,  was 
rich  in  phosphorus  and  contained  nucleoprotein,  Pekelharing's  pepsin 
was  free  from  phosphorus  and  yielded  no  nucleoprotein.  Friedenthal 
and  Miyamota  3  have  also  shown  that  the  pepsin  is  still  active  after 
the  splitting  off  of  the  nuclein  complex  (and  also  the  protein).  As  pepsin 
is  readily  precipitated  with  the  proteins  and  combines  therewith,  it  is 
difficult  to  decide  whether  pepsin  is  a  protein  substance  or  not,  and  the 
question  as  to  its  nature  is  still  undecided,  just  as  is  the  case  with  other 
enzymes.  As  ordinarily  known,  pepsin,  at  least  in  an  impure  form,  is 
soluble  in  water  and  glycerin.  It  is  precipitated  by  alcohol,  but  is  only 
slowly  destroyed  thereby.  In  aqueous  solution  its  action  is  quickly 
destroyed  on  heating  to  boiling.  According  to  Biernacki  4  pepsin 
in  neutral  solutions  is  destroyed  by  heating  to  55°  C.  In  the  dry  state 
it  can  be  heated  to  over  100°  C.  without  losing  its  activity.  In  the 
presence  of  2  p.  m.  HC1  a  temperature  of  55°  C.  is  not  injurious,  and  the 
compound  with  acid  is  more  resistant  than  the  free  pepsin  (Grober5). 
Pepsin  in  acid  solution  is  destroyed  by  heating  to  65°  C.  for  five  minutes. 


'Glaessner,  Hofmeister's  Beitrage,  1;  Klug,  Pfluger's  Arch.,  92;  Reach,  Hofmeis- 
ter's  Beitrage,  4;  Pekelharing,  Arch,  des  scienc,  biolog.,  St.  P6tersbourg  11;  Pawlow- 
Festband,  1904;  Bergmann,  Skand.  Arch.  f.  Physiol.,  18. 

2  Zeitschr.  f.  Biologie,  44. 

3  Brucke,  Wien.  Sitzungsber,.  43;  Sundberg,  Zeitschr.  f.  physiol.  Chem.,  9;  Schou- 
mow-Simanowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  33;  Pekelharing,  Zeitschr.  f.  physiol. 
Chem.,  22  and  35;  Xencki  and  Sieber,  ibid.,  32;  Friedenthal  and  Miyamota,  Centrabl. 
f.  Physiol.,  15,  785. 

« Zeithschr.  f .  Biologie,  28. 

6  Arch.  f.  exp.  Path.  u.  Pharm.,  51. 


468  DIGESTION. 

On  adding  peptone  or  certain  salts  the  pepsin  may  be  heated  to  70c  C. 
for  the  same  time  without  destruction. 

The  behavior  of  pepsin  on  heating  its  acid  solution  is  influenced  not 
only  by  the  degree  of  acidity,  but  by  the  duration  of  heating  and  also 
by  the  amount  of  other  bodies  in  the  solution.  If  an  acid  (0.2  per  cent 
HC1)  infusion  of  the  calf's  stomach  be  warmed  for  several  days  to  about 
40  or  45°  C,  a  part  of  the  pepsin  is  destroyed,  but  we  obtain  in  this 
manner  an  infusion  which  still  dissolves  proteins  but  has  no  rennin  action 
(Hammaesten  1).  The  pepsin  from  different  animals  acts  differently 
in  this  regard  and  the  pepsin  of  the  pike  stomach  is  very  quickly  destroyed 
at  37-40°  C. 

Pepsin  is  extraordinarily  sensitive  to  the  action  of  alkalies,  not  only 
caustic,  and  carbonated,  but  also  against  the  hydroxides  of  the  alka- 
line earths.  It  is  easily  made  inactive  by  these  substances.  If  the 
action  of  the  alkali  is  not  too  strong  then,  as  shown  by  Pawlow  and 
Tichomieow,2  the  enzyme  can  in  part  be  reactivated  by  the  addition 
of  acid  if  the  greater  part  (about  four-fifths),  of  the  alkalinity  be  neutral- 
ized by  the  addition  of  acid  and  then  after  some  hours  more  acid  be  added. 
If  the  entire  quantity  of  acid  be  added  at  one  time  the  reactivation  does 
not  take  place. 

The  only  property  which  is  characteristic  of  pepsin  is  that  it  dissolves 
protein  bodies  in  acid  but  not  in  neutral  or  alkaline  solutions,  with  the 
formation  of  proteoses,  peptones,  and  other  products. 

The  methods  for  the  preparation  of  relatively  pure  pepsin  depend, 
as  a  rule,  upon  its  property  of  being  thrown  down  with  finely  divided 
precipitates  of  other  bodies,  such  as  calcium  phosphate  or  cholesterin. 
The  rather  complicated  methods  of  Beucke  and  Sundbeeg  are  based 
upon  this  property.  Pekelhaeing  makes  use  of  a  prolonged  dialysis 
and  precipitation  with  0.2  p.  m.  HC1. 

Very  permanent  pepsin  solutions,  from  which  the  enzyme  with  con- 
siderable protein  can  be  precipitated  by  alcohol,  may  be  prepared  by 
extraction  with  glycerin.  Solutions  having  a  strong  action  may  also 
be  prepared  by  making  an  infusion  of  the  gastric  mucosa  of  an  animal 
in  acidified  water  (2-5  p.  m.  HC1).  This  is  unnecessary,  as  we  can  obtain 
pure  gastric  juice  according  to  Pawlow's  method,  and  also  because  very 
active  commercial  preparations  of  pepsin  can  be  bought  in  the  market. 

The  Action  of  Pepsin  on  Proteins.  Pepsin  is  inactive  in  neutral  or 
alkaline  reactions,  but  in  acid  liquids  it  dissolves  coagulated  protein 
bodies.  The  protein  always  swells  and  becomes  transparant  before 
it  dissolves.  Unboiled  fibrin  swells  up  in  a  solution  containing  1  p.  m. 
HO,  forming  a  gelatinous  mass,  and  does  not  dissolve  at  ordinary  tem- 

1  Zeitschr.  f.  physiol.  Chem.,  56.  2  Ibid.,  54. 


PEPSIN.  469 

perature  within  a  couple  of  days.  Upon  the  addition  of  a  little  pepsin, 
however,  this  swollen  mass  dissolves  quickly  at  ordinary  temperatures. 
Hard-boiled  egg  albumin,  cut  in  thin  pieces  with  sharp  edges,  is  not 
perceptibly  changed  by  dilute  acid  (2-4  p.  m.  HC1)  at  the  temperature 
of  the  body  in  the  course  of  several  hours.  But  the  simultaneous  pres- 
ence of  pepsin  causey  the  edges  to  become  clear  and  transparent,  blunt 
and  swollen,  and  the  protein  gradually  dissolves. 

From  what  has  been  said  above  in  regard  to  pepsin,  it  follows  that 
proteins  may  be  employed  as  a  means  of  detecting  pepsin  in  liquids. 
Ox-fibrin  may  be  employed  as  well  as  coagulated  egg  albumin,  which 
latter  is  used  in  the  form  of  slices  with  sharp  edges.  As  the  fibrin  is 
easily  digested  at  the  normal  temperature,  while  the  pepsin  test  with 
egg  albumin  requires  the  temperature  of  the  body,  and  as  the  test  with 
fibrin  is  somewhat  more  delicate,  it  is  often  preferred  to  that  with  egg 
albumin.  When  we  speak  of  the  "  pepsin  test  "  without  further  explana- 
tion we  ordinarily  understand  it  as  the  test  with  fibrin. 

This  test,  nevertheless,  requires  care.  The  fibrin  used  should  be 
ox-fibrin  and  not  pig-fibrin,  which  last  is  dissolved  too  readily  with  dilute 
acid  alone.  The  unboiled  ox-fibrin  may  be  dissolved  by  acid  alone 
without  pepsin,  but  this  generally  requires  more  time.  In  testing  with 
unboiled  fibrin  at  normal  temperature,  it  is  advisable  to  make  a  control 
test  with  another  portion  of  the  same  fibrin  with  acid  alone.  Since  at 
the  temperature  of  the  body  unboiled  fibrin  is  more  easily  dissolved  by 
acid  alone,  it  is  best  always  to  wTork  with  boiled  fibrin. 

As  pepsin  has  not  thus  far  been  prepared  in  a  positively  pure  condi- 
tion, it  is  impossible  to  determine  the  absolute  quantity  of  pepsin  in  a 
liquid.  It  is  possible  only  to  compare  the  relative  amounts  of  pepsin 
in  two  or  more  liquids,  which  may  be  done  in  several  ways. 

The  older  method,  that  of  Brucke,  consists  in  diluting  the  two  pepsin  solu- 
tions to  be  compared  with  certain  proportions  of  1  p.  m.  hydrochloric  acid,  so 
that  when  the  amount  of  pepsin  contained  in  the  original  solution  is  equal  to  1, 
each  solution  contains  a  degree  of  dilution,  p,  corresponding  to  1,  §,  |,  \,  ^, 
etc.  A  flock  of  fibrin  or  a  piece  of  hard-boiled  egg  is  added  to  each  test  and  the 
time  noted  when  each  test  begins  to  digest  and  when  it  ends.  The  relative 
amount  of  pepsin  is  calculated  from  the  rapidity  of  digestion  as  follows:  The  tests 
P=i,  I)  A,  of  one  series  is  digested  in  the  same  time  as  tests  p  =  l,  \,  \  of  the 
other  series,  hence  the  first  solution  contained  four  times  as  much  pepsin. 
Grutzner  1  has  improved  this  test  by  using  fibrin  colored  with  carmine,  and  on 
comparing  with  carmine  solutions  of  known  dilution  he  determines  colorimetrically 
the  rapidity  of  digestion. 

Mett's  Method.  Draw  up  white  of  egg  in  a  glass  tube  1-2  millimeters  in 
diameter,  coagulate  it  by  plunging  it  into  water  at  95°  C,  and  cut  the  ends  off 
sharply;  then  add  two  tubes  to  each  test-tube  with  a  few  cubic  centimeters 
of  the  acid  pepsin  solution;  allow  them  to  digest  at  body  temperature,  and  after 
a  certain  time,  generally,  after  ten  hours,  measure  the  lineal  extent  of  the  digested 


1  Grutzner,  Pfliiger's  Arch.,  8  and  106.     See  also  A.  Korn,  ''  Ueber  Methoden  Pepsin 
quantitativ  zu  bestimmen,"  Inaug.-Dissert.,  Tubingen,  1902. 


470  DIGESTION. 

layer  of  albumin  in  the  various  tests,  bearing  in  mind  that  the  digested  layer  at 
each  end  must  not  be  longer  than  6-7  millimeters.  The  quantity  of  pepsin  in 
the  comparative  tests  is  as  the  square  of  the  millimeters  of  the  albumin-column 
dissolved  in  the  same  time.  Thus  if  in  one  case  2  millimeters  of  albumin  were 
dissolved  and  in  the  other  3  millimeters,  then  the  quantity  of  pepsin  is  as  4:9. 
If  the  fluid  removed  from  the  stomach,  which  is  rich  in  bodies  having  a  disturb- 
ing influence  upon  pepsin  digestion,  is  to  be  tested,  then  the  liquid  must  be  first 
properly  diluted  with  hydrochloric  acid  (Nierenstein  and  Schiff1). 

Objections  have  been  raised  against  these  methods  from  several  sides,  and  they 
are  in  fact  very  uncertain.  Huppert  and  E.  Schutz  measure  the  relative 
quantities  of  pepsin  from  the  amount  of  secondary  proteoses  formed  under  cer- 
tain conditions.  The  proteoses  were  determined  by  the  polariscope.  J.  Schutz 
determines  the  total  proteose-nitrogen,  and  Spriggs  2  finds  that  the  change  in 
the  viscosity  is  a  measure  of  the  amount  of  pepsin. 

Volhard  and  Lohlein  3  use  an  acid  casein  solution  for  the  pepsin  determina- 
tion, and  determine,  after  precipitation  with  sodium  sulphate,  the  acidity  of  the 
nitrate  of  the  digested  test  as  well  as  of  the  original  control  solution.  The  casein 
is  precipitated  as  an  acid  compound  by  the  sulphate,  and  the  filtrate  separated 
from  the  precipitate  contains  less  acid  than  the  original  solution.  In  propor- 
tion as  the  digestion  progresses  less  substance  is  precipitated  by  the  sulphate, 
and  the  acidity  of  the  filtrate  becomes  correspondingly  higher.  The  increase 
in  acidity  in  the  different  portions  varies  within  certain  limits  as  the  square  root 
of  the  quantity  of  ferment. 

Jacoby  suggested  a  method  which  is  based  on  the  fact  that  a  cloudy  solution 
of  ricin  becomes  clear  by  the  action  of  pepsin-hydrochloric  acid,  and  indeed  with 
varying  rapidity  with  different  quantities  of  pepsin.  This  method,  which  requires 
further  testing,  seems  to  be  delicate  and  is  of  value,  as  is  doubtless  the  following 
method  of  Fuld  and  Levison.4  This  is  based  on  the  property  that  edestan  can 
be  precipitated  from  acid  solution  by  NaCl,  but  not  the  proteoses  formed  therefrom. 

A  solution  of  1  p.  m.  edestin  in  hydrochloric  acid  (jro  normal)  is  prepared 
whereby  the  edestin  is  changed  into  edestan.  The  activity  of  a  gastric  juice  (or 
a  pepsin-hydrochloric  acid  solution)  is  tested  in  the  following  manner:  the  solu- 
tion to  be  tested  is  placed  in  decreasing  quantities  in  a  series  of  test-tubes  and 
allowed  to  act  upon  an  equal  quantity  of  the  edestan  solution,  2  cc.,  and  the 
minimum  of  juice  determined  which  is  necessary  to  digest  the  solution,  within 
one-half  an  hour  and  at  room  temperature,  so  that  on  the  addition  of  solid 
NaCl  and  shaking  no  precipitate  occurs.  Gross  5  suggested  a  similar  method  by 
using  an  acid  casein  solution  and  precipitating  with  sodium  acetate. 

The  rapidity  of  the  pepsin  digestion  depends  on  several  circumstances. 
Thus  different  acids  are  unequal  in  their  action;  hydrochloric  acid  shows 
in  slight  concentration,  0.8-1.8  p.  m.,  a  more  powerful  action  than  any 
other  acid,  whether  inorganic  or  organic.  In  greater  concentration  other 
acids  may  have  a  powerful  action;  but  no  constant  relation  has  been 
found  between  the  strength  of  various  acids  and  their  action  in  pepsin 
digestion,  and  the  reports  of  the  action  of  different  acids  are  contradic- 

1  Mett,  see  Pawlow,  I.e.;  28;  Nierenstein  and  Schiff,  Berl.  klin.  Wochenschr.,  40; 
Jastrowitz,  Bioch.  Zeitschr.,  2. 

2  Huppert  and  Schutz,  Pfluger's  Arch.,  80;  J.  Schutz,  Zeitschr.  f.  physiol.  Chem., 
30;  Spriggs,  ibid.,  35. 

3  Hofmeister's  Beitrage,  7. 

4  Jftcoby,  Bioch.  Zeitschr.,  1;  Fuld  and  Levison,  ibid.  G. 
6  Berl.  klin.  Wbchensclir.,  45. 


PEPSIN   DIGESTION.  471 

tory.1  Sulphuric  acid,  it  seems,  has  a  weaker  action  than  the  other 
inorganic  acids.  The  degree  of  acidity  is  also  of  the  greatest  importance. 
With  hydrochloric  acid  the  degree  of  acidity  is  not  the  same  for  differ- 
ent protein  bodies.  For  fibrin  it  is  0.8-1  p.  m.,  for  myosin,  casein,  and 
vegetable  proteins  about  1  p.  m.,  for  coagulated  egg  albumin,  on  the 
contrary,  about  2.5  p.  m.  In  regard  to  the  dependence  of  the  extent 
of  transformation  upon  the  quantity  of  enzyme  and  the  time  of  diges- 
tion we  refer  to  page  58.  The  kind  of  protein  is  of  importance,  for 
example,  for  besides  what  was  said  above  in  regard  to  the  fibrin,  hard- 
boiled  egg  albumin  is  much  easier  digested  by  an  acidity  of  1-2  p.  m. 
HC1  than  liquid  egg  albumin,  which  is  rather  resistant  to  the  action 
of  gastric  juice.  The  accumulation  of  products  of  digestion  has  a  retard- 
ing action  on  digestion  (page  65),  although,  according  to  Chittenden 
and  Amerman,2  the  removal  of  the  digestion  products  by  means  of  dialysis 
does  not  essentially  change  the  relation  between  the  proteoses  and  true 
peptones.  Pepsin  acts  more  slowly  at  low  temperatures  than  it  does  at 
higher  ones.  It  is  even  active  in  the  neighborhood  of  0°  C,  but  with 
increasing  temperature  the  rapidity  of  digestion  also  increases  until 
about  40°  C,  when  the  maximum  is  reached.  If  the  swelling  up  of  the 
protein  is  prevented,  as  by  the  addition  of  neutral  salts,  such  as  NaCl, 
in  sufficient  amounts,  or  by  the  addition  of  bile  to  the  acid  liquid, 
digestion  can  be  prevented  to  a  greater  or  less  extent.  Foreign  bodies 
of  different  kinds  produce  dissimilar  effects,  in  which  naturally  the 
variable  quantities  in  which  they  are  added  are  of  the  greatest  impor- 
tance. Salicylic  acid  and  carbolic  acid,  and  especially  sulphates 
(Pfleiderer),  retard  digestion,  while  arsenious  acid  promotes  it  (Chit- 
tenden), and  hydrocyanic  acid  is  relatively  indifferent.  Salts  of  the 
alkali  and  alkaline  earth  metals  have  a  strong  retarding  action  in  strong 
concentration.  By  experiments  with  salt  solutions  so  strongly  diluted 
that  the  action,  on  account  of  the  strong  dissociation,  was  brought  about 
by  ions  and  not  by  the  electrolytically  neutral  molecules  (min.  -fa  and 
max.  I  normal  salt  solutions),  J.  Schutz  3  found  that  the  anions  had  a 
much  greater  retarding  action  upon  pepsin  digestion  than  the  cations. 
Of  these  latter  the  sodium  cation  had  the  strongest  retarding  action. 
Alcohol  in  large  quantities  (10  per  cent  and  above)  disturbs  the  digestion, 
while  small  quantities  act  indifferently.  Metallic  salts  in  very  small 
quantities   may   indeed   sometimes   accelerate   digestion,    but   otherwise 


1  See  Wr6blewski,  Zeitschr.  f.  physiol.  Chem.,  21,  and  especially  Pfleiderer,  Pfliiger's 
Arch.,  66,  which  also  gives  references  to  other  works;  Larin,  Biochem.  Centralbl.,  1, 
484;  and  A.  Pick,  Wein.  Sitzungsber.,  M.  N.  Klasse,  112. 

*  Journ.  of  Physiol.  14. 

1  Hofmeister's  Beitriige,  5. 


472  DIGESTION. 

they  tend  to  retard  it.  The  action  of  metallic  salts  in  different  cases 
can  be  explained  in  various  ways,  but  they  often  seem  to  form  with  pro- 
teins insoluble  or  difficultly  soluble  combinations.  The  alkaloids  may 
also  retard  the  pepsin  digestion  (Chittenden  and  Allen1).  A  very 
large  number  of  observations  have  been  made  in  regard  to  the  action 
of  foreign  substances  on  artificial  pepsin  digestion,  but  as  these  observa- 
tions have  not  given  any  direct  result  in  regard  to  the  action  of  the 
same  substances  in  natural  digestion,  as  well  as  upon  secretion  and 
absorption,  we  will  not  discuss  them  here. 

The  Products  of  the  Digestion  of  Proteins  by  Means  of  Pepsin  and  Acid. 
In  the  digestion  of  nucleoproteins  or  nucleoalbumins  an  insoluble  residue 
of  nuclein  or  pseudonuclein  always  remains,  although  under  certain 
circumstances  a  complete  solution  may  occur.  Fibrin  also  yields  an 
insoluble  residue,  which  consists,  at  least  in  great  part,  of  nuclein, 
derived  from  the  form-elements  inclosed  in  the  blood-clot.  This  residue 
which  remains  after  the  digestion  of  certain  proteins  was  called  dyspep- 
tone  by  Meissner.  This  name  is  therefore  not  only  unnecessary  but 
indeed  erroneous,  as  this  residue  does  not  consist  of  bodies  related  to  the 
peptones.  In  the  digestion  of  proteins,  substances  similar  to  acid  albu- 
minates, parapeptone  (Meissner2),  antialbumate,  and  antialbumid 
(Kuhne),  may  also  be  formed.  On  separating  these  bodies  the  filtered 
liquid,  neutralized  at  boiling-point,  contains  proteoses  and  peptones  in 
the  old  sense,  while  the  so-called  Kuhne  true  peptone  and  the  other 
cleavage  products  are  obtained  only  after  a  longer  and  more  intense 
digestion.  The  relation,  between  the  proteoses,  changes  very  much  in 
different  cases  and  in  the  digestion  of  the  proteins.  For  instance,  a  larger 
quantity  of  primary  proteoses  is  obtained  from  fibrin  than  from  hard- 
boiled  egg  albumin  or  from  the  proteins  of  meat;  and  the  different 
proteins,  according  to  the  researches  of  Klug,3  yield  on  pepsin  diges- 
tion unequal  quantities  of  the  various  digestive  products.  In  the  diges- 
tion of  unboiled  fibrin  an  intermediate  product  may  be  obtained  in  the 
earlier  stages  of  the  digestion — a  globulin  which  coagulates  at  55°  C 
(Hasebroek4).  For  information  in  regard  to  the  different  proteoses 
and  peptones  which  are  formed  in  pepsin  digestion  see  pages  127  to  136. 

Action   of  Pepsin-Hydrochloric  Acid   on  Other   Bodies.     The   gelatin- 
forming  substances  of  the  connective  tissue,  of  the  cartilage,  and  of  the 


1  .Studies  from  the  Lab.  Physiol.  Chem.  Yale  University,  1,  76.     See  also  Chitten- 
den and  Stewart,  ibid.  3,  60. 

2  Tlie  works  of  Meissner  on  pepsin  digestion  are  found  in  Zeitschr.  f.  rat.  Med.,  7, 
8,  10,  12,  and  14. 

»  Pfluger's  Arch.,  65. 

4  Zeitschr.  f.  physiol.  Chem.,  11. 


PEPSIN   DIGESTION.  473 

bones,  from  which  last  the  acid  dissolves  only  the  inorganic  substances, 
is  converted  into  gelatin  by  digesting  with  gastric  juice.  The  gelatin  is 
further  changed  so  that  it  loses  its  property  of  gelatinizing  and  is  con- 
verted into  gelatoses  and  peptone  (see  page  120).  True  mucin  (from  the 
submaxillary)  is  dissolved  by  the  gastric  juice,  yielding  substances  similar 
to  peptone,  and  a  reducing  substance  similar  to  that  obtained  by  boil- 
ing with  a  mineral  acid.  •  Mucoids  from  tendons,  cartilage,  and  bones 
dissolve,  according  to  Posner  and  Gies,1  in  pepsin-hydrochloric  acid, 
but  leave  a  residue  which  amounts  to  about  10  per  cent  of  the  original 
material  and  which,  as  it  seems,  consists  in  great  part,  if  not  entirely, 
of  a  combination  of  proteid  with  glucothionic  acid  (Chapters  VI  and 
VII).  The  solution  contains  primary  and  secondary  mucoproteoses 
and  mucopeptones.  The  former  contain  glucothionic  acid,  but  the  latter 
do  not.  Elastin  is  dissolved  more  slowly  and  yields  the  previously  men- 
tioned substances  (page  117).  Keratin  and  the  epidermal  formations 
are  insoluble.  The  nucleins  are  dissolved  with  difficulty,  and  the  cell 
nuclei,  therefore,  remain  in  great  part  undissolved  in  the  gastric  juice. 
According  to  London  2  and  his  collaborates  the  nucleic  acids  are  not 
attacked  in  the  stomach.  The  animal  cell-membrane  is,  as  a  rule,  more 
easily  dissolved  the  nearer  it  stands  to  elastin,  and  it  dissolves  with 
greater  difficulty  the  more  closely  it  is  related  to  keratin.  The  mem- 
brane of  the  plant-cell  is  not  dissolved.  Oxyhemoglobin  is  changed  into 
hsematin  and  protein,  the  latter  undergoing  further  digestion.  It  is 
for  this  reason  that  blood  is  changed  into  a  dark-brown  mass  in  the 
stomach.  The  gastric  juice  does  not  act  upon  fat,  but,  on  the  contrary, 
dissolves  the  cell-membrane  of  fatty  tissue,  setting  the  fat  free.  Gastric 
juice  has  no  action  on  starch  or  the  simple  varieties  of  sugar.  The 
statements  in  regard  to  the  ability  of  gastric  juice  to  invert  cane-sugar 
are  very  contradictory.  At  least  this  action  of  the  gastric  juice  is  not 
constant,  and  if  it  is  present  at  all,  it  is  probably  due  to  the  action  of  the 
acid. 

Pepsin  alone,  as  above  stated,  has  no  action  on  proteins,  and  an  acid  of  the 
intensity  of  the  gastric  juice  can  only  very  slowly,  if  at  all,  dissolve  coagulated 
albumin  at  the  temperature  of  the  body.  Pepsin  and  acid  together  not  only 
act  more  quickly,  but  qualitatively  they  act  otherwise  than  the  acid  alone,  at 
least  upon  dissolved  protein.  This  has  led  to  the  assumption  of  the  presence  of 
a  pepsin-hydrochloric  acid  whose  existence  and  action  are  only  hypothetical. 
As  pepsin  digestion,  it  seems,  yields  finally  the  same  products  as  the  hydrolytic 
cleavage  with  acids,  we  can  say  for  the  present  only  that  this  enzyme  acts  like 
other  catalysts  in  very  powerfully  accelerating  a  process  which  would  also  pro- 
ceed without  the  catalvte. 


1  Amer.  Journ.  of  Physiol.,  11. 

*  Zeitschr.  f.  physiol.  Chem.  70,  72. 


474  DIGESTION. 

Rennin  or  (thymosin  is  the  enzyme,  which  is  especially  character- 
ized by  the  fact  that  it  coagulates  milk  or  casein  solutions  containing 
lime  in  neutral  or  indeed  faintly  alkaline  reaction.  It  must  probably 
be  considered  as  a  proteolytic  enzyme.  Rennin  is  habitually  found  in 
the  neutral,  watery  infusion  of  the  fourth  stomach  of  the  calf  and  sheep, 
especially  in  an  infusion  of  the  fundus  part.  In  other  mammals  and  in 
birds  it  is  seldom  found,  and  in  fishes  hardly  ever  in  the  neutral  infusion. 
In  these  cases,  as  in  man  and  the  higher  animals,  a  rennin-forming  sub- 
tance,  a  rennin  zymogen,  occurs,  which  is  converted  into  rennin  by  the 
action  of  an  acid  (Hammarsten)  .  Hedin  has  obtained  a  retarding 
solution  by  treating  a  neutral  infusion  of  the  stomachs  of  various  animals 
with  dilute  ammonia  and  then  neutralizing.  These  solutions  entirely 
or  partly  retard  the  action  of  the  rennin  from  the  same  animal  and  is 
destroyed  by  acid  with  the  setting  free  of  rennin.  Hedin  therefore 
considers  the  rennin  zymogen  as  a  combination  between  rennin  and  an 
inhibitory  substance,  in  which  combination  the  inhibitory  body  is 
destroyed  by  treatment  with  acid;  consequently  the  rennin  appears  in 
an  active  form. 

According  to  Bang  the  rennin  of  the  human  and  pig  stomachs  differs 
from  that  of  the  calf's  stomach  in  being  much  more  resistant  to  acids, 
more  easily  destroyed  by  alkalies,  and  that  its  action  is  much  more 
accelerated  by  calcium  chloride  than  that  from  the  calf's  stomach.1 
Active  rennin  occurs  in  the  human  stomach  under  physiological  condi- 
tions, but  may  be  absent  under  special  pathological  conditions.2 

According  to  the  experience  of  Hammarsten  the  rennin  of  the  pike 
and  of  the  dog  differs  from  that  of  the  calf,  and  Hedin  3  finds  in  the  specific 
kind  of  inhibitory  action  of  rennin  produced  by  means  of  ammonia 
treatment  as  well  as  by  immune  serum,  a  proof  that  the  rennin  enzyme 
of  different  kinds  of  animals  differ  more  or  less  from  each  other.  In 
regard  to  this  inhibition  see  pages  62-64. 

Enzymes  having  a  rennin  action  has  also  been  found  in  the  blood 
and  several  organs  of  higher  animals  as  well  as  in  invertebrates.  Sim- 
ilar enzymes  are  also  very  widely  distributed  in  the  plant  kingdom  and 
numerous  micro-organisms  have  the  ability  to  produce  rennin. 


1  Deutsch.  med.  Wochenschr.,  1899,  and  Pfliiger's  Arch.,  79. 

2  Schumburg,  Virchow's  Arch.,  97.  A  good  review  of  the  literature  may  be  found 
in  Szydlowski,  Beitrage  zur  Kenntnis  des  Labenzym  nach  Beobaehtungen  an  Saug- 
lingen,  Jahrb.  f.  Kinderheilkunde  (N.  !\),  34.  See  also  Lorcher,  Pfliiger's  Arch.,  69. 
which  also  contains  the  pertinent  literature.  An  excellent  review  of  the  literature 
on  rennin  and  its  action  may  be  found  in  E.  Fuld,  Ergebnisse  der  Physiol.,  1,  Abt.  1, 
468. 

3  Hammarsten,  Upsala  Liikaref.  forh.  8,  78  (1872).  Zeitschr.  f.  physiol.  Chem. 
56,  18  (1908),  68, 119  (1910);  Hedin,  ibid.  72,  187,  74,  242,  76,  355  (1911),  77,  229  (1912). 


REN N IN.  475 

The  law  given  on  page  58  in  which  the  time  of  coagulation  is 
inversely  proportional  to  the  amount  of  enzyme,  is  true  for  calf  rennin 
(Fuld  !)  and  for  sheep  rennin  (Hedin2).  The  other  rennins  investi- 
gated do  not  follow  this  law  at  37°  C,  which,  according  to  van  Dam,  is 
due  in  the  case  of  the  pig  rennin  to  its  less  resistance  toward  the  alkali 
of  the  milk.3 

Rennin  is  just  as  difficult  to  prepare  in  a  pure  state  as  the  other 
enzymes.  The  purest  rennin  enzyme  thus  far  obtained  did  not  give  the 
ordinary  protein  reactions.  On  heating  its  solution  rennin  is  more  or 
less  quickly  destroyed,  depending  upon  the  length  of  heating  and  upon 
the  concentration.  If  an  active  and  strong  infusion  of  the  gastric  mucosa 
of  the  calf's  stomach  in  water  containing  3  p.  m.  HC1  is  heated  to  40-45° 
C.  for  48  hours,  the  rennin  or  nearly  all,  is  destroyed,  while  the  pepsin 
remains.  A  pepsin  solution  free  from  rennin  can  be  obtained  in  this 
way. 

A  much-discussed  question  is,  whether  the  digestion  of  protein  and 
the  rennet  action  are  brought  about  by  two  special  enzymes,  or  represent 
two  different  enzyme  actions,  or  whether  there  is  only  one  enzyme,  the 
pepsin,  which  has  both  actions. .  The  supporters  of  this  last  view  dispose 
of  the  question  in  different  ways.  Some,  like  Pawlow  and  Parast- 
schuk,  consider  the  rennet  action  simply  as  the  reverse  of  the  synthetical 
action  of  pepsin,  a  view  which  is  improbable  in  the  highest  degree. 
Others,  such  as  Sawjalow4  and  Gewin,  consider,  on  the  contrary,  that 
the  coagulation  of  milk  is  only  a  pepsin  action  and  indeed  as  the  first 
step  in  the  beginning  of  proteolysis,  namely,  the  beginning  of  peptic 
digestion  of  casein.  Rokoczy  5  believes  in  the  presence  of  two  enzymes 
in  the  calf's  stomach,  one  of  which,  the  rennin,  disappears  on  the  increas- 
ing age  of  the  animal. 

The  simultaneous  occurrence  in  the  animal  and  plant  kingdoms  of 
enzymes  having  a  proteolytic  and  rennet  action  and  the  parallelism  of 
the  pepsin  and  rennet  action  indicates  an  identity  of  both  enzymes  and 
enzyme  actions.  This  parallelism  in  fact  does  not  prove  much,  because 
it  has  mostly  been  studied  in  acid  reaction,  while  rennet  is  character- 
istically active  in  neutral  or  faintly  alkaline  reaction. 

At  the    same    time    Hammarsten  6    finds  that  in  acid  reactions  no 


1  Hofmeister's  Beitrage  2. 
1  Not  published  investigations. 
8  Zeitschr.  f.  physiol.  Chem.  64,  316  (1910). 

4  The  recent  literature  on  this  question  can  be  found  in  Hammarsten,  Zeitschr. 
physiol.  Chem.,  56,  18  (1909). 
*Ibid.  68,  421  (1910),  73,  453  (1911). 
8  Zeitschr.  f.  physiol.  Chem.  68,  119  (1910),  which  also  contains  the  recent  literature. 


470  DIGESTION. 

parallelism  exists  in  the  two  enzyme  actions  with  extracts  of  the  dog's 
and  calf's,  stomach,  and,  also  on  testing  the  two  enzyme  actions  upon  the 
same  casein  solution  no  parallelism  was  present.  The  pathological  cases 
in  man,  if  the  observations  are  reliable,  where  only  one  enzyme  action 
occurs,  seems  to  dispute  the  identity  of  the  action  of  these  two  enzymes. 
This  opposition  is  also  shown  by  the  fact  that  pepsin,  so  far  as  known, 
only  has  a  digestive  action  in  the  presence  of  free  H  ions,  while  the 
coagulation  of  milk  occurs  in  the  absence  of  these  and  indeed  in  the 
presence  of  HO  ions.  Among  other  facts  which  contravene  the  identity 
is  the  fact  that  a  pepsin  solution  can  be  prepared  which  has  a  digestive 
action  but  cannot  coagulate  milk,  and  the  reverse,  namely,  rennet  solu- 
tions can  be  made  which  coagulate  milk  but  do  not  have  digestive  action 
in  acid  reaction  (Hammarsten1).  The  observations  of  Ducceschi,2 
that  pepsin  but  no  rennin  occurs  in  the  stomach  of  the  Didelphys,  also 
conflict  with  the  identity  of  the  two  enzymes. 

The  views  of  Nencki  and  Sieber3  take  a  certain  reconciliary  posi- 
tion. According  to  them  pepsin  forms  a  gigantic  molecule  which  has 
various  side-chains,  one  of  which  has  digestive  action  in  acid  solution 
while  the  others  coagulate  milk.  This. view  coincides  well  with  most 
of  the  observations  made  thus  far. 

In  regard  to  the  formation  of  plasteins  under  the  influence  of  rennin 
solutions  and  other  enzyme  solutions,  see  Chapters  I  and  II. 

Gastric  Lipase  (stomach  steapsin).  F.  Volhard4  made  the  dis- 
covery that  the  gastric  juice  has  a  strong  fat-splitting  action  only  when 
the  fat  is  in  a  fine  emulsion,  as  in  the  yolk  of  the  egg,  in  milk  or  in  cream. 
Considerable  controversy  has  arisen  in  regard  to  the  importance  of  the 
splitting  of  fat,  and  the  occurrence  of  a  special  gastric  lipase  is  indeed 
disputed.  From  numerous  observations  it  follows  without  question 
that  in  man  and  many  animals  a  gastric  lipase  occurs  and  is  secreted 
with  the  gastric  juice.  Nevertheless  the  extent  of  fat  splitting  in  the 
stomach  is  generally  not  very  great.  In  its  action  this  lipase  follows 
Schutz's  rule  and  in  its  other  properties  it  seems  to  vary  in  different 
animals. 

The  question  whether  the  cover  cells,  principally,  or  the  chief  cells 


1  Zeitschr.  f.  physiol.  Chem.,  56. 

2  Centralbl.,  f.  Physiol.  22,  784. 
J  Zeitschr.  f.  physiol.  Chem.,  32. 

4  Volhard,  Munch,  med.  Wochenschr.,  1900,  and  Zeitschr.  f.  klin.  Med.,  42,  43. 
See  also  Stade,  Hofmeister's  Beitrage,  3;  A.  Fromme,  ibid.,  7;  A  Zinsser,  ibid.;  H. 
Engel.,  ibid.;  and  Inouye,  Arch.  f.  Verdauangskrank.,  9;  Falloise,  Arch,  internat.  d. 
Physiol.,  3  and  4;  London,  Zeitschr.  f.  physiol,  Chem.,  50;  Levites,  ibid.,  49;  Laqueur. 
Hofmeister's  Beitrage,  8,  281;  Heinsheimer,  Deutsch.  med.  Wochenschr.,  32,  and 
Arbeiten  aus  d.  pathol.  Institute,  Berlin  (Hirschwald,  1906). 


FORMATION  OF  HYDROCHLORIC  ACID.  477 

also,  or  both,  take  part  in  the  formation  of  free  acid  is  disputed.1  There 
can  be  no  doubt  that  the  hydrochloric  acid  of  the  gastric  juice  origin- 
ates in  the  chlorides  of  the  blood,  because,  as  is  well  known,  a  secretion 
of  perfectly  typical  gastric  juice  takes  place  in  the  stomachs  of  fasting 
animals  or  those  which  have  starved  for  some  time.  As  the  chlorides 
of  the  blood  are  derived  from  the  food,  it  is  easily  understood,  as  shown 
by  Cahn,2  that  in  dogs  after  a  sufficiently  long  common-salt  starva- 
tion, the  stomach  secreted  a  gastric  juice  containing  pepsin,  but  no  free 
hydrochloric  acid.  On  the  administration  of  soluble  chlorides,  a  gastric 
juice  containing  hydrochloric  acid  was  immediately  secreted.  The 
conditions  are  not  so  simple,  because  in  the  first  case  not  only  does  the 
amount  of  hydrochloric  acid  diminish  but,  as  shown  by  Wohlgemuth 
and  then  by  Kudo,  the  quantity  of  juice  diminishes  greatly,  and  on  the 
introduction  of  NaCl  the  quantity  of  juice  secreted  increases.  Accord- 
ing to  Pugliese3  the  gastric  juice  in  starvation,  after  a  certain  time, 
has  a  neutral  reaction,  and  the  introduction  of  NaCl  does  not  now  change 
its  properties.  In  the  secretion  of  free  acid  it  is  assumed  by  Pugliese 
that  the  gland  cells,  which  decompose  the  chloride,  have  sufficient 
amounts  of  protein  at  their  disposal.  On  the  introduction  of  alkali 
iodides  or  bromides,  Kulz,  Nencki  and  Schoumow-Simanowski  4  have 
shown  that  the  hydrochloric  acid  of  the  gastric  juice  is  replaced  by  HBr, 
and  to  a  less  extent  by  HI.  The  secretion  of  free  hydrochloric  acid 
from  the  alkaline  blood  has  been  explained  in  various  ways,  but  as  yet 
no  satisfactory  theory  has  been  suggested.5 

In  regard  to  the  secretion  of  pepsin  we  must  recall  that  this  last 
is  not  already  produced,  but  is  formed  from  a  preliminary  step,  a  pep- 
sinogen or  propepsin.  Langley  6  has  positively  shown  the  existence 
of  such  a  substance  in  the  mucous  coat.  This  substance,  propepsin, 
shows  a  comparatively  strong  resistance  to  dilute  alkalies  (a  soda  solu- 


1  See  Heidenhain,  Pfliiger's  Arch.,  18  and  19,  and  Hermann's  Handbuch,  5,  part  I, 
"  Absonderungsvorgange;"  Klemensiewicz,  Wien.  Sitzungsber,.  71;  Frankel,  Pfliiger's 
Arch.,  48  and  50;  Contejean.  1.  c;  Kranenburg,  Archives  Teyler,  Ser.  II,  Haarlem, 
1901;  and  Mosse,  Centralbl.  f.  Physiol.,  17,  217;  Fitzgerald,  Proc.  Roy.  Soc.  B.  82, 
83;  L6pez-Suarez,  Bioch.  Zeitschr.  46,  490  (1912). 

2  Zeitschr.  f.  physiol.  Chem.,  10. 

1  Wohlgemuth,  Arbeiten  aus  d.  pathol.  Institute,   Berlin,   1906;    Kudo,   Bioch. 
Zeitschr.  16,  217  (1909),  Pugliese,  Maly's  Jahresb.,  36,  394. 

4  Kulz,  Zeitschr.  f.  Biologie,  23;  Nencki  and  Schoumow,  Arch,  des  sciences  biol. 
de  St.  Petersboura,  3. 

5  Koeppe,  Pfliiger's  Arch.,  62;  Benrath  and  Sachs,  ibid.,  109;  Maly,  see  v.  Bunge's 
Lehrbuch  der  physiol.  u.  pathol.  Chem.,  4.  Aufl.,  1898;  Schwarz,  Hofmeister's  Bei- 
trage.  5, 

6  SchilT,  Lecons,  sur  la  physiol.  de  la  digestion,  1867,  2;  Langley  and  Edkins,  Journ. 
of  Physiol.,  7. 


478  DIGESTION. 

tion  of  5  p.  m.)  which  easily  destroy  pepsin  (Langley).  Pepsin,  on  the 
other  hand,  withstands  better  than  propepsin  the  action  of  carbon  dioxide, 
which  quickly  destroys  the  latter.  The  occurrence  of  a  rennin  zymogen 
and  possibly  also  of  a  steapsinogen,  in  the  mucous  coat  has  been  men- 
tioned above. 

The  question  in  what  cells  the  two  zymogens,  especially  the  pro- 
pepsin, are  produced,  has  been  extensively  discussed  for  several  years. 
Formerly,  it  was  the  general  opinion  that  the  cover  cells  were  pepsin 
cells,  but  since  the  investigations  of  Heidenhain  and  his  pupils,  Langley 
and  others,  the  formation  of  pepsin  has  been  attributed  to  the  chief 
cells.1 

The  Pyloric  Secretion.  That  part  of  the  pyloric  end  of  the  dog's 
stomach  which  contains  no  fundus  glands  was  dissected  by  Klemensie- 
wicz,  one  end  being  sewed  together  in  the  shape  of  a  blind  sac  and  the 
other  sewed  into  the  stomach.  From  the  fistula  thus  created  he  was 
able  to  obtain  the  pyloric  secretion  of  a  living  animal,  later  the  secretion 
from  a  pyloric  fistula  has  been  obtained  in  other  ways.  This  secretion 
is  alkaline,  viscous,  jelly-like,  rich  in  mucin,  of  a  specific  gravity  of 
1.009-1.010,  containing  16.5-20.5  p.  m.  solids.  It  habitually  con- 
tains pepsin,  which  has  been  proved  by  Heidenhain  by  observations 
on  a  permanent  pyloric  fistula,  and  the  amount  may  sometimes  be  con- 
siderable. Contejean  investigated  the  pyloric  secretion  in  other  ways, 
and  finds  that  it  contains  both  acid  and  pepsin.  The  alkaline  reaction 
of  the  secretions  investigated  by  Heidenhain  and  Klemensiewicz 
is  due,  according  to  Contejean,  to  an  abnormal  secretion  caused  by  the 
operation,  because  the  stomach  readily  yields  an  alkaline  juice  instead 
of  an  acid  one  under  abnornal  conditions.  The  reports  of  Heidenhain 
and  Klemensiewicz  have  nevertheless  been  substantiated  by  Aker- 
mann,   Kresteff,  Schemiakine  and  others.2 

The  secretion  of  gastric  juice  under  different  conditions  may  vary 
considerably.  The  statements  concerning  the  quantity  of  gastric  juice 
secreted  in  a  certain  time  are  therefore  unreliable.  Rosemann  ob- 
served, on  sham  feeding  in  dogs,  a  secretion  of  917  cc.  in  the  course  of 
Z\  hours — a  considerable  quantity.  Kudo  s  found  more  pepsin  in  the 
secreted  juice  when  the  quantity  of  juice  was  less. 

The  Chyme  and  the  Digestion  in  the  Stomach.  By  means  of  the 
chemical  stimulation  caused  by  the  food,  a  copious  secretion  of  gastric 


1  See  footnote  1,  p.  477. 

'Heidenhain  and  Klemensiewicz,  1.  c;  Contejean,  1.  c.,  Chapter  II,  and  Skand. 
Arch  f.  Physiol,  6;  Akermann,  ibid.,  5;  Kresteff,  Maly's  Jahresber.,  30;  Schemia- 
kine Arch,  des  scienc.  biolog.  de  St.  P£tersbourg,  10. 

1  Rosemann,  Pfluger's  Arch.  118;  Kudo,  Bioch.  Zeitschr.  16. 


CHYME  AND  DIGESTION  IN  THE  STOMACH.  479 

juice  occurs,  which  gradually  mixes  with  the  swallowed  food,  and  digests 
it  more  or  less  strongly.  The  material  in  the  stomach  during  digestion, 
which  has  a  pasty  or  thick  consistency,  and  is  called  chyme,  is  not  a 
homogeneous  mixture  of  the  ingesta  with  the  various  digestive  fluids, 
gastric  juice,  saliva,  and  gastric  mucus,  but  the  conditions  seem  to 
be  more  complicated. 

From  the  investigations  of  several  workers,1  on  the  movements  of 
the  stomach,  we  conclude  that  this  organ  in  carnivora  and  also  in  man 
consists  of  two  physiologically  different  parts,  the  pylorus  and  the 
fundus.  The  greater  fundus  part,  which  serves  essentially  as  a  reservoir, 
may  be  a  rhythmic,  strong  contraction  of  the  muscle,  acting  like  a 
sphincter  between  it  and  the  pylorus  part,  be  separated  from  the  latter, 
and  according  to  some  observers  so  completely  so  that  during  contrac- 
tion scarcely  anything  passes  from  the  fundus  to  the  pylorus  part. 
Differing  from  the  fundus  part  the  pylorus  is  the  seat  of  very  powerful 
contractions  by  which  its  contents  are  intimately  mixed  with  gastric 
juice  and  are  also  driven  through  the  pyloric  valve  into  the  intestine. 

The  contents  of  the  pylorus  part  have  an  acid  reaction,  and  a  strong 
pepsin  digestion  takes  place  in  the  contents,  which  are  thoroughly  mixed 
with  gastric  juice.  The  contents  of  the  fundus,  on  the  contrary,  show 
a  different  behavior,  for  here,  as  Ellenberger  first  showed,  a  special 
stratification  of  the  various  solid  food-stuffs  takes  place. 

By  very  instructive  investigations  on  different  animals  (frogs,  rats, 
rabbits,  guinea-pigs,  and  dogs)  Grutzner  2  later  showed  that  when 
the  aminals  are  fed  with  food  having  different  colors,  and  the  stomach 
removed  after  a  certain  time,  and  the  contents  frozen,  the  frozen  sec- 
tions show  a  regular  stratification  of  the  contents.  These  layers  are 
so  arranged  that  the  food  first  taken  is  found  in  direct  contact  with  the 
mucosa,  while  the  food  taken  later  is  enclosed  by  that  partaken  of  first, 
and  this  prevents  contact  with  the  walls  of  the  stomach.  The  empty 
stomach,  whose  walls  touch  each  other,  is  so  filled  that,  as  a  rule,  the 
foodstuffs  taken  later  are  in  the  middle  of  the  older  food. 

Because  of  this  fact  only  the  foodstuffs  which  lie  close  to  the  surface 
of  the  mucous  membrane  undergo  the  process  of  peptic  digestion,  and 
it  is  principally  these  ingesta,  which  lie  on  the  surface  and  are  laden  with 
pepsin  and  mixed  with  gastric  juice,  which  are  shoved  to  the  pylorus 
end,   here  mixed  and  digested,   and  finally  moved  into  the  intestine 


1  Hofireister  and  Schutz,  Arch.  f.  exp.  Path.  u.  Pharm.,  20;  Moritz.  Zeitschr.  f. 
Biologie,  32;  Cannon,  Amer.  Journ.  of  Physiol.,  1;  Sehemiakine,  1.  c;  Cathcart, 
Journ.  of  Physiol.,  1911,  42. 

1  See  Ellenberger,  Pfliiger'a  Arch.,  114,  and  Scheunert,  ibid.,  144;  Grutzner,  ibid., 
106. 


480  DIGESTION. 

The  fundus  part  is  therefore  less  a  digestion-organ  than  a  storage-organ, 
and  in  the  interior  of  the  same,  the  food  may  remain  for  hours  without 
coming  in  contact  with  a  trace  of  gastric  juice. 

What  has  been  said  above  applies  at  least  to  solid  food.  We  have 
no  extensive  observations  on  the  behavior  of  fluids  or  semifluid  food. 
According  to  Grutzner,  in  these  cases,  as  well  as  in  the  above-mentioned 
experiments,  the  swallowed  foodstuffs  are  not  irregularly  mixed  together. 
Fluids  quickly  leave  the  stomach,  which  is  also  the  case  with  a  mixture 
of  solid  and  fluid  food. 

Milk  is  an  exception  because  it  coagulates  and  the  clot  remains  in  the 
stomach  while  the  whey  quickly  leaves  the  stomach. 

The  fact  that  only  that  part  of  the  ingesta  lying  on  the  mucous 
membrane  is  mixed  with  gastric  juice,  while  the  mass  in  the  interior  is 
not  acid  in  reaction,  is  of  special  importance  for  the  digestion  of  starches 
in  the  stomach.  By  this  we  can  explain  why  the  salivary  diastase, 
although  sensitive  toward  acids,  can  continue  its  action  for  a  long  time 
in  the  contents  of  the  stomach.  That  this  is  true  was  first  found  by 
Ellenberger  and  Hofmeister  and  then  by  Cannon  and  Day  *  by 
special  experiments  upon  animals.  The  occurrence  of  sugar  and  dex- 
trin in  the  contents  of  the  human  stomach  has  been  repeatedly  observed. 
In  carnivora,  whose  saliva  shows  scarcely  any  diastatic  action,  it  is 
a  'priori  not  expected  that  there  should  be  a  diastatic  action  in  the 
stomach,  but  the  conditions  are  different  in  herbivora,  where  an  abun- 
dant digestion  of  starch  takes  place  in  the  various  stomachs,  according 
to  the  different  species. 

The  gastric  contents  which  have  been  prepared  in  the  pylorus  part 
are  passed  through  the  pylorus  into  the  intestine  intermittently.  This' 
material  is  generally  fluid,  but  it  is  possible  that  pieces  of  solid  food 
may  also  occur,  and  this  has  often  been  observed.  Thin  or  plastic 
food  leaves  the  stomach  earlier  than  solid  food,  and  it  is  obvious  that  the 
time  in  which  the  stomach  unburdens  itself  depends  naturally  upon 
the  coarseness  or  fineness  of  the  food.  This  depends  essentially  upon 
the  reflex  action  of  the  stomach  or  intestine,  causing  an  opening  or 
closing  of  the  pylorus,  which  action  is  dependent  upon  the  quantity  and 
character  of  the  food,  the  amount  of  fat,  and  the  degree  of  acidity  in 
the  contents  of  the  stomach  and  intestine.  The  emptying  of  the  food 
into  the  small  intestine  causes,  as  shown  by  Pawlow,  a  closing  of  the 
pylorus  by  chemo-reflex  in  which  the  hydrochloric  acid  and  the  fat  take 
part,  and  we  thus  find  in  this  regard  an  alternate  action  between  the 
stomach  and  duodenum. 


1  EllenberRer  and  Hofmeister,  Maly's  Jahresb.,  15  and  16;  Cannon  and  Day,  Amer. 
Journ.  of  Physiol..  9. 


DIGESTION   IN  THE  STOMACH.  481 

This  alternate  action,  according  to  Cannon  l  is  due  to  the  fact  that 
the  acid  in  the  pylorus  which  acts  upon  the  sphincter  and  makes  pos- 
sible the  passage  of  the  fluid  chyme  by  the  contraction  of  the  muscles 
of  the  stomach.  In  the  intestine  the  acid  has  a  reverse  stimulation 
upon  the  sphincter  and  causes  a  contraction  of  the  same.  As  soon  as 
the  acid  is  neutralized  the  contractions  of  the  sphincter  cease  and  the 
passage  of  new  portions  of  the  chyme  occur.  If  the  flow  of  bile  and  pan- 
creatic juice  is  prevented,  and  the  neutralization  of  the  acid  contents 
of  the  stomach  in  the  intestine  is  retarded,  then  the  stomach  does  not 
eject  its  contents  so  often.  The  duration  of  gastric  digestion  varies 
according  to  conditions,  and  in  consequence  the  reports  of  observers  are 
widely  divergent.  Beaumont2  found  in  his  extensive  observations 
on  the  Canadian  hunter  St.  Martin  that  the  stomach,  as  a  rule,  is 
emptied  1£-5|  hours  after  a  meal,  depending  upon  the  character  of  the 
food. 

The  time  in  which  different  foods  leave  the  stomach  also  depends 
upon  their  digestibility.  Respecting  the  unequal  digestibility  in  the 
stomach  we  must  differentiate  between  the  rapidity  with  which  the  food- 
stuffs are  chemically  transformed  and  that  with  which  they  leave  the 
stomach  and  pass  into  the  intestine.  This  distinction  is  especially 
important,  and  it  is  evident  that  the  main  factors  governing  speed  of 
digestion  and  the  time  required  before  the  food  leaves  the  stomach  are  the 
kind  of  food  and  the  fineness  of  its  subdivision,  and  its  action  upon  the 
gastric  secretion,  upon  the  pyloric  reflexes,  etc. 

The  observations  of  Boldyreff  and  others3  on  the  action  of  fats 
and  fatty  acids  and  not  too  dilute  hydrochloric  acid  (stronger  than  0.2 
per  cent)  are  conclusive  concerning  the  manner  in  which  the  properties 
of  the  food  act  upon  the  gastric  secretion  and  upon  the  digestion  in  the 
stomach  as  a  whole.  Irrespective  of  the  reducing  action  of  the  fats 
upon  the  extent  and  digestive  power  of  the  gastric  juice  Boldyreff 
found  after  food  very  rich  in  fat  that  the  bile,  pancreatic  juice  and  intes- 
tinal juice  migrate  from  the  intestine  into  the  stomach  so  that  the  diges- 
tion in  the  stomach  in  these  cases  is  essentially  brought  about  by  the 
pancreatic  juice. 

We  have  numerous  investigations  on  the  rapidity  with  which  the 
food  is  digested  in  the  stomach  of  dogs,  but  we  must  especially  mention 


1  Amer.  Journ.  of  Physiol.,  20. 

2  The  Physiology  of  Digestion,  1833. 

3  Boldyreff,  Pfliiger's  Arch.,  121,  140;  Migay,  Maly's  Jahresb.  39;  Best  and 
Cohnheim,  Zeitschr.  f.  Physiol.  Chem.  69;  Cathcart,  Journ.  of  Physiol.  42.  See 
also  Abderhalden  and  Medigreeeanu,  Zeitschr.  f.  physiol.  Chem.,  57. 


482  DIGESTION. 

the  researches  of  E.  Zunz,1  London  2  and  his  co-workers.  London, 
Polowzowa  and  Sagelmann  have  observed  that  all  the  foodstuffs  do 
not  leave  the  stomach  with  the  same  rapidity,  indeed,  by  feeding  with 
bread  (Polowzowa),  the  carbohydrates  leave  more  quickly  than  the 
protein,  and  with  a  mixture  of  gliadin  and  beef-fat  (Sagelmann)  the 
protein  left  the  stomach  more  quickly  than  the  fat.  This  is  in  accord 
with  the  recent  observations  of  London  and  Sivr^  which  show  that  the 
fats  remain  longest  in  the  stomach,  the  starches  the  shortest  and  meat 
takes  a  middle  position.3  According  to  these  authors  the  stomach  has 
a  sort  of  "  selective  capacity,"  but  this  is  strongly  disputed  by  Schetjn- 
ert  and  Grimmer.4  Nevertheless  the  researches  of  Cannon  5  on  cats, 
making  use  of  another  method,  have  shown  that  this  is  true.  After 
preliminary  hunger  the  animals  received  different  food,  such  as  meat, 
fat,  and  carbohydrate  mixed  with  bismuth  subnitrate  and  then  with 
the  aid  of  the  Rontgen  rays  the  time  was  noted  when  the  food  passed 
into  the  intestine.  The  carbohydrate  leaves  the  stomach  first,  the 
proteins  next,  and  the  fats  last.  If  the  carbohydrate  is  given  before 
the  protein  food,  then  it  leaves  the  stomach  with  ordinary  rapidity; 
while  if  protein  food  and  then  carbohydrate  is  given  the  passage  of  the 
carbohydrate  is  retarded.  A  mixture  of  protein  food  and  carbohydrates 
leaves  the  stomach  more  slowly  than  carbohydrates  alone,  but  faster 
than  protein  food  alone.  The  fat,  which  remains  in  the  stomach  for  a 
long  time  and  leaves  the  stomach  only  in  amounts  which  are  absorbed 
or  removed  from  the  duodenum,  retards  the  passage  of  the  protein  foods 
as  well  as  the  carbohydrates.  Tangl  and  Erdelyi  6  have  found  in 
regard  to  the  different  kinds  of  fat,  that  a  fat  leaves  the  stomach  the  slower 
according  to  the  he:ght  of  its  melting-point.  According  to  London  and 
Schwarz,7  with  mixed  protein  feeding,  the  digestion  in  the  stomach 
is  regulated  by  that  kind  of  protein,  which,  when  alone,  is  removed  from 
the  stomach  the  slowest. 

The  reason  why  different  foodstuffs  leave  the  stomach  with  unequal  rapidity 
is  explained  by  Cannon  by  the  above-mentioned  action  of  the  hydrochloric  acid 

1  E.  Zunz,  Hofrr.eister's  Beitrage,  3;  Annal  de  la  soc.  roy.  des  scienc.  med.  Bruxelles 
12,  13,  and  Memoires  publ.  par  l'Acad.  roy.  Belg.,  1906,  1907,  and  1908.  Intern. 
Beitr.  zu.  Path.  u.  Ther.  der  Ernahrungsstorungen  2;  Bull,  de  l'Acad.  roy.  de  med. 
de  Belgique,  24  (1910). 

1  The  numerous  works  of  London  and  co-workers  will  be  found  in  Zeitschr.  f. 
physiol.  Chem.,  45-53,  55-58,  60-74. 

1  London  with  Polowzowa,  Zeitschr.  f.  physiol.  Chem.,  49,  with  Sagelmann,  ibid., 
62;  London  and  SivTe,  ibid.  60  (1909). 

4  Scheunert,  Zeitschr.  f.  physiol.  Chem.,  51;  Grimmer,  Bioch.  Zeitschr.,  3. 

6  Amer.  Journ.  of  Physiol.,  12  and  20;  Amer.  Journ.  Med.  Sciences,  138,  504  (1909). 

•Bioch.  Zeitschr.  34,  94  (1911). 

T  Zeitschr.  f.  physiol.  Chem.  68,  (1910). 


DIGESTION    IN   THE  STOMACH.  483 

upon  the  pyloric  sphincter.  The  proteins  combine  with  the  hydrochloric  acid 
and  hence  its  action  upon  the  sphincter  becomes  weaker,  while  this  is  not  the 
case  with  the  carbohydrates.  If  the  carbohydrates  are  moistened  with  alkali 
tin  y  Leave  the  Btomach  more  slowly  than  usual,  and  the  acid  proteins,  on  the  con- 
trary, leave  the  stomach  earlier  than  other  proteins. 

As  our  knowledge  of  the  digestibility  of  the  different  foods  in  the 
stomach  is  slight  and  uncertain,  so  also  our  knowledge  of  the  action  of 
other  bodies,  such  as  alcoholic  drinks,  bitter  principles,  spices,  etc.,  on 
the  natural  digestion  is  very  uncertain  and  imperfect.  The  difficulties 
which  stand  in  the  way  of  this  kind  of  investigation  are  very  great,  and 
therefore  the  results  obtained  thus  far  are  often  ambiguous  or  conflict 
with  each  other.  For  example,  certain  investigators  have  observed  that 
small  quantities  of  alcohol  or  alcoholic  drinks  do  not  prevent  but  rather 
facilitate  digestion;  others  observed  only  a  disturbing  action,  while 
still  others  report  having  found  that  the  alcohol  first  acts  somewhat  as 
a  disturbing  agent,  but  afterward,  when  it  is  absorbed,  produces  and  abun- 
dant secretion  of  gastric  juice,  and  thereby  facilitates  digestion.  The 
accelerating  action  of  alcohol  upon  the  flow  of  gastric  juice  has  been 
mentioned  on  page  464. 

In  regard  to  the  importance  of  the  stomach  we  used  to  be  of  the 
general  opinion  that  an  abundant  peptonization  of  protein  does  not 
occur  in  the  stomach,  and  that  the  food  rich  in  protein  is  only 
chiefly  prepared  in  the  stomach  for  the  real  digestion  in  the  intestine. 
That  the  stomach,  at  least  the  fundus,  acts  in  the  first  place  as  a  storage 
chamber,  follows  from  the  shape  of  this  organ,  especially  in  certain 
animals,  and  this  function  becomes  especially  prominent  in  certain  new- 
born animals,  as  dogs  and  cats.  In  these  animals  the  gastric  secretion 
contains  acid  but  no  pepsin,  and  the  casein  of  the  milk  is  precipitated 
by  the  acid  alone  as  solid  lumps  or  as  a  solid  coagulum  filling  the 
stomach.  Gradually  small  quantities  of  this  coagulum  pass  into  the 
intestine  and  an  overburdening  of  the  intestine  is  thus  prevented.  In 
other  animals,  as  the  snake  and  certain  fishes  which  swallow  entire  animals, 
the  major  part  of  the  digestive  work  goes  on  in  the  stomach.  The 
importance  of  the  stomach  for  digestion  cannot  therefore  be  established 
in  all  instances.  It  varies  in  different  animals  and  differs  even  in  indi- 
vidual animals  of  the  same  species,  depending  upon  the  fineness  or  coarse- 
ness of  the  food,  upon  the  greater  or  less  rapidity  with  which  pepton- 
ization takes  place,  and  also  upon  the  rapid  or  slow  increment  in  the 
quantity  of  hydrochloric  acid,  etc. 

In  regard  to  the  extent  of  chemical  digestive  work,  i.e.,  in  the  first 
place  the  destruction  of  protein  in  the  stomach,  we  have  numerous 
researches,  some  carried  out  by  the  use  of  older  methods  and  others 
by  using  newer  and  more  reliable  methods.  Among  these  latter  we 
must  mention  those  of  Zunz,  London  and  collaborators,  Tobler,  Lanq 


484  DIGESTION. 

and  Cohnheim.1  These  investigations  refer  to  the  conditions  in  dogs, 
and  as  shown  by  Rosenfeld  2  in  horses,  and  by  Lotsch  3  in  pigs,  that 
the  conditions  are  different  in  other  animals.  The  following  description 
applies  only  to  dogs. 

In  the  dog  Abderhalden,  London  and  co-workers4  have  shown 
that  in  the  stomach  proteoses  and  peptones  are  formed,  but  no  amino- 
acids,  or  at  least  not  in  any  mentionable  quantity.  The  scanty  occurrence 
-of  amino-acids  is  substantiated  by  the  observations  of  Zunz  and  others  5 
that  the  amount  of  amino-nitrogen  titratable  with  formol  in  the  stomach 
contents,  is  only  small. 

In  like  manner  we  must  agree  in  the  belief  that  a  part  of  the  protein 
always  leaves  the  stomach  undigested  and  that  the  principal  mass,  about 
80  per  cent,  passes  into  the  intestine  more  or  less  digested.  Besides  this 
it  also  seems  as  if  the  peptones  occur  in  the  pylorus  part  to  a  greater 
extent  than  the  proteoses,  while  in  the  fundus  part  the  reverse  is  the 
case.  Of  the  dissolved  protein  of  the  entire  stomach-contents  about 
60  per  cent  exists  as  proteoses.  Opinions  are  also  contradictory  in 
regard  to  the  absorption  of  the  decomposition  products  of  the  proteins 
in  the  stomach.  While  several  investigators,  like  Tobler,  Lang,  Cohn- 
heim, Zunz  and  others  accept  such  an  absorption,  London  and  co-workers 
positively  deny  this. 

The  digestion  of  sundry  foods  is  not  dependent  on  one  organ  alone, 
but  is  divided  among  several.  For  this  reason  it  is  to  be  expected  that 
the  various  digestive  organs  can  act  for  one  another  to  a  certain  extent, 
and  that  therefore  the  work  of  the  stomach  could  be  taken  up  more  or 
less  by  the  intestine.  This  in  fact  is  the  case.  Thus  the  stomachs  of 
dogs  and  cats  have  been  completely  extirpated  or  nearly  so  (Czerny, 
Carvallo  and  Pachon,  London  and  collaborators),  or  that  part 
necessary  in  the  digestive  process  has  also  been  eliminated  by  plugging 
the  pyloric  opening  (Ludwig  and  Ogata),  and  in  both  cases  it  was  pos- 
sible to  keep  the  animal  alive,  well  fed,  and  strong  for  a  shorter  or 
longer  time.      The  extirpation  of  the  stomach  has  also  been  repeatedly 

1  Tobler,  Zeitschr.  f.  physiol.  Chem.,  45;  Lang,  Bioch.  Zeitschr.,  2;  Cohnheim, 
Munch,  med.  Wochenschr.,  1907.  In  regard  to  the  works  of  Zunz,  London,  and 
collaborators,  see  footnotes  1,  2  and  3,  p.  482. 

2  Rosenfeld,  Ueber  die  Eiweissverdauung  im  Magen  des  Pferdes,  Inaug.-Dissert., 
Dresden,  1908. 

'  Lotsch.  Zur  Kenntnis  der  Verdauung  von  Fleisch  im  Magen  und  Dunndarm  des 
Schweines,  Inaug.-Dissert.  Freiburg  i.  Sa.,  1908;  see  also  Abderhalden,  Klingemann 
and  Pappenhusen,  Zeitschr.  f.  physiol.  Chem.  71,  411  (1911). 

4  Abderhalden  and  London,  with  Kautsch,  Zeitschr.  f.  physiol.  Chem.,  48,  with 
L.  Baurnann,  ibid.,  51,  and  with  v.  Korosy,  ibid.,  51. 

:  Intern.  Beitr.  zu  Path.  u.  Ther.  d.  Ern.-Stor.  2;  London  and  Rabinowitsch, 
Zeitschr.  f.  physiol.  Chem.  74. 


DIGESTION   IN  THE   STOMACH.  485 

performed  on* human  beings  with  the  same  results.1  In  these  cases  it  is 
evident  that  the  digestive  work  of  the  stomach  was  taken  up  by  the 
intestine;  but  all  food  cannot  be  digested  in  these  cases  to  the  same 
extent,  and  the  connective  tissue  of  meat  especially  is  sometimes  found 
to  a  considerable  extent  undigested  in  the  excrements. 

It  is  a  well-known  fact  that  the  contents  of  the  stomach  may  be 
kept  without  decomposing  for  some  time  by  means  of  hydrochloric  acid, 
while,  on  the  contrary,  when  the  acid  is  neutralized  a  fermentation 
commences  by  which  lactic  acid  and  other  organic  acids  are  formed. 
According  to  Cohn,  an  amount  of  hydrochloric  acid  above  0.7  p.  m. 
completely  arrests  lactic-acid  fermentation,  even  under  otherwise  favor- 
able circumstances,  and  according  to  Strauss  and  Bialocour  the  limit 
of  lactic-acid  fermentation  lies  at  1.2  p.  m.  hydrochloric  acid  united 
to  organic  bodies.  The  hydrochloric  acid  of  the  gastric  juice  has  unques- 
tionably an  antifermentative  action,  and  also,  like  all  dilute  mineral 
acids,  an  antiseptic  action.  This  action  is  of  importance,  as  many  path- 
ogenic micro-organisms  may  be  destroyed  by  the  gastric  juice.  The 
common  bacillus  of  cholera,  certain  streptococci,  etc.,  are  killed  by  the 
gastric  juice,  while  others,  especially  as  spores,  are  unacted  upon.  The 
fact  that  gastric  juice  can  diminish  or  retard  the  action  of  certain  tox- 
albumins,  such  as  tetanotoxine  and  diphtheria  toxine,  is  also  of  great 
interest  (Nencki,  Sieber,  and  Schoumowa2). 

Because  of  this  antifermentative  and  antitoxic  action  of  gastric  juice 
it  is  considered  that  the  principal  importance  of  this  juice  lies  in 
its  antiseptic  action.  The  fact  that  intestinal  putrefaction  is  not 
increased  on  the  extirpation  of  the  stomach,  as  derived  from  experi- 
ments made  on  man  and  animal,3    does  not  uphold  this  view. 

Since  the  hydrochloric  acid  of  the  gastric  juice  prevents  the  con- 
tents of  the  stomach  from  fermenting,  with  the  generation  of  gas,  those 
gases  which  occur  in  the  stomach  probably  depend,  at  least  in  great 
measure,  upon  the  swallowed  air  and  saliva,  and  upon  those  gases  gen- 
erated in  the  intestine  and  returned  through  the  pyloric  valve.  Planer 
found  in  the  stomach-gases  of  a  dog  66-68  per  cent  N,  23-33  per  cent 

1  Czerny,  cited  from  Bunge,  Lehrbuch  d.  physiol.  u.  path.  Chem.  4.  Aufl.,  Theil  2, 
173;  Carvallo  and  Pachon,  Arch.  d.  Physiol.  (5),  7;  Ogata,  Arch.  f.  (Anat.  u.)  Physiol. 
1883;  Grohe,  Arch.  f.  exp.  Path.  u.  Pharm.  49;  London  and  collaborators,  Zeitschr. 
f.  physiol.  Chem.  74,  328  (1911)  ;  in  regard  to  the  case  in  man,  see  Schlatter  in 
Wr6blewski,  Centralbl.  f.  Physiol.  11,  p.  665,  and  the  surgical  journals. 

2  Cohn,  Zeitschr.  f.  physiol.  Chem.,  14;  Strauss  and  Bialocour,  Zeitschr.  f.  klin. 
Med.,  28.  See  also  Kiihne,  Lehrb.,  57;  Bunge,  I.ehrb.  d.  Physiol.,  4.  Aufl.,  148  and 
159;  Hirschfeld,  Pfliiger's  Arch.,  47;  Nencki,  Sieber,  and  Schoumowa,  Centralbl.  f. 
Bacterid.,  etc.,  23.  In  regard  to  the  action  of  gastric  juice  upon  pathogenic  microbes 
we  must  refer  the  reader  to  hand-books  of  bacteriology. 

3  See  Carvallo  and  Pachon.  1.  c,  and  Schlatter  in  Wr6blewski,  1.  c. 


486  DIGESTION. 

CO2,  and  only  a  small  quantity,  0.8-6.1  per  cent,  of  oxygen.  Schier- 
beck  !  has  shown  that  a  part  of  the  carbon  dioxide  is  formed  by  the 
mucous  membrane  of  the  stomach.  The  tension  of  the  carbon  dioxide 
in  the  stomach  corresponds,  according  to  him,  to  30-40  mm.  Hg  in  the 
fasting  condition.  It  increases  after  partaking  food,  independently  of 
the  kind  of  food,  and  may  rise  to  130-140  mm.  Hg  during  digestion. 
The  curve  of  the  carbon-dioxide  tension  in  the  stomach  is  the  same  as 
the  curve  of  acidity  in  the  different  phases  of  digestion,  and  Schier- 
beck  also  found  that  the  carbon-dioxide  tension  is  considerably  increased 
by  pilocarpine,  but  diminished  by  nicotine.  According  to  him,  the 
carbon  dioxide  of  the  stomach  is  a  product  of  the  activity  of  the  secretory 
cells. 

After  death,  if  the  stomach  still  contains  food,  autodigestion  goes 
on  not  only  in  the  stomach,  but  also  in  the  neighboring  organs,  during 
the  slow  cooling  of  the  body.  This  leads  to  the  question,  Why  does  the 
stomach  not  digest  itself  during  life?  Ever  since  Pavy  has  shown  that 
after  tying  the  smaller  blood-vessels  of  the  stomach  of  dogs  the  cor- 
responding part  of  the  mucous  membrane  was  digested,  efforts  have 
been  made  to  find  the  cause  in  the  neutralization  of  the  acid  of  the  gas- 
tric juice  by  the  alkali  of  the  blood.  That  the  reason  for  the  non- 
digestion  during  life  is  to  be  sought  for  in  the  normal  circulation  of  the 
blood  cannot  be  contradicted;  but  the  reason  is  not  to  be  found  in  the 
direct  neutralization  of  the  acid.  The  investigations  of  Fermi  and 
Otte  2  show  that  the  blood  circulation  acts  in  an  indirect  manner  by  the 
normal  nourishment  of  the  cell  protoplasm,  and  this  is  the  reason  why 
the  digestive  fluids,  the  gastric  juice  as  well  as  the  pancreatic  juice,  act 
differently  upon  the  living  protoplasm  as  compared  with  the  dead.  We 
know  nothing  about  this  resistance  of  the  living  protoplasm.  Some 
claim  that  it  is  closely  connected  with  occurrence  of  different  inhibitory 
substances  in  the  gastric  mucosa.  Of  these  the  substance  found  by 
Weinland  is  thermolabile  while  that  of  Danilewsky,  Hansel  and 
Schwarz  is  resistant  toward  heat.3  Without  mentioning  the  still  un- 
known nature  of  these  bodies,  the  neutral  gastric  juice,  as  well  as  an 
acid  infusion  of  the  mucosa,  has  such  a  strong  digestive  action  that  the 
inhibiting  action  of  the  mentioned  substances  can  only  be  shown  under 
special  conditions,  and  it  is  therefore  difficult  to  conceive  how  these  sub- 
stances could  have  a  protective  action  in  life. 

1  Planer,  Wien.  Sitzungsber.,  42;  Schierbeck,  Skand.  Arch.  f.  Physiol.,  3  and  5. 

2  Pavy,  Phil.  Transactions,  153,  Part  I,  and  Guy's  Hospital  Reports,  13;  Otte, 
Travaux  du  laboratoire  de  l'lnstitut  de  Physiol,  de  Liege,  5,  1896,  which  also  contains 
the  literature. 

1  Wf-inland,  Zeitschr.  f.  Biologie,  44;  Hansel,  Biochem.  Centralbl.,  1,  p.  404, 
and  2,  p.  320;  Schwartz,  Hofmeister's  Beitrage,  6. 


EXAMINATION    OF  THE   GASTRIC   CONTENTS.  487 

Under  pathological  conditions  irregularities  in  the  secretion  may 
occur.  The  quantity  of  enzymes  may  be  diminished  and  both  enzymes 
or,  as  found  in  certain  cases,  one  (the  rennin),  may  be  absent.  The 
hydrochloric  acid  may  also  be  absent  or  may  exist  in  very  small  amounts. 
A  pathological  high  degree  of  acidity  of  the  pure  juice  is  not  very  prob- 
able, while  on  the  contrary  a  hypersecretion  of  gastric  juice  in  different 
forms  does  occur. 

In  testing  the  gastric  juice  or  the  filtered  stomach  contents,  diluted 
with  digestive  hydrochloric  acid,  for  pepsin,  we  make  use  of  the  pepsin 
tests  given  on  pages  469,  470.  In  testing  for  rennin  the  liquid  must  be 
first  carefully  neutralized,  and  1-2  cc.  of  this  liquid  added  to  10  cc. 
milk.  In  the  presence  of  appreciable  quantities  of  rennin,  the  milk 
should  coagulate  at  room  temperature  within  10-20  minutes  without 
changing  its  reaction.  The  addition  of  lime  salts  is  unnecessary,  and 
may  readily  lead  to  erroneous  conclusions. 

In  many  cases  it  is  especially  important  to  determine  the  degree  of 
acidity  of  the  gastric  juice.  This  may  be  done  by  the  ordinary  titration 
methods.  Phenolphthalein  must  not  be  used  as  an  indicator,  as  too 
high  results  are  produced  in  the  presence  of  large  quantities  of  proteins. 
Good  results  may  be  obtained,  on  the  contrary,  by  using  very  delicate 
litmus  paper.  Although  the  acid  reaction  of  the  contents  of  the  stomach 
may  be  caused  simultaneously  by  several  acids,  still  the  degree  of  acidity 
is  here,  as  in  other  cases,  expressed  in  only  one  acid,  e.g.,  HO.  Gen- 
erally the  acidity  is  designated  by  the  number  of  cubic  centimeters  of 
N/10  sodium  hydroxide  required  to  neutralize  the  several  aeids  in 
100  cc.  of  the  liquid  of  the  stomach.  An  acidity  of  43  per  cent  means 
that  100  cc.  of  the  liquid  of  the  stomach  required  43  cc.  of  N/10  sodium 
hydroxide  to  neutralize  it. 

It  is  also  important  to  be  able  to  ascertain  the  nature  of  the  acid  or 
acids  occurring  in  the  contents  of  the  stomach.  For  this  purpose,  and 
especially  for  the  detection  of  free  hydrochloric  acid,  a  great  number  of  color 
reactions  have  been  proposed  which  are  all  based  upon  the  fact  that  the 
coloring  substance  gives  a  characteristic  color  with  very  small  quanti- 
ties of  hydrochloric  acid,  while  lactic  acid  and  the  other  organic  acids 
do  not  give  these  colorations,  or  only  in  a  certain  concentration,  which 
can  hardly  exist  in  the  contents  of  the  stomach.  These  reagents  are  a 
mixture  of  ferric-acetate  and  potassium-sulphocyanide  solutions 
(Mohr's  reagent  has  been  modified  by  several  investigators),  methyl- 
aniline-violet,   TROPiEOLIN  00,   CONGO  RED,   MALACHITE-GREEN,  PHLORO- 

glucinol-vanillin,  dimethylaminoazobenzene,  and  others.  As  reagents 
for  free  lactic  acid,  Uffelmann  suggests  a  strongly  diluted,  amethyst-blue 
solution  of  ferric  chloride  and  carbolic  acid,  or  a  strongly  diluted 
nearly  colorless  solution  of  ferric  chloride.  These  give  a  yellow 
color  with  lactic  acid,  but  not  with  hydrochloric  acid  or  with  volatile 
fatty  acids. 

The  value  of  these  reagents  in  testing  for  free  hydrochloric  acid  or  lactic 
acid  is  still  disputed.  Among  the  reagents  for  free  hydrochloric  acid  it  seems 
Steensma's  '  modification  of  Gunzburg's  test  with  phloroglucinol-vanilhn,  and 

1  Bioch.  Zeitschrift,  8. 


488  DIGESTION. 

the  test  with  tropseolin  00,  performed  at  a  moderate  temperature  as  suggested  by 
Boas,  and  the  test  with  dimethylamincazobenzene,  which  is  the  most  delicate, 
seem  to  be  the  most  valuable.  If  these  tests  give  positive  results,  then  the  presence 
of  hydrochloric  acid  may  be  considered  as  proved.  A  negative  result  does  not 
eliminate  the  presence  of  hydrochloric  acid,  as  the  delicacy  of  these  reactions 
has  a  limit,  and  also  the  simultaneous  presence  of  protein,  peptones,  and  other 
bodies  influences  the  reactions  more  or  less.  The  reactions  for  lactic  acid  may 
also  give  negative  results  in  the  presence  of  comparatively  large  quantities  of 
hydrochloric  acid  in  the  liquid  to  be  tested.  Sugar,  sulphocyanides,  and  other 
bodies  may  act  with  these  reagents  like  lactic  acid. 

In  testing  for  lactic  acid  it  is  safest  to  shake  the  material  with  ether  and  test 
the  residue  after  the  evaporation  of  the  solvent.  On  the  evaporation  of  the  ether 
the  residue  may  be  tested  in  several  ways.  Boas  utilizes  the  property  possessed 
by  lactic  acid  of  being  converted  into  aldehyde  and  formic  acid  on  careful  oxida- 
tion with  sulphuric  acid  and  manganese  dioxide.  The  aldehyde  is  detected  by 
its  forming  iodoform  with  an  alkaline  iodine  solution  or  by  its  forming  aldehyde- 
mercury  with  Nessler's  reagent.  Croner  and  Cronheim:  x  have  suggested 
another  method. 

The  quantitative  estimation  consists  in  the  formation  of  iodoform  with  N/10 
iodine  solution  and  caustic  potash,  adding  an  excess  of  hydrochloric  acid  and 
titrating  with  a  N/10  sodium-arsenite  solution,  and  retitrating  with  iodine  solu- 
tion, after  the  addition  of  starch-paste,  until  a  blue  coloration  is  obtained.  This 
method  presupposes  the  use  of  ether  entirely  free  from  alcohol.  For  details  see 
the  original  publication  and  the  modification  of  this  method  suggested  by 
Jerusalem.2 

In  order  to  be  able  to  judge  correctly  of  the  value  of  the  different 
reagents  for  free  hydrochloric  acid,  it  is  naturally  of  greatest  importance 
to  be  clear  in  regard  to  what  we  mean  by  free  hydrochloric  acid.  It  is 
a  well-known  fact  that  hydrochloric  acid  combines  with  proteins,  and  a 
considerable  part  of  the  hydrocholoric  acid  may  therefore  exist  in  the 
contents  of  the  stomach,  after  a  meal  rich  in  proteins,  in  combination 
with  them.  This  hydrochloric  acid  combined  with  proteins  cannot 
be  considered  as  free,  and  it  is  for  this  reason  that  certain  investigators 
consider  such  methods  as  those  of  Sjoqvist,  which  will  be  described 
below,  as  of  little  value.  However,  it  must  be  remarked  that,  according 
to  the  unanimous  experience  of  many  investigators,  the  hydrochloric 
acid  combined  with  proteins  is  physiologically  active  and  in  this  regard 
we  must  refer  to  the  recent  investigations  of  Alb.  Muller  and  J.  Schutz.3 
Those  reactions  (color  reactions)  which  only  respond  to  actually  free 
hydrochloric  acid  do  not  show  the  physiologically  active  hydrochloric 
acid.  The  suggestion  of  determining  the  "  physiologically  active " 
hydrochloric  acid  instead  of  the  "  free  "  seems  to  be  correct  in  principle; 
and  as  the  conceptions  of  free  and  of  physiologically  active  hydrochloric 
acid  are  not  the  same,  it  must  always  be  well  defined  whether  one  wishes 
to   determine   the   actually   free   or   the    physiologically   active   hydro- 


1  Boas.  Deutsch.  med.  Wochenschr.,  1803,  and  Munchener  med.  Wochenschr.  1893, 
Croner  and  Cronheim,  Berl.  klin.  Wochenschr.,  1905.  See  also  Thomas,  Zeitschr.  f. 
physiol.  Chem.,  50. 

2  Bioch.  Zeitschr.,  12. 

8  Alb.  Muller,  Deutsch.  Arch.  f.  klin.  Med.,  88,  and  Pfluger's  Arch.,  116;  J.  Schiitz. 
Wien,  klin.  Wochenschr.,  20.  and  Wien.  med.  Wochenschr.,   1900  (older  literature). 


SECRETION  OF  BRUNNER'S  GLANDS.  489 

chloric  acid  before  any  conclusions  are  drawn  as  to  the  value  of  a  certain 
reaction. 

The  acid  reaction  may  be  partly  due  to  free  acid,  partly  to  acid  salts  (mono- 
phosphates), and  partly. to  both.  According  to  Leo,1  one  can  test  for  acid  phos- 
phates by  calcium  carbonate,  which  is  not  neutralized  therewith,  while  the  free 
acids  are.  If  the  gastric  content  has  a  neutral  reaction  after  shaking  with  cal- 
cium carbonate,  and  the  carbon  dioxide  is  driven  out  by  a  current  of  air,  it  con- 
tains only  free  acid;  if  it  has  an  acid  reaction,  acid  phosphates  are  present, 
and  if  it  is  less  acid  than  before,  it  contains  both  free  acid  and  acid  phosphate. 
It  must  not  be  forgotten  that  a  faint  acid  reaction  may,  after  treatment  with  cal- 
cium carbonate,  also  be  due  to  the  protein.  This  method  can  likewise  be  applied 
in  the  estimation  of  free  acid. 

Various  titration  methods  have  been  suggested  for  the  estimation  of  the 
free  hydrochloric  acid,  but  these  cannot  yield  conclusive  results  for  the  reasons 
given  in  Chapter  I.  For  this  determination  physico-chemical  methods  (page  74), 
are  necessary,  but  they  have  not  been  used  to  any  great  extent  for  clinical  pur- 
poses. Holmgren  2  has  suggested  a  method  for  estimating  hydrochloric  acid 
based  upon  the  adsorption  phenomenon. 

A  great  number  of  methods  have  been  suggested  for  the  quantitative  estima- 
tion of  the  total  acidity,  among  which  we  must  mention  those  of  K.  Morner  and 
Sjoqvist,  which  are  extensively  used.  As  the  value  of  a  special  determination 
of  the  free  and  total  hydrochloric  acid  is  doubtful,  or  at  least  disputed,  and  also  as 
the  question  is  chiefly  of  clinical  interest  we  must  refer  to  the  hand-books  of  clinical 
investigations  of  v.  Jaksch,  Eulenburg,  Kolle,  and  Weintraud  and  of  Sahli. 
The  same  applies  to  the  tests  for  volatile  fatty  acids. 

m.     THE  GLANDS  OF  THE  MUCOUS  MEMBRANE  OF  THE  INTESTINE  AND 

THEIR   SECRETIONS. 

The  Secretion  of  Brunner's  Glands.  These  glands  are  partly  con- 
sidered as  small  pancreatic  glands  and  partly  as  mucous  or  salivary 
glands.  Their  importance  is  not  the  same  in  all  animals.  According 
to  Grutzner  they  are  in  dogs  closely  related  to  the  pyloric  glands  and 
contain  pepsin.  This  also  coincides  with  the  observations  of  Glaessner 
and  of  Ponomarew,  which  differ  from  each  other  only  in  that  Pono- 
marew  finds  that  the  secretion  is  inactive  in  alkaline  reaction  and  con- 
tains only  pepsin,  while  Glaessner  claims  it  is  active  in  both  acid  and 
alkaline  reaction  and  that  it  contains  pseudopepsin.  According  to 
Abderhalden  and  Rona  the  pure  duodenal  secretion  of  the  dog  contains 
a  proteolytic  enzyme  which  does  not  belong  to  the  trypsin  type  but 
rather  to  the  pepsin  variety.  The  statements  as  to  the  occurrence  of  a 
diastatic  enzyme  in  Brunner's  glands  are  disputed.  Scheunert  and 
Grimmer  3  indeed  found  diastatic  enzyme  in  the  duodenal  glands  of  the 
horse,  ox,  pig  and  rabbit,  but  no  proteolytic  or  rennin  enzyme. 

Centralbl,  f.  d.  med.  Wissensch.,  1889,  p.  481;  Pfliiger's  Arch.,  48,  and  Berlin, 
klin.  Wochenschr.,  1905,  p.  1491. 

2  Deutsch.  med.  Wochenschr.,  1911,  p.  247. 

3  Grutzner,  Pfluger's  Arch.,  12;  Glaessner,  Hofmeister's  Breitrage,  1;  Ponomarew, 
Biochem.  Centralbl.,  1,  351;  Abderhalden  and  Rona,  Zeitschr.  f.  physiol.  Chem., 
47;  Scheunert  and  Grimmer,  cited  in  Bioch.  Centralbl.,  5,  673. 


490  DIGESTION. 

The  Secretion  of  Lieberkuhn's  Glands.  The  secretion  of  these  glands 
has  been  studied  with  the  aid  of  a  fistula  in  the  intestine  according  to 
the  method  of  Thiry  and  Vella  or  of  Pawlow.  According  to  Boldy- 
reff,1  in  dogs,  with  an  empty  stomach,  a  scanty  secretion  lasting  about 
15  minutes  occurs  at  regular  intervals  for  about  two  hours.  According 
to  Boldyreff  the  intestinal  juice  is  obtained  from  a  Thiry- Villa  fistula 
outside  of  the  digestion  period  without  any  apparent  stimulation.  Accord- 
ing to  this  experimenter,  during  gastric  digestion  the  juice  is  periodically 
but  less  abundantly  secreted  as  the  time  interval  is  much  longer,  namely 
three,  four  or  five  hours.  Otherwise  it  is  generally  admitted  that  the 
partaking  of  food  causes  the  secretion,  or  if  this  is  continuous,  as  in 
lambs  (Pregl),  it  increases  the  secretion.  The  researches  of  Dele- 
zenne  and  Frouin  show  without  question  that  the  passage  of  chyme 
into  the  intestine  increases  the  secretion  of  the  intestinal  juice.  The 
acid  causes  a  formation  of  secretin  (see  below),  and  this  produces, 
according  to  the  above  investigators,  a  secretion  of  intestinal  juice. 
Among  the  chemically  active  substances  causing  a  secretion  we  must 
mention  acids  in  general  and  gastric  juice.  Soaps,  chloral,  ether  and 
on  intravenous  injection,  also  intestinal  juice  or  an  extract  of  the  intes- 
tinal mucosa  (Frouin),  are  chemical  excitants  of  intestinal  juice. 
Several  salts,  NaCl,  Na2SOi,  and  others,  may  cause  an  abundant  secre- 
tion of  fluid  into  the  intestine  when  injected  intravenously  or  subcu- 
taneously,  as  well  as  after  direct  application  to  the  peritoneal  surface 
of  the  intestine.  This  action  can  be  arrested  by  the  antagonistic, 
inhibiting  action  of  a  lime  salt  (MacCallum).  Pilocarpine,  which  has 
the  power  of  increasing  the  activity  of  secretions,  does  not  increase  the 
secretion  in  lambs,  and  in  dogs  it  does  not  seem  to  be  always  active 
(Gamgee2). 

Mechanical  irritation  of  the  intestinal  mucosa  increases  the  secre- 
tion in  dogs  (Thiry)  as  well  as  in  man  (Hamburger  and  Hekma),  but 
it  is  still  doubtful  whether  we  here  have  a  perfectly  physiological  juice. 
In  the  cases  observed  by  Hamburger  and  Hekma3  the  flow  of  fluid  was 
greatest  at  night  as  well  as  between  five  and  eight  o'clock  in  the  after- 
noon, and  was  lowest  between  two  and  five  o'clock  in  the  afternoon. 
The  quantity  of  this  secretion  in  the  course  of  twenty-four  hours  has 
not  been  exactly  determined. 


1  Thiry,  Wien,  Sitz.-Ber.,  50;  Vella,  Molleschott's  Untersuch.,  13;  Boldyreff, 
Zeitschr.  f.  physiol.  Chem.,  50,  Centralbl.  f.'Physiol.  24,  93  (1910). 

2  Delezenne  and  Frouin,  Compt.  rend.  soc.  biol.,  56;  Frouin,  ibid.,  56  and  58; 
MacCallum,  University  of  California  Publications,  1,  1904;  Gamgee,  Physiol.  Chem- 
istry, 2,  410  (literature). 

3  Journ.  de  Physiol,  et  d.  path,  gen.,  1902  and  1904. 


INTESTINAL  JUICE.  491 

According  to  Delezenne  and  Frouin,  if  any  mechanical  irritation 
is  prevented,  the  fluid  flowing  spontaneously  from  a  fistula  in  a  dog 
is  ten  times  more  abundant  in  the  duodenum  than  that  in  the  middle 
or  lower  part  of  the  jejunum.  In  the  upper  part  of  the  small  intestine 
of  the  dog,  on  the  contrary,  this  secretion  is  scanty,  slimy,  and  gelatin- 
ous; in  the  lower  part  it  is  more  fluid,  with  gelatinous  lumps  or  flakes 
(Rohmann).  Intestinal  juice  has  a  strong  alkaline  reaction  toward 
litmus,  generates  carbon  dioxide  on  the  addition  of  an  acid,  and  contains 
(in  dogs)  nearly  a  constant  quantity  of  NaCl  and  Na2CC>3,  4.8-5  and  4-5 
p.  m.  respectively  (Gumilewski,  Rohmann1).  The  intestinal  juice 
of  the  lamb  corresponded  to  an  alkalinity  of  4.54  p.  m.  Na2C03.  It 
contains  protein  (Thiry  found  8.01  p.  m.),  the  quantity  decreasing 
with  the  duration  of  the  elimination.  The  quantity  of  solids  varies. 
In  dogs  the  quantity  of  solids  is  12.2-24.1  p.  m.  and  in  lambs  29.85  p.  m. 
The  specific  gravity  of  the  intestinal  juice  of  the  dog,  according  to  the 
observations  of  Thiry,  is  1.010-1.0107,  and  in  lambs  1.0143  (Pregl). 
The  intestinal  juice  from  lambs  contains  18.097  p.  m.  protein,  1.274  p. 
m.  proteoses  and  mucin,  2.29  p.  m.  urea,  and  3.13  p.  m.  remaining  organic 
bodies. 

We  have  the  investigations  of  Demant,  Turby  and  Manning,  H. 
Hamburger  and  Hekma  and  Nagano2  on  the  human  intestinal  juice. 
Human  intestinal  juice  has  a  low  specific  gravity,  nearly  1.007,  about 
10-14  p.  m.  solids,  and  is  strongly  alkaline  toward  litmus.  The  con- 
tent of  alkali  calculated  as  sodium  carbonate  is  2.2  p.  m.,  according 
to  Nagano,  Hamburger  and  Hekma,  and  5.8-6.7  p.  m.  Na  CI.  The 
determination  of  the  freezing-point  was  —0.62°  (Hamburger  and  Hekma). 

The  intestinal  juice  of  the  dog  contains,  according  to  Boldyreff 
a  lipase  which  acts  especially  upon  emulsified  fat  (milk),  and  is  different 
from  pancreas  lipase,  in  that  its  action  is  not  accelerated  by  bile. 
Jansen  3  found  that  the  lipase  was  secreted  from  a  Thiry- Vella  fistula 
especially  under  the  influence  of  bile  and  acid.  The  intestinal  juice  of 
animals  and  man  also  contains  an  enzyme,  erepsin,  discovered  by  O. 
Cohnheim,  which  does  not  ordinarily  have  a  splitting  action  upon  native 
proteins,  but  upon  proteoses  and  peptones.  It  also  possibly  contains 
a  nuclease,  and  it  also  has  a  faint  amylolytic  action.  The  juice,  and  to 
a  high  degree  the  mucous  coat,  contains  iwertase  and  maltase,  which 


1  Gumilewski,  Pfliiger's  Arch.,  39.  Rohmann,  ibid.,  41. 

2  Demant,  Virchow's  Arch.,  75;  Turby  and  Manning,  Centralbl.  f.  d.  med.  Wis- 
senschaft,  1892,  945;  Hamburger  and  Hekma,  1.  c;  Nagano,  Mitt,  aus  d.  Grenzgeb. 
d.  Med.  u.  Chir.,  9. 

1  Boldyreff,  Archiv.  d.  sciences  biolog,  de  St.  P6tersbourg,  11;  Jansen,  Zeitschr. 
f.  physiol.  Chem.  58,  400  (1910). 


492  DIGESTION. 

fact  has  been  substantiated  by  the  observations  of  Paschutin,  Brown" 
and  Heron,  Bastianelli,  and  Tebb.1  A  lactose-inverting  enzyme, 
a  lactase,  also  occurs,  as  shown  by  Rohmann  and  Lappe,  Patjtz  and 
Vogel,  Weinland,  and  Orban,2  in  new-born  infants  and  young  ani- 
mals, and  also  in  grown  mammals  which  were  fed  upon  a  milk  diet  (see 
Chapter  I,  page  52).  The  lactase  can  be  obtained  more  abundantly 
from  the  mucosa  than  from  the  juice  and  according  to  some  occurs  only 
in  the  cells.  The  claims  as  to  the  occurrence  of  a  glucoside  splitting 
enzyme  are  disputed  (Frouin,  Omi  3) . 

Besides  erepsin  and  the  other  enzymes  mentioned,  the  intestinal 
mucosa  also  contains  substances  which  have  an  inhibitory  action  upon 
pepsin  and  trypsin.  (Danilewsky  and  Weinland4),  also  enterokinase 
or  a  mother-substance  of  the  same,  and  finally  also  the  so-called  'pro- 
secretin. These  two  last-mentioned  bodies,  which  are  closely  connected 
with  the  secretion  of  pancreatic  juice,  will  be  discussed  in  connection 
with  this  digestive  fluid. 

The  various  enzymes  are  not  formed  in  equal  quantities  in  all  parts 
of  the  intestine.  Diastase  and  invertase  occur,  according  to  Boldy- 
reff,  all  through  the  intestine,  while  the  lipase  on  the  contrary  does  not 
occur  in  the  lower  parts.  The  kinase  occurs  only  in  the  upper  part  of 
the  intestine  (Boldyreff,  Bayliss  and  Starling,  Delezenne).  Ac- 
cording to  Hekma  the  kinase  occurs  in  all  parts  of  the  intestine,  but 
most  abundantly  in  the  duodenum  and  the  upper  part  of  the  jejunum. 
The  enzymes,  Falloise  claims,  generally  occur  in  greatest  abundance 
in  the  upper  parts  of  the  intestine;  but  the  erepsin  occurs  to  a  greater 
extent  in  the  jejunum  than  in  the  duodenum.  According  to  the  investi- 
gations of  Vernon  the  behavior  of  erepsin  is  not  the  same  in  different 
animals.  In  cats  and  hedge-hogs  the  duodenum  is  richer  in  erepsin  than 
the  jejunum  and  ileum;  in  rabbits  it  is  the  reverse,  namely,  the  ileum 
is  much  richer  than  the  duodenum.  The  secretin,  according  to  Bay- 
liss and  Starling,  is  formed  entirely  in  the  upper  part  of  the  intestine.. 
The  epithelium-cells  of  the  glands  or  the  mucous  membrane  are  generally 
considered  as  the  seat  of  formation  of  the  enzymes,  and  the  same  is 
true   also  for  the   enterokinase,   according  to  Bayliss   and  Starling,. 


Paschutin,  Centralbl.  f.  d.  med.  Wissensch.,  1870,  561;  Brown  and  Heron,  Annal. 
d.  Chem.  u.  Pharm.,  204;  Bastianelli,  Moleschott's  Untersuch,  zur  Naturlehre,  14 
(this  contains  all  the  older  literature).  See  also  Miura,  Zeitschr.  f.  Biologie,  32;  Wid- 
dicombe,  Journ.  of  Physiol.,  28;  Tebb,  ibid.,  15. 

2  Rohmann  and  Lappe,  Ber.  d.  deutsch,  chem.  Gessellch.,  28;  Pautz  and  Vogel, 
Zeitschr.  f.  Biologie.  32;  Weinland,  ibid.,  38;  Orban,  Maly's  Jahresber.,  29. 

3  Frouin  and  Thomas,  Arch,  internat.  de  Physiol.,  7;  Omi,  Das  Verhalten  dea 
Salizins  irn  tierschen  Organisrnus,  Inaug.-Dissert.  Breslau,  1907. 

*  See  footnote  3,  p.  486. 


EREPSIN.  493 

Hekma,  Falloise,  and  others,  which,  however,  Delezenne  says.1  is 
formed  in  the  leucocytes  and  Peyer's  glands. 

Erepsin.  This  enzyme,  discovered  by  O.  Cohnheim,  has  no  direct 
action  upon  native  proteins  with  the  exception  of  casein,  but  has  the 
power  of  splitting  proteoses,  peptones  and  certain  polypeptides.  In 
this  change  mono-  as  well  as  diamino-acids  are  produced.  Erepsin 
occurs  in  the  mucous  membrane  and  in  the  intestinal  juice  of  man  as 
well  as  of  dogs;  the  mucous  membrane  seems  to  be  richer  than  the  juice 
(Salaskin,  Kutscher  and  Seemann2).  An  enz3me  like  erepsin  also 
occurs  in  the  pancreas  (Bayliss  and  Starling,  Vernon),  and  this  has 
the  power  of  acting  upon  casein,  but  not,  or  only  faintly,  upon  fresh 
fibrin.  This  erepsin  is  probably  identical  with  the  enzyme  nvclease, 
discovered  by  F.  Sachs  in  the  pancreas,  which  acts  upon  nucleic  acids, 
while  Nakayama  claims  that  erepsin  differs  from  trypsin  in  having  a 
cleavage  action  upon  nucleic  acids.  Intestinal  erepsin  is  not  inhibited, 
according  to  Glaessner  and  Stauber,  by  blood-serum  and  differs  in 
this  regard  from  trypsin.  Erepsin  shows  a  great  similarity  to  the  intra- 
cellular enzymes  active  in  autolysis,  and  according  to  Vernon  and  others 
erepsins  occur  in  the  various  tissues  of  invertebrates  as  well  as  verte- 
brates. These  tissue  erepsins,  which  are  closely  related  to  the  auto- 
lytic  enzymes,  if  they  are  not  identical  with  them,  behave  somewhat 
differently  from  the  intestinal  erepsin  and  are  not  identical  therewith. 
Enzymes,  having  an  action  similar  to  erepsin,  occur,  Vines  believes,3 
in  all  plants  so  far  investigated. 

Erepsin  becomes  inactive  on  heating  to  59°.  It  works  best  in 
alkaline  solution,  but  has  hardly  any  action  in  faint  acid  reaction.  In 
this  regard,  as  well  as  by  the  fact  that  only  a  little  ammonia  is  split  off 
by  its  action  upon  peptone  substances,  it  differentiates  itself  from  cer- 
tain of  the  autolytic  enzymes  studied  so  far.  The  optimum  of  alkalinity 
is,  according  to  Euler,4  at  least  in  the  splitting  of  a  polypeptide,  much 
lower  than  the  optimum  for  tryptic  digestion. 

The  secretion  of  the  glands  in  the  large  intestine  seems  to  con- 
sist chiefly  of  mucus.     Fistulas   have   also   been   introduced   into   these 


1  Boldyreff,  Arch.  d.  scienc.  biolog,  de  St.  Petersbourg,  11;  Bayliss  and  Starling, 
Journ.  of  Physiol.,  29,  30;  Hekma,  1.  c;  Falloise,  see  Biochem.  Centralbl.,  4,  p.  153; 
Vernon,  Journ.  of  Physiol.,  33;  Delezenne,  Compt.  rend.  soc.  biolog.,  54  and  56. 

2  Cohnheim,  Zeitschr.  f.  Physiol.  Chem.,  33,  35,  36,  and  47;  Salaskin,  ibid.,  35; 
Kutscher  and  Seemann,  ibid.,  35. 

*  Bayliss  and  Starling,  Journ.  of  Physiol.,  30;  Vernon,  ibid.,  30  and  33.  See  also 
Cohnheim  and  Pletnew,  Zeitschr.  f.  physiol.  Chem.  69;  F.  Sachs,  Zeitschr.  f.  physiol. 
Chem.,  46;  Nakayama,  ibid.,  41;  Glaessner  and  Stauber,  Bioch.  Zeitschr.  25;  Vines, 
Annals  of  Botany,  18,  19,  and  23. 

4  Zeitschr.  f.  physiol.  Chem.,  51 


494  DIGESTION. 

parts  of  the  intestine,  which  are  chiefly,  if  not  entirely,  to  be  considered 
as  absorption  organs.  The  investigations  on  the  action  of  this  secretion 
on  nutritive  bodies  have  not  as  yet  yielded  any  positive  results. 

IV.  THE  PANCREAS  AND  PANCREATIC  JUICE. 

In  invertebrates,  which  have  no  pepsin  digestion  and  which  also 
have  no  formation  of  bile,  the  pancreas,  or  at  least  an  analogous  organ, 
seems  to  be  the  essential  digestive  gland.  On  the  contrary,  an  anatom- 
ically characteristic  pancreas  is  absent  in  certain  vertebrates  and  in 
certain  fishes.  Those  functions  which  should  be  regulated  by  this  organ 
seem  to  be  performed  in  these  animals  by  the  liver,  which  may  be 
rightly  called  the  hepatopancreas.  In  man  and  in  most  vertebrates 
the  formation  of  bile,  and  of  certain  secretions,  containing  enzymes 
important  for  digestion,  is  divided  between  the  two  organs,  the  liver  and 
the  pancreas. 

The  pancreatic  gland  is  similar  in  certain  respects  to  the  parotid 
gland.  The  secreting  elements  of  the  former  consist  of  nucleated  cells 
whose  basis  forms  a  mass  rich  in  proteins,  which  expands  in  water  and 
in  which  two  distinct  zones  exist.  The  outer  zone  is  more  homogene- 
ous, the  inner  cloudy,  due  to  a  quantity  of  granules.  The  nucleus  lies 
about  midway  between  the  two  zones,  but  this  position  may  change 
with  the  varying  relative  size  of  the  two  zones.  According  to  Heiden- 
hain  *  the  inner  part  of  the  cells  diminishes  in  size  during  the  first  stages 
of  digestion,  in  which  the  secretion  is  active,  while  at  the  same  time 
the  outer  zone  enlarges  owing  to  the  absorption  of  new  material.  In 
the  later  stage,  when  the  secretion  has  decreased  and  the  absorption 
of  the  nutritive  bodies  has  taken  place,  the  inner  zone  enlarges  at  the 
•expense  of  the  outer,  the  substance  of  the  latter  having  been  converted 
into  that  of  the  former.  Under  physiological  conditions  the  glandular 
cells  are  undergoing  a  constant  change,  at  one  time  consuming  from  the 
inner  part  and  at  another  time  growing  from  the  outer  part.  The 
inner  granular  zone  is  converted  into  the  secretion,  and  the  outer,  more 
homogeneous  zone,  which  contains  the  repairing  material,  is  then  con- 
verted into  the  granular  substance.  The  so-called  islands  of  Langer- 
hans  are  related  to  the  internal  secretion  or  contain  a  substance  taking 
part  in  the  transformation  of  the  sugar  of  the  animal  body.2 

The  chief  portion  of  protein  substances  contained  in  the  gland  con- 
sists, it  seems,  of  a  protein  insoluble  in  water  or  neutral  salt  solution  and 


1  Pfluger's  Arch.,  10. 

1  See  Diamare  and  Kuliabko,  Centralbl.  f.  Physiol.,  18  and  19;  Rennie,  ibid.,  18; 
Sauerbeck,  Yirchow's  Arch.,  177,  Suppl. 


PANCREAS  AND  PANCREATIC  JUICE.  495 

of  nucleoproteins,  while  the  globulin  and  albumin  occur  only  to  a  slight 
extent  as  compared  with  the  nucleoproteins.  Among  the  compound 
proteins  is  the  substance  studied  and  isolated  by  Umber  but  previously 
discovered  by  Hammarsten  l  and  called  a-proteid.  This  nucleopro- 
tein  contains,  as  an  average,  1.G7  per  cent  P,  1.29  per  cent  S,  17.12  per 
cent  N,  and  0.13  per  cent  Fe.  It  yields,  according  to  Hammarsten, 
tf-proteid  on  boiling,  which  is  much  richer  in  phosphorus  than  the  nucleo- 
protein.  The  native  proteid  (a)  is  the  mother-substance  of  guanylic 
acid;  according  to  Umber  it  dissolves  on  pepsin  digestion  without  leaving 
any  residue,  and  yields  on  trypsin  digestion  guanylic  acid  on  one  side 
and  proteoses  and  peptones  on  the  other.  It  can  be  extracted  from  the 
gland  by  a  physiological  salt  solution,  and  is  precipitated  by  acetic  acid. 
Besides  this  compound  protein  the  pancreas  must  contain  at  least  one 
other  protein  which  is  the  mother-substance  of  the  thymonucleic  acid 
obtainable  from  the  pancreas. 

Besides  these  protein  substances  the  gland  also  contains  several 
enzymes,  or  more  correctly  zymogens,  which  will  be  discussed  later. 
Among  the  extractive  bodies,  which  are  probably  in  part  formed  by 
post-mortem  changes  and  chemical  action,  we  must  mention  leucine- 
tyrosine,  purine  bases  in  variable  quantities,2  inosite,  lactic  acid,  volatile 
fatty  acids  and  fats.  The  mineral  bodies  vary  considerably  in  quantity, 
not  only  in  animals  and  man  but  also  in  men  and  women  (Gossmann). 
'The  calcium  seems,  according  to  Gossmann,  to  exist  in  much  greater 
amount  than  the  magnesium.  According  to  the  investigations  of  Mag- 
nus-Levy the  human  pancreas  contains  278  p.  m.  solids  with  106  p.  m. 
fat  and  156  p.  m.  protein.  Gossmann3  found  in  man  17.92  p.  m.  ash 
and  in  women  13.05  p.  m. 

Besides  the  previously-mentioned  (Chapter  VII)  relation  to  the  trans- 
formation of  sugar  in  the  aminal  body,  the  pancreas  has  the  property 
of  secreting  a  juice  especially  important  in  digestion. 

Pancreatic  Juice.  This  secretion  may  be  obtained  by  adjusting  a 
fistula  in  the  excretory  duct,  according  to  the  methods  suggested  by 
Bernard,  Ludwig,  and  Heidenhain,  and  perfected  by  Pawlow,4 

In  herbivora,  such  as  rabbits,  whose  digestion  is  uninterrupted,  the 
secretion  of  the  pancreatic  juice  is  continuous.  In  carnivora,  it  seems, 
on  the  contrary,  to  be  intermittent  and  dependent  on  the  digestion. 

1  Umber,  Zeitschr.  f.  klin.  Med.,  40  and  43;  Hammarsten,  Zeitschr.  f.  physioL 
Chem.,  19. 

2  See  Kossel,  Zeitschr.  f.  physiol.  Chem.,  8. 

1  Magnus-Levy,  Bioch  Zeitschr.  24;  Gossmann,  Maly's  Jahresb.  30. 

*  Bernard,  Lecons  de  Physiol.,  2,  190;  Ludwig,  see  Bernstein,  Arbeiten,  ad.  physioL 
Anstalt  zu  Leipzig,  1869;  Heidenhain,  Pfluger's  Arch.,  10,  604;  Pawlow,  The  Work 
of  the  Digestive  Glands,  (translated  by  Thompson,  Philadelphia,  1910),  and  Ergebnisse 
der  Physiologie,  1,  Abt.  1. 


496  DIGESTION. 

During  starvation  the  secretion  almost  stops,  but  commences  again 
after  partaking  of  food  and  reaches  its  maximum,  it  is  claimed  by 
Bernstein,  Heidenhain,  and  others,  within  the  first  three  hours. 

Pawlow  and  his  pupils,  especially  Schepowalnikoff,  have  shown 
that  the  above-mentioned  (page  492)  enterokinase  activates  the  trypsino- 
gen  into  trypsin.  These  observations  were  later  confirmed  by  others, 
by  Delezenne  and  Frouin,  Popielski,  Camus  and  Gley,  Bayliss  and 
Starling,  Zunz,  and  have  been  further  studied.  The  pure  juice  con- 
tains, at  least  as  a  rule,  only  trypsinogen,  and  no  trypsin.  By  mixing 
with  the  intestinal  juice,  or  by  contact  with  the  intestinal  mucosa,  the 
trypsinogen  is  converted  into  trypsin  by  the  kinase.  Enterokinase, 
which  itself  has  no  action  upon  proteins,  and  therefore  is  not  a  pro- 
teolytic enzyme,  is  not  well  known.  It  is  made  inactive  by  heating  and 
is  therefore  considered  by  many  (including  Pawlow)  as  an  enzyme. 
Others,  on  the  contrary,  like  Hamburger  and  Hekma,  Dastre  and 
Stassano,  deny  the  enzyme  nature  of  enterokinase  because  they  find 
that  a  certain  quantity  of  intestinal  juice  will  activate  only  a  certain 
quantity  of  trypsin.  Enterokinase  has  been  found  in  man  and  all 
mammals  investigated.  According  to  most  investigators  it  is  formed 
in  the  glands  or  the  cells  of  the  intestinal  mucosa,  while  according  to 
Delezenne  it  comes  from  Peyer's  patches  and  from  the  lymph-glands 
and  leucocytes,  hence  impure  fibrin  containing  leucocytes  acts  as  a  kin- 
ase. These  deductions  of  Delezenne  are  disputed  by  Bayliss  and" 
Starling,  Hekma  and  others. 

If  we  accept  the  view  that  the  juice  secreted  after  partaking  food 
is  regularly  free  from  trypsin,  still  under  other  circumstances  the  juice 
may  contain  trypsin.  Thus,  according  to  Camus  and  Gley,  the  juice 
secreted  under  the  influence  of  secretin  (see  below)  is  not  always  free 
from  trypsin,  and  Zunz  found  that  Witte's  peptone  or  pilocarpine 
causes  a  secretion  of  juice  which  often  contained  trypsin  and  was  directly 
active.  According  to  Camus  and  Gley  not  only  does  an  exterior  activa- 
tion of  the  trypsinogen  in  the  juice  take  place,  but  also  in  the  interior 
of  the  gland.  An  auto-activation  of  the  juice  in  certain  cases  is  also 
accepted  by  others  (Sawitsch1). 

The  activation  of  the  trypsinogen  into  trypsin  may,  in  life,  be  brought  about 
— as  the  researches  of  Herzen,  which  have  been  substantiated  by  Gachet  and 
Pachon,  Bellamy,  Mendel  and  Rettger,  have  shown — not  only  in  the  intestine, 
but  also  in  the  gland  itself.  This  activation  of  the  trypsinogen  in  the  gland  itself 
is  caused  in  a  still  undiscovered  manner  by  a  body  of  unknown  nature  formed  in  the 
spleen,  which  is  congested  during  digestion.     Such  a  "  charging  "  of  the  pancreas 

1  Camus  and  Gley,  Journ.  de  Physiol,  et  de  Pathol,  gem,  1907;  Zunz,  Recherches 
sur  l'activation  de  sac  pancreatique  par  les  Sels.,  Bruxelles,  1907;  Sawitsch,  Zentralbl. 
f.  d.  ges.  Physiol,  u.  Path,  des  Stoffwechsels,  1909. 


TRYPSINOGEN.  497 

by  the  sploon  has  beeti  repeatedly  suggested  by  Schiff,1  but  this  has  recently 
been  denied  by  Prym.  According  to  this  experimenter  the  extirpation  of  the 
spleen  causes  no  change  in  the  properties  of  the  pancreatic  juice,  and  the  intra- 
venous injection  of  spleen  infusion  is  also  without  action  on  a  splenectomized 
dog  with  permanent  pancreatic  fistula.  The  observations  of  Herzen  that  a 
spleen  infusion  has  a  strong  activating  action  upon  a  weak  pancreas  infusion 
were  substantiated  by  Prym,2  but  he  claims  that  this  is  due  essentially  to  micro- 
organsims.  Besides  this  the  spleen  itself  contains  proteolytic  enzymes  (page 
-371). 

The  conversion  of  the  trypsinogen  into  trypsin  in  the  removed  gland 
or  in  an  infusion  under  the  influence  of  air  and  water  and  also  by  other 
bodies  has  been  known  for  a  long  time.  According  to  Vernon  the  tryp- 
sin itself  has  a  strong  activating  action  upon  trypsinogen,  and  in  this 
regard  it  is  more  active  than  enterokinase.  The  correctness  of  this 
statement  is  still  denied  by  Bayliss  and  Starling  and  by  Hekma.  The 
ordinary  view  of  Heidenhain,  that  the  transformation  of  trypsinogen 
into  trypsin  is  also  brought  about  by  acids,  has  been  found  to  be  incor- 
rect by  Hekma.3  Besides  the  enterokinase  and  the  micro-organisms, 
there  are  other  activators  of  the  trypsinogen.  As  first  shown  by 
Delezenne  and  then  by  Zunz,  by  further  investigations  the  lime  salts 
have  a  special  power  in  activating  trypsinogen.4  These  last  do  not  act 
immediately,  but  only  after  some  time,  for  example,  a  couple  of  hours, 
and  then  they  activate  suddenly.  The  lime  salts  are  not  necessary 
for  the  digestive  action  of  the  juice,  and  when  the  activation  has  once 
taken  place,  they  can  be  removed  without  any  harm.  They  probably 
have  a  similar  action  as  in  the  coagulation  of  the  blood.  According 
to  Delezenne  the  lime  salts  have  the  same  importance  in  the  activa- 
tion of  the  rennin-zymogen  of  the  juice  as  in  the  activation  of  the 
trypsinogen.  This  enzyme  is  also  activated  by  enterokinase.  The 
erepsin  of  the  pancreatic  juice  (page  493)  occurs  as  an  active  enzyme. 

We  are  not  quite  clear  whether  the  two  other  enzymes,  the  diastase 
and  lipase,  are  secreted  as  such  or  as  zymogens.  It  seems,  nevertheless, 
that  both  are  in  part  secreted  as  complete  enzymes. 

In  the  human  embryo  the  trypsinogen  and  the  erepsin  (as  well  as  also  the 
pepsin)  appear  in  the  fourth  and  fifth  fcetal  month.  The  enterokinase  appears 
at  the  same  time  or  shortly  after  the  trypsinogen.5 

1  Bellamy,  Journ.  of  Physiol.,  27;  Mendel  and  Rettger,  Amer.  Journ.  of  Physiol.,  7. 
A  very  complete  reference  to  the  literature  may  be  found  in  Menia  Besbokaia  Du 
rapport  fonctionell  entre  le  pankreas  et  la  rate,  Lausanne,  1901. 

2  Pfliiger's  Arch.,  104.,  and  107. 

3  Vernon,  Journ.  of  Physiol.,  28;  Hekma,  Kon.  Akad.  v.  Wetenschappen  te 
Amsterdam,  1903,  and  Arch.  f.  (Anat.  u)  Physiol.,  1904;  Bayliss  and  Starling,  Journ. 
of  Physiol.,  30 

4  Delezenne,  Compt.  rend.  soc.  biol.,  59,  60,  62,  63;  Zunz,  footnote  1,  p.  496. 
s  Ibrahim.,  Bioch.  Zeitschr.  22,  24  (1909). 


498  DIGESTION. 

The  way  in  which  the  trypsinogen  is  converted  into  trypsin  is  still 
unknown  and  is  the  subject  of  dispute.  According  to  one  view,  proposed 
by  Pawlow  and  defended  by  Bayliss  and  Starling,  the  trypsinogen 
is  transformed  into  trypsin  by  the  action  of  the  kinase.  In  the  opinion 
of  Delezenne,  Dastre,  and  Stassano,  and  others,1  the  trypsin,  on  the 
contrary,  is  a  combination  of  the  kinase  and  trypsinogen,  analogous  to 
the  cytotoxines,  which,  according  to  Ehrlich's  side-chain  theory,  are 
combinations  between  a  complement  and  an  amboceptor.  (See  page 
69.) 

The  specific  excitants  for  the  secretion  of  pancreatic  juice  are, 
according  to  Pawlow  and  his  collaborators,  acids  of  various  kinds- 
hydrochloric  acid  as  well  as  lactic  acid — and  fats,  the  latter  acting 
probably  by  virtue  of  the  soaps  produced  therefrom.  Alkalies  and 
alkali  carbonates  have,  on  the  contrary,  a  retarding  action.  It  appears 
that  the  acids  act  by  irritating  the  mucosa  of  the  duodenum.  Accord- 
ing to  London  and  Schwarz  the  secretion  can  also  be  excited  from  the 
entire  jejunum  and  the  upper  part  of  the  ileum.  The  secretion  becomes 
weaker  the  further  away  the  exciting  source  is  from  the  duodenum.2 
Water,  which  causes  a  secretion  of  acid  gastric  juice,  likewise  becomes, 
indirectly,  a  stimulant  for  the  pancreatic  secretion,  but  may  also  be 
an  independent  exciter.  The  psychical  moment  may,  at  least  in  the 
first  place,  have  an  indirect  action  (secretion  of  acid  gastric  juice), 
and  the  food  seems  otherwise  to  have  an  action  on  pancreatic  secretion 
by  its  action  on  the  secretion  of  gastric  juice. 

The  most  important  excitant  for  the  secretion  of  juice  is  hydrochloric 
acid,  but  opinions  are  not  in  unison  as  to  the  manner  in  which  the  acid 
acts.  Pawlow's  school  claims  that  the  acid  acts  reflexly  upon  the 
intestine,  causing  a  secretion  of  juice.  That  a  reflex  action  is  here  pro- 
duced is  not  contradicted  by  the  investigations  of  Popielski,  Wert- 
heimer  and  Lepage,  Fleig,3  and  others.  According  to  the  researches 
of  Bayliss  and  Starling,  which  have  been  confirmed  by  Camus,  Gley, 
Fleig,  Herzen,  Delezenne,  and  others,  a  second  factor  must  also  be 
active  here.  Bayliss  and  Starling  have  shown  that  a  body  which 
they  have  called  secretin  can  be  extracted  from  the  intestinal  mucosa 
by  a  hydrochloric-acid  solution  of  4  p.  m.,  and  this  when  introduced  into 
the  blood  produces  a  secretion  of  pancreatic  juice,  bile,  and  in  the 
opinion  of  some  investigators  also  of  saliva  and  intestinal  juice.     The 

1  Bayliss  and  Starling,  Journ.  of  Physiol.,  30  and  32,  which  also  cities  the  other 
investigators  and  also  O.  Cohnheim,  Bioch.  Centralbl.  1,  169  and  S.  Rosenberg,  ibid., 
2,  708. 

*  Zeitschr.  f.  physiol.  Chem.  68,  346  (1910)  which  also  contains  the  literature. 

'  Fleig,  Centralbl.  f.  Physiol.,  16,  681,  and  Compt.  rend.  soc.  biol.,  55.  See  also 
footnote  1. 


SECRETION  OF  PANCREATIC  JUICE.  499 

secretin,  which  according  to  Bayliss  and  Starling,1  is  the  same  in  all 
vertebrates  examined,  is  not  destroyed  by  heat;  it  is  therefore  not 
identical  with  ciitorokinase,  and  is  not  considered  an  enzyme.  It  is 
formed  from  another  substance,  prosecretin,  by  the  action  of  acids. 
According  to  Delezenne  and  Pozerski  secretin  occurs  as  such  in  the 
intestinal  mucosa,  and  the  acids  act  only  by  the  elimination  of  certain 
bodies  having  a  retarding  action.  According  to  Popielski  secretin 
action  is  different  from  acid  action;  and  the  secretin  action  can  also  be 
obtained  by  Witte's  peptone.  He  believes  that  the  secretin  is  not 
a  specific  constituent  of  the  intestine  but  a  body  widely  distributed. 
(Iizelt  disputes  the  occurrence  of  a  specific  secretin  and  he  compares 
this  body  to  peptone.  Gley  has  obtained  a  solution  which  had  a  stronger 
secreting  action  than  secretin  by  macerating  the  mucosa  with  proteoses.2 
v.  Furth  and  Schwarz3  also  call  attention  to  the  uncertainty  of  our 
knowledge  as  to  the  nature  of  secretin.  According  to  them  secretin 
is  probably  a  mixture  of  bodies,  among  which  probably  the  choline,  found 
by  them  in  the  intestinal  walls,  acts  the  role  of  an  exciter  of  secretion. 

A  second  means  of  causing  secretion  is  the  fat,  which  probably  only 
acts  after  it  has  been  saponified.  Oil-soap  directly  introduced  into  the 
duodenum  brings  about  a  strong  secretion  of  pancreatic  juice  (Sawitsch, 
Babkin4),  and  at  the  same  time  a  flow  of  bile,  gastric  juice,  and  the 
secretion  of  Brunner's  glands  occurs.  The  pancreatic  juice  secreted 
under  these  circumstances  has  about  the  same  amount  of  enzymes  as 
the  juice  secreted  after  partaking  of  food. 

We  know  very  little  as  to  how  the  soaps  act.  Fleig  5  found  that  by  macera- 
tion of  the  mucosa  of  the  upper  part  of  the  duodenum  with  soap  solution,  a  sub- 
stance goes  into  solution  which  he  calls  sapokrinin,  and  which  when  introduced 
into  the  blood  brings  about  a  strong  secretion  of  pancreatic  juice.  This  sapok- 
rinin, which  is  derived  from  a  prosapokrinin.  is  not  an  enzyme  and  is  not  identical 
with  secretin.  After  the  action  of  chloral  hydrate  an  abundant  secretion  occurs 
in  the  duodenum  (Wertheimer  and  Lepage),  which  Falloise  considers  as  pro- 
duced by  a  special  secretin,  chloral  secretin.  The  secretion  of  pancreatic  juice 
can  also  be  increased  by  alcohol,  and  Fleig  6  claims  to  have  obtained  a  secretin, 
ethyl  secretin,  by  macerating  the  intestinal  mucosa  with  alcohol.  Further  investiga- 
tions are  necessary  of  all  these  so-called  secretins. 


1  Journ.  of  Physiol.,  29. 

2  Delezenne  and  Pozerski,  Compt.  rend.  soc.  biol.,  56;  Popielski,  Centralbl.  f. 
Physiol.,  19;  Pfluger's  Arch.  128;  Gizelt,  Pfluger's  Arch.  123;  Gley,  Compt.  Rend.  151, 
345. 

3  v.  Furth  and  Schwarz,  Pfluger's  Arch.  124  (literature  on  secretin). 

4  Arch  des  scienc.  biol.  de  St.  Petersbourg,  11,  and  Zeitschr.  f.  physiol.  Chem.,  56. 
6  Compt.  rend.  soc.  biol.,  55,  and  Journ.  de  Physiol,  et  de  Pathol,  gen.,  1904. 

6  Wertheimer  and  Lepage,  Compt.  rend.  soc.  biol.,  52;  Fleig,  ibid.,  55;  Falloise, 
Bull.  Acad.  Roy.  Belg.,  1903. 


500  DIGESTION. 

The  estimation  as  to  the  quantity  of  pancreatic  juice  secreted  in  the 
twenty-four  hoars  differs  very  much.  According  to  the  determina- 
tions of  Pawlow  and  his  collaborators,  Kuwschinski,  Wassiliew,  and 
Jablonsky,1  the  average  quantity  (with  normally  acting  juice)  from  a 
permanent  fistula  in  dogs  is  21.8  cc.  per  kilo  in  the  twenty-four  hours. 

The  pancreatic  juice  of  the  dog  is  a  clear,  colorless,  and  odorless 
alkaline  fluid  which  when  obtained  from  a  temporary  fistula  is  very 
rich  in  proteins,  sometimes  so  rich  that  it  coagulates  like  the  white  of 
the  egg  on  heating.  Besides  proteins,  the  juice  also  contains  the  three 
above-mentioned  enzymes  (or  their  zymogens),  amylopsin,  perhaps  also 
maltase,  trypsin,  steapsin,  also  an  enzyme  similar  to  erepsin,  and  besides 
these  a  rennin,  which  was  first  observed  by  Ruhne.  Besides  the  above- 
mentioned  bodies  the  pancreatic  juice  invariably  contains  small  quan- 
tities of  leucine,  fat,  and  soaps.  As  mineral  constituents  it  contains 
chiefly  alkali  chlorides  and  considerable  alkali  carbonate,  some  phos- 
phoric acid,  lime,  magnesia,  and  iron. 

The  quantity  of  solids  in  the  pancreatic  juice  of  the  dog  varies,  as 
found  by  Mazurkiewicz,  Babkine  and  Sawitsch,2  according  to  the 
rapidity  of  secretion  and  the  kind  of  excitant.  As  a  rule  the  amount 
of  solids  is  in  inverse  proportion  to  the  rapidity  of  secretion.  The  juice 
secreted  after  the  action  of  acids  has  the  lowest  amount  of  solids,  9-37.4 
p.  m.  The  juice  after  taking  food  is  more  concentrated,  about  60-70 
p.  m.  and  that  after  vagus  stimulation  often  contains  90  p.  m.  solids. 
The  juice  analyzed  by  C.  Schmidt3  from  a  temporary  fistula  contained 
99-116  p.  m.  solids.     The  quantity  of  mineral  bodies  was  8.8  p.  m. 

The  mineral  constituents  consisted  chiefly  of  NaCl,  7.4  p.  m.,  which  is  remark- 
able because  the  juice  contains  such  a  large  amount  of  alkali  carbonate.  In  the 
juice  examined  by  De  Zilwa  4  the  quantity  of  alkali  in  the  secretin  juice  was 
5-7.9  p.  m.  and  in  the  pilocarpin  juice  2.9  -5.3  p.  m.  Na2C03. 

In  the  pancreatic  juice  of  rabbits  11-26  p.  m.  solids  have  been  found,  and 
in  that  from  sheep  14.3-36.9  p.  m.  In  the  pancreatic  juice  of  the  horse  9-15.5 
p.  m.  solids  have  been  found;  in  that  of  the  pigeon,  12-14  p.  m. 

The  human  physiological  pancreatic  secretion  from  a  fistula  has  been 
investigated  by  Glaessner.5     The   secretion  was  clear,  foamed  readily, 

1  Arch,  des  sciences  de  St.  Petersbourg,  2,  391.  The  previous- claims  of  Bidder  and 
Schmidt,  and  others  may  be  found  in  Kiihne,  Lehrbuch,  114. 

2  Mazurkiewicz,  1.  c;  Babkin  and  Sawitsch,  Zeitschr,  f.  physiol.  Chem.,  56. 
8  Cited  from  Maly  in  Hermann's  Handbuch  der  Physiol.,  5,  Theil  II,  189. 

4  Journ.  of  Physiol.,  31. 

6  Zeitschr.  f.  physiol.  Chem.,  40.  See  also  Ellinger  and  Kohn,  ibid.,  45,  and  the 
investigations  upon  cystic  fluids  from  the  pancreas  by  Schumm,  ibid.,  36,  and  Murray 
and  Gies,  American  Medicine,  4,  1902;  Glaessner  and  Popper,  Deutsch.  Arch.  f.  klin. 
Med.  04,  46;  see  also  Wohlgemuth,  Bioch.  Zeitschr.  39;  Bradley,  Journ.  of  Biol. 
Chem.  6. 


AMYLOPSIN.     STEAPSIN.  501 

had  a  strong  alkaline  reaction  even  toward  phenolphthalein,  and  con- 
tained globulin  and  albumin  but  no  proteoses  and  peptones.  The  specific 
gravity  was  1.0075  and  the  freezing-point  depression  was  A  =—0.46- 
0.49°.  The  solids  were  12.44-12.71  p.  m.,  the  total  protein  1.28-1.74 
p.  m.,  and  the  mineral  bodies  5.66-6.98  p.  m.  The  secretion  contained 
trypsinogen,  which  was  activated  by  the  intestinal  juice.  Diastase  and 
lipase  were  present;  inverting  enzymes,  on  the  contrary,  were  not.  The 
daily  quantity  of  juice  was  500-800  cc.  The  quantity  of  secretion,  of 
ferments,  and  of  alkalinity  was  lowest  in  starvation,  but  soon  rose  with 
the  taking  of  food,  and  reached  its  maximum  in  about  four  hours. 

Amylopsin,  or  pancreatic  diastase,  which,  according  to  Korowin 
and  Zweifel,  is  not  found  in  new-born  infants  and  does  not  appear 
until  more  than  one  month  after  birth,  seems,  although  not  identical 
with  ptyalin,  to  be  closely  related  to  it.  Amylopsin  acts  very  energetic- 
ally upon  boiled  starch,  and  according  to  Ivuhne  also  upon  unboiled 
gtarch,  especially  at  37  to  40°  C,  and  according  to  Vernon  1  best  at 
35°  C.  It  forms,  similarly  to  the  action  of  saliva,  besides  dextrin,  chiefly 
isomaltose  and  maltose,  with  only  very  little  glucose  (Musculus  and 
v.  Mering,  Kulz  and  Vogel2).  The  glucose  is  probably  formed  by  the 
action  of  the  invertin  existing  in  the  gland  and  juice.  The  pancreatic 
juice  of  the  dog  in  fact,  contains,  according  to  Bierry  and  Terroine,3 
maltase,  its  action  becomes  apparent  only  after  very  faint  acidification 
cf  the  juice.  According  to  Rachford  the  action  of  the  amylopsin  is 
not  reduced  by  very  small  quantities  of  hydrochloric  acid,  but  is  dimin- 
ished by  larger  amounts.  Vernon,  Grutzner,  and  Wachsmann  find 
that  the  action  is  indeed  accelerated  by  very  small  quantities  of  hydro- 
chloric acid,  0.045  p.  m.,  while  alkalies  in  very  small  amounts  have  a 
retarding  action.  This  retarding  action  of  alkalies  and  hydrochloric 
acid  may  be  stopped  by  bile  (Rachford).  Wohlgemuth  as  well  as 
Minami4  find  that  the  action  of  diastase  is  increased  to  a  high  degree 
by  bile.  The  active  constituent  of  the  bile  was  soluble  in  water  and 
alcohol  but  was  not  identical  with  the  bile  salts  or  cholesterin.  The 
statements  in  regard  to  the  action  of  lecithin  are  contradictory. 

Steapsin,  or  Fat-splitting  Enzyme.  The  action  of  the  pancreatic 
juice  on  fats  is  twofold.     First,  the  neutral  fats  are  split  into  fatty  acids 


1  Korowin,  Maly's  Jahresber.,  3;  Zweifel,  footnote  2,  p.  456,  Kuhne,  Lehrbuch, 
117;  Vernon,  Journ.  of  Physiol.,  27. 

2  See  footnote  5,  p.  456. 

3  See  Tebb.  Journ.  of  Physiol.,  15;  Bierry  and  Terroine,  Compt.  rend.  soc.  biolog., 
58;  Bierry,  ibid.,  62. 

4  Rachford,  Amer.  Journ.  of  Physiol.,  2;  Vernon.   1.  c;  Grutzner,  Pfliiger's  Arch. 
91;  Wohlgemuth,  Bioch.  Zeitschr.  21,  447  (1909);  Minami,  ibid.,  39,  339  (1912). 


502  DIGESTION. 

-and  glycerin,  which  is  an  enzymotic  process;    and  secondly,  it  has  also 
the  property  of  emulsifying  the  fats. 

The  action  of  the  pancreatic  juice  in  splitting  the  fats  may  be  shown 
in  the  following  way:  Shake  olive-oil  with  caustic  soda  and  ether, 
siphon  off  the  ether  and  filter  if  necessary,  then  shake  the  ether  repeatedly 
with  water  and  evaporate  at  a  gentle  heat.  In  this  way  is  obtained  a 
residue  of  fat  free  from  fatty  acids,  which'  is  neutral  and  which  dissolves 
in  acid-free  alcohol  and  is  not  colored  red  by  alkanet  tincture.  If  such 
fat  is  mixed  with  perfectly  fresh  alkaline  pancreatic  j  uice  or  with  a  freshly 
prepared  infusion  of  the  fresh  gland  and  treated  with  a  little  alkali  or 
with  a  faintly  alkaline  glycerin  extract  of  the  fresh  gland  (9  parts  glyc- 
erin and  1  part  1  per  cent  soda  solution  for  each  gram  of  the  gland), 
and  some  litmus  tincture  added  and  the  mixture  warmed  to  37°  C, 
the  alkaline  reaction  will  gradually  disappear  and  an  acid  one  take  its 
place.  This  acid  reaction  depends  upon  the  conversion  of  the  neutral 
fats  by  the  enzyme  into  glycerin  and  free  fatty  acids.  A  very  much 
used  method  consists  in  determining  the  acidity  of  the  mixture  by  means 
of  titration  before  and  after  the  action  of  the  juice  or  the  infusion. 

The  action  of  the  pancreatic  juice  in  splitting  fats  is  a  process  analo- 
gous to  that  of  saponification,  the  neutral  fats  being  decomposed,  by 
the  addition  of  the  elements  of  water  into  fatty  acids  and  glycerin 
according  to  the  following  equation.  C3H5.O3.R3  (neutral  fat)+3H20  = 
C3H5.O3.H3  (glycerin)  +  3  (H.O.R)  (fatty  acid).  This  depends  upon 
a  hydrolytic  splitting,  which  was  first  positively  proved  by  Bernard 
and  Berthelot.  The  pancreas  enzyme  also  decomposes  other  esters, 
just  as  it  does  the  neutral  fats  (Nencki,  Baas,  Loevenhart  x  and 
others).  The  fat-splitting  action  of  the  lipase  is,  according  to  Paw- 
low,  Bruno  and  many  others  2  aided  in  its  action  by  the  bile.  Rosen- 
heim and  Shaw-Mackenzie  found  that  the  lipase  action  was  accel- 
erated by  ha3molytic  substances,  as  well  as  by  normal  serum;  this 
accelerating  action  was  inhibited  by  cholesterin.  The  accelerating 
substance  of  the  serum  was  dialyzable  and  resistant  to  heat.  Rosen- 
heim was  able  to  divide  the  lipase  existing  in  a  glycerin  extract  of  the 
pig  pancreas  into  enzyme  and  co-enzyme  (page  52);  in  diluting  with 
water  a  precipitate  occurred  which  contained  the  real  thermolabile 
enzyme  while  the  dialyzable,  heat  resisting  co-enzyme  remained  in  the 


1  Bernard,  Ann.  de  chim.  et  physique  (3),  25;  Berthelot,  Jahresber,  d.  Chem., 
1855,  733;  Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  20;  Baas,  Zeitschr.  f.  physiol.  Chem., 
14,  416;  Loevenhart,  Journ.  of  Biol.  Chem.,  2;  Terroine  an  1  Z.  Morel,  Compt.  rend. 
80C.  biol.,  65,  66. 

2  Bruno,  Arch,  des  scienc  biol.  de  St.  P6tersbourg,  7;  see  also  Loevenhart  and 
Souder,  Journ.  of  biol.  Chem.,  2;  v.  Furth  and  Schutz,  Hofmcister's  Beitrage,  9;  Ter- 
roine, Bioch.  Zeitschr.  23;  Compt.  rend.  soc.  biol.  68,  439,  518,  666,  754  (1910). 


TRYPSIN.  503 

filtrate.1  In  regard  to  the  synthetic  action  of  pancreatic  lipase  see 
page  60. 

The  fatty  acids  which  are  split  off  by  the  action  of  the  pancreatic 
juice  combine  in  the  intestine  with  the  alkalies,  forming  soaps,  which 
have  a  strong  emulsifying  action  on  the  fats,  and  thus  the  pancreatic 
juice  becomes  of  great  importance  in  the  emulsification  and  the  absorp- 
tion of  the  fats. 

Trypsin.  The  action  of  the  pancreatic  juice  in  digesting  proteins 
was  first  observed  by  Bernard,  but  first  proved  by  Corvisart.2  It 
depends  upon  a  special  enzyme  called,  by  Kuhne,  trypsin.  This  enzyme 
as  previously  explained,  does  not  occur  in  the  gland  as  such,  but  as 
trypsinogen.  According  to  Albertoni3  this  zymogen  is  found  in 
the  gland  in  the  last  third  of  the  intra-uterine  life.  Enzymes  more  or 
less  like  trypsin  occur  in  other  organs,  and  are  very  widely  diffused  in 
the  vegetable  kingdom,4  in  yeast  and  in  higher  plants,  and  are  also  formed 
by  various  bacteria.  The  enzymes  similar  to  trypsin  occurring  in  the 
plant  kingdom  are,  according  to  Vines,  a  mixture  of  peptases,  which 
transform  the  proteins  into  peptone,  and  ereptases,  which  split  the  pep- 
tones into  amino-acids. 

As  we  know  of  so-called  antienzymes  for  other  enzymes,  so  we  also  have  anti- 
trypsins, and  not  only  in  the  intestinal  canal  but  also  in  the  blood-serum  (see  page 
63).  The  results  as  to  the  possibility  of  producing  antitrypsins  by  immuniza- 
tion, is  still  disputed. 

Trypsin,  like  other  enzymes,  has  not  been  prepared  in  a  pure  con- 
dition. Nothing  is  positively  known  in  regard  to  its  nature,  but  as 
obtained  thus  far  it  shows  a  variable  behavior  (Kuhne,  Klug,  Levene, 
Mays,  and  others).  At  least  it  does  not  seem  to  be  a  nucleoprotein,  and 
trypsin  has  also  been  obtained  which  did  not  give  the  biuret  test  (Klug, 
Mays,  Schwarzschild).  Trypsin  dissolves  in  water  and  glycerin,  while 
Kuhne's  trypsin  was  insoluble  in  glycerin.  It  is  very  sensitive  to  heat, 
and  even  the  body  temperature  gradually  decomposes  it  (Vernon,  Mays). 
In  neutral  solution  it  becomes  inactive  at  45°  C.  In  dilute  soda  solu- 
tion of  3-5  p.  m.  it  is  still  more  readily  destroyed  (Biernacki,  Vernon  5). 

1  Journ.  of  Physiol.  40  (1910). 

2  Gaz.  hebciomadaire,  1857,  Nos.  15,  16,  19,  cited  from  Bunge,  Lehrbuch,  4,  Aufl., 
185. 

3  See  Maly's  Jahresber.,  8,  254. 

4  In  this  connection  see  Vines,  Annals  of  Botany,  16,  17,  18,  19,  22,  and  23,  and 
Oppenheimer,  Die  Fermente,  1910. 

5  Kiihne,  Verh.  d.  naturh.-med.  Vereins  zu  Heidelberg  (N.  F.),  1,  3;  Klug,  Math, 
naturw.  Ber.  aus  Ungarn.,  18,  1902;  Levene,  Amer.  Journ.  of  Physiol.,  5;  Mays, 
Zeitschr.  f.  Physiol.  Chem.,  38;  Vernon,  Journ.  of  Physiol.,  28  and  29;  Biernacki, 
Zeitschr.  f.  Biologie,  28;  Schwarzschild,  Hofmeister's  Beitrage,  4. 


504  DIGESTION. 

The  presence  of  protein  or  proteoses  has,  to  a  certain  extent,  a  pro- 
tective action  on  heating  an  alkaline  trypsin  solution,  and  this  has 
been  substantiated  by  recent  investigations  of  Bayliss  and  Vernon. 
The  simpler  cleavage  products  have  a  still  greater  protective  action 
(Vernon1).  Trypsinogen,  according  to  the  unanimous  statements 
of  several  experimenters,  is  more  resistant  toward  alkalies  than  trypsin. 
Trypsin  is  gradually  destroyed  by  gastric  juice  and  even  by  digestive 
hydrochloric  acid  alone. 

The  preparation  of  pure  trypsin  has  been  tried  by  various  experimen- 
ters. The  most  careful  work  in  this  direction  was  done  by  Kuhne  and 
[Mays.  Various  methods  have  been  suggested  by  Mays,  but  we  cannot 
enter  into  a  discussion  of  them.  A  very  pure  preparation  can  be  obtained 
by  making  use  of  the  combined  salting  out  with  NaCl  and  MgSO-i.  A 
very  active  solution,  and  one  that  can  be  kept  for  a  long  time  (for  more 
than  twenty  years  according  to  Hammarsten),  can  be  obtained  by  extract- 
ing with  glycerin  (Heidenhain  2) .  An  impure  but  still  very  active 
infusion  can  be  obtained  after  a  few  days  by  allowing  the  finely  divided 
gland  to  stand  with  water  which  contains  5-10  cc.  chloroform  per  liter 
(Salkowski)  at  the  temperature  of  the  room.  Such  infusions  can  be 
obtained,  nearly  free  from  proteins,  by  dialyzing  with  running  water  after 
the  addition  of  toluene. 

Like  other  enzymes,  trypsin  is  characterized  by  its  action,  and  this 
action  consists  in  dissolving  protein  and  in  splitting  it  into  simpler  prod- 
ucts, mono-  and  diamino-acids,  tryptophane,  etc.,  in  alkaline,  neutral, 
and  indeed  in  very  faintly  acid  solutions.  This  action  has  been  so  far 
considered  as  characteristic  for  trypsin.  Recent  investigations  seem 
to  indicate  that  this  action  is  not  due  to  one  enzyme  alone,  but  to  the 
combined  action  of  several  enzymes. 

Although  contrary  to  May's  statement,  there  is  no  question  that 
there  occurs  in  the  pancreas  besides  trypsin,  an  enzyme  similar  to  erepsin 
(Bayliss  and  Starling,  Vernon3).  According  to  the  latter  this 
erepsin  has  a  strong  action  upon  peptone,  and  he  believes  that  the  pep- 
tone-splitting action  of  a  pancreas  infusion  is  in  great  part  due  to  the 
erepsin.  The  pancreas,  besides  these,  also  contains  a  nuclease  (see  page 
493),  whose  relation  to  pancreas  erepsin  has  not  been  determined. 

The  unity  of  trypsin  has  also  been  disputed  from  another  point  of  view. 
According  to  Pollak  the  trypsin  (in  the  ordinary  sense)  contains  a  second 
enzyme,  which  does  not  act  upon  protein,  but  only  upon  gelatin,  and  he  calls 


1  Bayliss,  Arch,  des  scienc.  biolog.  de  St.  Petersbourg.  11,  Suppl.;  Vernon,  Journ. 
of  Physiol.,  31. 

-  PfluKer's  Arch.,  10. 

*  Bayliss  and  Starling,  Journ.  of  Physiol.,  30;  Vernon,  ibid.,  30;  and  Zeitschr. .  f.. 
physiol.  Chem.,  50;  Mays,  ibid.,  49  and  51. 


ACTION  OF  TRYPSIN.  505 

this  enzyme  glutinase.  This  glutinasc  is  much  more  resistant  toward  acids 
than  trypsin,  and  by  proper  treatment  with  acids  Pollak  was  able  to  change  a 
pancreas  infusion  so  that  it  acted  upon  gelatin  and  not  upon  certain  proteins. 
The  correctness  of  these  observations  has,  indeed,  not  been  generally  accepted, 
and  it  is  disputed  by  Ascoli  and  Neppi.1  According  to  them  the  action  of  the 
trypsin  is  weakened  by  the  acid,  and  indeed  to  such  varying  degrees  for  differ- 
ent proteins  that  the  action  upon  albumin  is  lost  while  the  action  upon  gelatin 
is  noticeable.  Nevertheless,  we  here  have  a  warning  to  be  careful  as  to  the 
conclusions  drawn  from  results  where  impure  infusions  are  used.  For  many 
experiments  it  is  undoubtedly  advisable  to  use  the  natural  pancreatic  juice. 

The  following  reports  on  the  action  of  trypsin  applies  to  the  so- 
called  trypsin,  with  the  reservation  that  it  is  perhaps  not  a  unit  enzyme. 

The  action  of  trypsin  on  proteins  is  best  demonstrated  by  the  use  of 
fibrin.  Very  considerable  quantities  of  this  protein  body  are  dissolved 
by  a  small  amount  of  trypsin  at  37-40°  C.  It  is  always  necessary  to 
make  a  control  test  with  fibrin  alone,  with  or  without  the  addition  of 
alkali.  Fibrin  is  dissolved  by  trypsin  without  any  putrefaction;  the 
liquid  has  a  pleasant  odor  somewhat  like  bouillon.  To  completely 
exclude  putrefaction  a  little  thymol,  chloroform,  or  toluene  should  be 
added  to  the  liquid.  Tryptic  digestion  differs  essentially  from  peptic 
digestion,  irrespective  of  the  difference  in  the  digestive  products,  in  that 
the  first  takes  place  in  neutral  or  alkaline  reaction  and  not,  as  is  neces- 
sary for  peptic  digestion,  in  an  acidity  of  1-2  p.  m.  HC1,  and  further 
by  the  fact  that  the  proteins  dissolve  in  trypsin  digestion  without  pre- 
viously swelling  up. 

As  trypsin  not  only  dissolves  proteids,  but  also  other  protein  sul  - 
stances  such  as  gelatin,  this  latter  body  may  be  used  in  detecting  tryp- 
sin. The  liquefaction  of  strongly  disinfected'  gelatin  is,  according  to 
Fermi,  2  a  very  delicate  test  for  trypsin  or  tryptic  enzymes.  Various 
suggestions  for  the  use  of  gelatin  in  the  trypsin  test  have  been  made. 
In  consideration  of  the  observations  of  Ascoli  and  Neppi  that  a  trypsin 
may  not  act  upon  fibrin  or  other  proteids  but  still  digest  gelatin,  it  is 
advisable  never  to  make  use  of  gelatin  or  proteid  alone  in  testing  for 
trypsin,  but  always  the  two. 

For  the  quantitative  estimation  of  trypsin  by  measuring  the  rapidity  of 
digestion  we  generally  make  use  of  the  method  of  Mett,  described  under  pepsin 
digestion.  Another  method,  suggested  by  Weiss,  consists  in  determining  the 
nitrogen  in  the  filtrate  after  coagulation  with  heat  and  acetic  acid.  Lohlein 
recommends  the  titration  method  of  Volhard  as  used  in  pepsin  determinations, 
and  has  given  directions  for  its  use.  Jacoby  recommends  the  use  of  (ricin,  and 
Gross  suggests  a  method  based  upon  the  precipitation  of  casein  by  acid.     Bay- 


1  Pollak,  Hofmeister's  Beitrage,  6;  contradictory  statements  are  found  in  Ehren- 
reich,  cited  in  Bioch.  Centralbl.,  4;  Ascoli  and  Neppi,  Zeitschr.  f.  physiol.  Chem.,  56. 

2  Arch.  f.  Hyg.  12  and  55. 


506  DIGESTION. 

liss  follows  the  digestion  by  the  electrical  conductivity,  and  F.  Weiss  l  determines 
the  quantity  of  nitrogen  not  precipitated  by  tannic  acid.  The  formol  titration 
can  also  be  used  with  advantage  for  determining  the  decomposition  (page  166). 

The  reaction  has  a  great  influence  upon  the  rapidity  of  the  trypsin 
digestion.  Trypsin  acts  energetically  in  neutral,  or  still  better  in  alkaline, 
solutions,  and  according  to  older  statements,  best  in  an  alkalinity  of 
3-4  p.  m.  Na2C03|  but  the  nature  of  the  protein  is  also  of  importance. 
The  reports  in  regard  to  the  action  of  trypsin  in  various  reactions  are 
still  somewhat  disputed.2  The  action  of  the  alkali  depends  upon  the 
number  of  hydroxyl  ions  (Dietze,  Kanitz),  and  according  to  Kanitz3 
the  digestion  proceeds  best  in  those  solutions  which  are  1/70-1/200 
normal  in  regard  to  hydroxyl  ions.  Free  mineral  acids,  even  in  very 
small  quantities,  completely  prevent  the  digestion.  If  the  acid  is  not 
actually  free,  but  combined  with  protein  bodies,  then  the  digestion 
may  take  place  quickly  when  the  acid  combination  is  not  in  too  great 
excess  (Chittenden  and  Cummins).  Organic  acids  act  less  disturbingly, 
and  in  the  presence  of  0.2  p.  m.  lactic  acid  and  the  simultaneous  presence 
of  bile  and  common  salt,  the  digestion  may  indeed  proceed  more  quickly 
than  in  a  faintly  alkaline  liquid  (Lindberger).  The  assertion  of  Rach- 
ford  and  Southgate,  that  the  bile  can  prevent  the  injurious  action 
of  the  hydrochloric  acid,  and  that  a  mixture  of  pancreatic  juice,  bile, 
and  hydrochloric  acid  digests  better  than  a  neutral  pancreatic  juice, 
could  not  be  substantiated  by  Chittenden  and  Albro.  That  bile  has 
an  action  tending  to  aid  the  tryptic  digestion  has  been  shown  by  many 
investigators,  and  recently  by  Bruno,  Zuntz  and  Ussow  and  others.4 
Carbon  dioxide,  according  to  Schierbeck,5  has  a  retarding  action 
in  acid  solutions,  but  it  accelerates  the  tryptic  digestion  in  faintly 
alkaline  liquids.  Foreign  bodies,  such  as  potassium  cyanide,  may  pro- 
mote tryptic  digestion,  while  other  bodies,  such  as  salts  of  mercury,  iron, 
and  others  (Chittenden  and  Cummins),  or  salicylic  acid  in  large  quan- 
tities, may  have  a  preventive  action.     According  to  Weiss  6  the  halogen 

1  Weiss,  Zeitschr.  f.  physiol.  Chem.,  40;  Lohlein,  Hofmeister's  Beitrage,  7;  Jacoby, 
Bioch.  Zeitschr.,  10;  Gross,  Arch.  f.  exp.  Path.  u.  Pharm.,  58;  Bayliss,  Arch,  des 
scienc.  biol.  de  St.  Petersbourg,  11,  Suppl.;  and  Journ.  of  Physiol.,  36;  Weiss,  Zeitschr. 
f.  physiol.  Chem.  31,  78  (1900). 

2  See  Kudo,  Bioch.  Zeitschr.,  15. 

3  Kanitz,  Zeitschr.  f.  physiol.  Chem.,  37,  who  also  cites  Dietze. 

4  Chittenden  and  Cummins,  Studies  from  the  Physiol.  Chem.  Laboratory  of  Yale 
College,  New  Haven,  1885,  1,  100;  Lindberger,  Maly's  Jahresber.,  13;  Rachford  and 
Southgate,  Medical  Record,  «1895;  Chittenden  and  Albro,  Amer.  Journ.  of  Physiol.,  1, 
1898;  Rachford,  Journ.  of  Physiol.,  25;  Bruno,  1.  c;  Zuntz  and  Ussow,  Arch.  f. 
(Anat.  u.)  Physiol.,  1900. 

'■>  Skand.  Arch.  f.  Physiol.,  3. 

6  Weiss,  Zeitschr.  f.  physiol.  Chem.,  40;  See  also  Kudo,  Bioch.  Zeitschr.  15,  473 
(1908). 


PRODUCTS  OF  TRYPTIC  DIGESTION.  507 

alkali  salts  disturb  tryptic  digestion  only  slightly,  and  NaCl  seems  to 
have  the  strongest  action.  The  sulphates  have  a  much  stronger  retard- 
ing action  than  the  chlorides.  The  nature  of  the  proteins  is  also  of 
importance.  Unboiled  fibrin  is,  relatively  to  most  other  proteins,  dis- 
solved so  very  quickly  that  the  digestion  test  with  raw  fibrin  gives  an 
incorrect  idea  of  the  power  of  trypsin  to  dissolve  coagulated  protein 
bodies  in  general.  Boiled  fibrin  is  digested  with  much  greater  difficulty 
and  also  requires  a  higher  alkalinity:  8  p.  m.  Na2C03  (Vernon1). 
The  resistance  of  certain  native  protein  solutions,  such  as  blood-serum 
and  egg-white,  against  the  action  of  trypsin  is  remarkable.  In  regard 
to  the  inhibition  of  the  action  of  trypsin  see  Chapter  I,  page  63. 

The  Products  of  the  Tryptic  Digestion.  In  the  digestion  of  unboiled 
fibrin  a  globulin  which  coagulates  at  55-60°  C.  may  be  obtained  as  an 
intermediary  product  (Herrmann2).  Besides  this,  one  obtains  from 
fibrin,  as  well  as  from  other  proteins,  the  products  previously  men- 
tioned in  Chapter  II.  In  trypsin  digestion  the  cleavage  may  proceed 
so  far  that  the  mixture  fails  to  give  the  biuret  reaction.  This  does  not 
indicate,  as  E.  Fischer  and  Abderhalden  have  shown,  a  complete 
cleavage  of  the  protein  molecule  into  mono-  and  diamino-acids,  etc. 
In  tryptic  digestion,  as  shown  by  Abderhalden  and  Reinbold,  using 
the  protein  edestin,  and  by  Abderhalden  and  Voegtlin  3  with  casein, 
a  gradual  cleavage  of  the  protein  takes  place,  and  thereby  certain  amino- 
acids,  like  tyrosine  and  tryptophane,  are  readily  and  completely  split 
off,  while  others,  like  leucine,  alanine,  aspartic  acid,  and  glutamic  acid, 
are  slowly  and  less  readily  split  off,  and  others,  such  as  a-proline,  phenyl- 
alanine, and  glycocoll,  stubbornly  resist  the  cleavage  action  of  the  trypsin. 
The  polypeptide-like  bodies  discovered  by  Fischer  and  Abderhalden, 
which  are  produced  in  digestion,  and  which  do  not  give  the  biuret 
reaction,  are  the  atomic  complexes  which  resist  the  action  of  trypsin. 
These  peptoids  contain  the  pyrrolidine  carboxylic  acid  and  phenylal- 
anine groups  of  the  protein,  but  also  yield  other  monamino-acids  such 
as  leucine,  alanine,  glutamic  acid,  and  aspartic  acid.  Among  the  above- 
mentioned  products  we  find  on  the  autodigestion  of  the  gland  other 
substances,  such  as  oxyphenylethylamine  (Emerson),  which  is  pro- 
duced from  tyrosine  by  fermentive  CO2  cleavage,  also  uracil  (Levene), 
guanidine  (Kutscher  and  Otori),  the  purine  bases,  which  originate 
from  the  nuclein  bodies,  and  choline,  which  latter  is  formed  from  lecithin 


1  Journ.  of  Physiol.,  28. 

2  Hermann,  Zeitschr.  /.  physiol.  Chem.,  11. 

3  Abderhalden  and  Reinbold,  Zeitschr.  f.  physiol.  Chem.,  44  and  46,  with  Voegtlin, 
ibid.  53. 


508  DIGESTION. 

(Kutscher  and  Lohmann1).  If  putrefaction  is  not  completely  pre- 
vented, still  other  bodies  occur  which  will  be  considered  later  in  con- 
nection with  the  putrefactive  processes  in  the  intestine. 

The  Action  of  Trypsin  upon  other  Bodies.  The  nucleoproteins  and 
nucleins  are  so  digested  that  the  protein  complex  is  separated  from  the 
nucleic  acid  and  then  digested.  The  nucleic  acids  may,  nevertheless, 
be  somewhat  changed  (Araki),  which  is  probably  brought  about  by 
another  enzyme,  the  nuclease  (Sachs).  A  cleavage  of  nucleic  acids  with 
the  setting  free  of  phosphoric  acid  and  purine  bases  is,  according  to 
Iwanoff,2  not  brought  about  by  trypsin.  The  splitting  is  first  pro- 
duced by  the  action  of  nuclease  or  erepsin  (see  page  493).  Gelatin  is 
dissolved  and  digested  by  pancreatic  juice.  A  cleavage  with  the  sepa- 
ration of  glycocoll  and  leucine  does  not  occur  (Kuhne  and  Ewald),  or 
only  to  a  trivial  extent  (Reich-Herzberge  3). 

The  gelatin-forming  substance  of  the  connective  tissues  is  not  directly 
dissolved  by  trypsin,  but  only  after  it  has  been  treated  with  acids  or 
soaked  in  water  at  70°  C.  By  the  action  of  trypsin  on  hyaline  cartilage 
the  cells  dissolve,  leaving  the  nucleus.  The  matrix  is  softened  and 
shows  an  indistinctly  constructed  network  of  collagenous  substances 
(Kuhne  and  Ewald).  The  elastic  substance,  the  structureless  membranes, 
and  the  membrane  of  the  fat-cells,  are  also  dissolved.  Parenchymatous 
organs,  such  as  the  liver  and  the  muscles,  are  dissolved  all  but  the  nuclei , 
connective  tissue,  fat-corpuscles,  and  the  remainder  of  the  nervous 
tissue.  If  the  muscles  are  boiled,  then  the  connective  tissue  is  also 
dissolved.  Mucin  is  dissolved  and  split  by  trypsin,  while  chitin  and  horn 
substance  do  not  seem  to  be  acted  upon  by  the  enzyme.  Oxyhemoglobin 
is  decomposed  by  trypsin  with  the  splitting  off  of  hsematin.  Trypsin 
splits  off  large  amounts  of  hydriodic  acid  from  diiodotyrosine  (Oswald  4) . 
Trypsin  has  no  action  upon  fats  and  carbohydrates. 

The  action  of  trypsin  on  simply  constructed  substances  of  known 
constitution  such  as  acid-amides,  polypeptides,  is  of  especially  great 
interest.  In  this  regard  we  have  the  somewhat  earlier  investigations 
of  Gulewitsch,  Gonnermann,  and    Schwarzschild,5  but  the  investi- 


1  Fischer  and  Abderhalden,  Zeitschr.  f.  physiol.  Chem.,  39;  Emerson,  Hofmeister's 
Beitrage,  1;  Levene,  Zeitschr.  f.  physiol.  Chem.,  37;  Kutscher  and  Lohmann,  ibid. 
39;  Kutscher  and  Otori,  ibid.,  43,  and  Centralbl.  f.  Physiol.,  18. 

2  Iwanoff,  Zeitschr.  f.  physiol.  Chem.,  39,  which  also  contains  the  literature;  Sachs, 
ibid.,  46. 

3  Kuhne  and  Ewald,  Verh.  d.  naturh.-med.  Vereins  zu  Heidelberg  (N.  F.),  1;  Reich- 
Herzberge,  Zeitschr.  f.  physiol.  Chem.,  34. 

4  Zeitschr.  f.  physiol.  Chem.  62,  432  (1909). 

5  Hofmeister's  Beitrage,  4,  where  the  other  works  are  also  cited. 


PANCREATIC  EENNIN.  509 

gations  of  Fischer  and  of  Abderhalden  and  their  co-workers,1  are 
much  more  complete  and  important.     In  this  connection  see  page  62. 

The  behavior  of  the  polypeptides  with  trypsin,  or  closely  related 
enzymes,  is  of  the  greatest  interest  and  in  many  respects  very  im- 
portant. Thus  in  the  polypeptides  we  have  a  means  of  determining 
the  kind  of  enzyme,  whether  it  belongs  to  the  pepsin,  trypsin,  or  erepsin 
group.  We  know  of  no  polypeptide  which  is  split  by  pepsin;  trypsin 
splits  only  certain  polypeptides,  but  not  others,  while  the  erepsin  on  the 
contrary  seems  to  split  all  polypeptides,  occurring  in  nature,  which 
are  composed  of  aminoacids.  By  the  aid  of  the  polypeptide  reaction 
Abderhalden  and  co-workers  have  also  been  able  to  show  that  the 
trypsin-like  enzyme,  occurring  in  the  blood-plasma,  is  not  identical 
with  trypsin  because  it  does  not  attack  glycyl-Z-tyrosine,  which  is  split 
by  trypsin. 

Pancreatic  rennin  is  an  enzyme  found  in  the  gland  and  in  the  juice, 
which  coagulates  neutral  or  alkaline  milk  (Kuhne  and  Roberts  and 
others).  This  enzyme,  according  to  Pawlow's  school,  is  identical 
with  trypsin.  The  similarity  of  action  of  these  two  enzymes  and  the 
fact  that  both  are  activated  simultaneously  from  the  zymogens  by  enter- 
okinase  or  lime  salts  (Delezenne,  Wohlgemuth  2)  seem  to  point  to 
this  identity.  On  the  other  hand  the  optimum  of  the  enzyme  action 
for  the  pancreatic  rennin  is  60-65°  C.  (Vernon),  which  is  much  higher 
than  for  the  trypsin,  and  Glaessner  and  Popper  3  have  also  observed 
a  case  where  the  human  pancreatic  juice  contained  no  rennin  enzyme. 

According  to  Halliburton  and  Brodie,4  casein  is  converted  by  the  pancreatic 
juice  of  the  dog  into  "  pancreatic  casein,"  a  substance  which,  in  regard  to  solubility, 
stands  to  a  certain  extent  between  casein  and  paracasein  (see  Chapter  XIII), 
and  which  is  converted  into  paracasein  by  rennin.  Further  investigations  on 
the  action  of  this  enzyme  upon  milk  and  especially  upon  pure  casein  solutions  are 
very  desirable. 

Pancreatic  Calculi.  The  concrement  from  a  cystic  enlargement  of  Wirsung's 
duct  in  a  man,  as  analyzed  by  Baldoni,  contained  in  1000  parts  as  follows: 
Water  34.4,  ash  126.7,  protein  substances  34.9,  free  fatty  acids  133,  neutral  fats 
124,  cholesterin  70.9,  soaps  and  pigment  499.1,  parts.  Scheunert  and  Berg- 
holz  8  have  reported  a  pancreatic  calculi  in  the  ox. 

1  Fischer  and  Bergell,  Ber.  d.  d.  chem.  Gesellsch.,  36  and  37;  Fischer  and  Abder- 
halden, Sitzungsber.  der  Kgl.  Pr.  Akad.  d.  Wissensch.,  Berlin,  1905.  The  works  of 
Abderhalden  and  co-workers  cannot  be  specially  cited,  but  may  be  found  in  Zeitschr. 
f.  physiol.  Chem.,  47,  48,  49,  51,  52,  53,  54,  55,  and  57. 

2  Kuhne  and  Roberts,  Maly's  Jahresber.,  9;  see  also  Edkins,  Journ.  of  Physiol., 
12  (literature);  Delezenne,  Compt.  rend.  soc.  biol.,  62  and  63;  Wohlgemuth,  Bioch. 
Zeitschr.,  2. 

3  Vernon,  Journ.  of  Physiol.,  12;  Glaessner  and  Popper,  Deutsch.  Arch.  f.  klin. 
Med.,  94. 

*  Journ.  of  Physiol.,  20. 

5  Baldoni,  Maly's  Jahresb.,  29,  353;  Scheunert  and  Bergholz.,  Zeitschr.  f.  physiol. 
Chem.,  52. 


510  DIGESTION. 

Besides  the  enzymes  which  have  been  discussed  in  connection  with 
the  pancreatic  juice,  the  gland  also  contains  others,  among  which  can  be 
mentioned  the  enzyme  which,  according  to  Stoklasa  and  his  collab- 
orators, occurs  principally  in  organs  and  tissues  and  which  decomposes 
sugar  into  alcohol  and  carbon  dioxide,  like  zymase.  Opinions  as  to  the 
importance  of  the  pancreas  for  glycolysis  are  diverse,  and  we  therefore 
refer  the  reader  to  what  has  been  previously  stated  on  this  subject  in 
Chapter  VII,  pages  407  and  408. 

V.     THE   CHEMICAL  PROCESSES   IN   THE  INTESTINE. 

The  action  which  belongs  to  each  digestive  secretion  may  be  essen- 
tially changed  under  certain  conditions  by  being  mixed  with  other 
digestive  fluids  for  various  reasons,  and  also  by  the  action  of  the 
enzymes  upon  each  other; 1  and  since  the  digestive  fluids  which  flow 
into  the  intestine  are  mixed  with  still  another  fluid,  the  bile,  it  will  be 
readily  understood  that  the  combined  action  of  all  these  fluids  in  the 
intestine  makes  the  chemical  processes  going  on  therein  very  complicated. 

As  the  acid  of  the  gastric  juice  acts  destructively  on  ptyalin,  this 
enzyme  has  no  further  diastatic  action,  even  after  the  acid  of  the  gastric 
juice  has  been  neutralized  in  the  intestine.  Roger  and  Simon  2  claim 
to  have  observed  in  saliva  made  inactive  by  the  gastric  juice,  a  reac- 
tivation caused  by  the  pancreatic  juice,  but  these  investigations  do 
not  seem  to  be  fully  conclusive.  The  bile  has,  at  least  in  certain  animals, 
a  slight  diastatic  action,  which  in  itself  can  hardly  be  of  any  great 
importance,  but  which  shows  that  the  bile  has  not  a  preventive,  but 
rather  a  beneficial  influence  on  the  energetic  diastatic  action  of  the  pan- 
creatic juice.  Several  experimenters3  have  observed  a  beneficial  action 
of  the  bile  on  the  diastatic  action  of  the  pancreas  infusion.  To  this 
may  be  added  that  the  micro-organisms  which  habitually  occur  in  the 
intestine  and  sometimes  in  the  food  have  partly  a  diastatic  action  and 
partly  produce  a  lactic-acid  and  butyric-acid  fermentation.  The 
maltose  which  is  formed  from  the  starch  seems  to  be  converted  into 
glucose  in  the  intestine.  It  seems  conclusively  that  the  cellulose  cannot 
be  digested  in  the  organism  of  the  dog.4  Lohrisch  found  that  on  an 
average  of  50  per  cent  of  the  introduced  cellulose  and  hemicellulose  was 
digested  in  human  beings  and  yielded  the  corresponding  sugar.     That 

1  See  Wr6ble\vski  and  collaborators,  Hofmcister's  Beitrage,  1. 

2  Compt.  rend.  soc.  biol.,  02. 

3  Martin  and  Williams,  Proceed,  of  Roy.  Soc,  45  and  48;  Bruno,  footnote  2,  p. 
502;  Buglia,  Bioch. Zeitschr.  25. 

*  Scheunert,  cited  from  Bioch.  Centralbl.  10,  71;  see  also  Lorhisch,  Zeitschr.  f.  pbysiol. 
Chem.  69,  143  (1910)  as  well  as  Bioch.  Centralbl.  8,  334. 


CHEMICAL  PROCESSES  IN  THE  [NTESTINE.  511 

cellulose  undergoes  a  fermentation  in  the  intestine  by  the  action  of  micro- 
organisms, producing  marsh-gas,  acetic  acid,  and  butyric  acid,  has 
been  especially  shown  by  Tappeiner;  still  it  is  not  known  to  what  extent 
the  cellulose  is  destroyed  in  this  way.1 

The  bile  has,  as  shown  by  Moore  and  Rockwood  2  and  then  espe- 
cially by  Pfluger,  the  property  to  a  high  degree  of  dissolving  fatty 
acids,  especially  oleic  acid,  which  itself  is  a  solvent  for  other  fatty  acids, 
and  hence,  as  will  be  seen  later,  it  is  of  great  importance  in  the  absorp- 
tion of  fat.  It  is  also  of  great  importance  that  the  bile,  as  previously 
stated,  not  only  activates  the  steapsinogen,  but  that,  as  first  shown  by 
Nencki  and  Rachford,3  it  accelerates  the  fat-splitting  action  of  the 
steapsin.  According  to  v.  Furth  and  Schutz4  the  bile-salts  are  the 
active  constituents  of  the  bile  in  this  cleavage,  and  the  fatty  acids  set 
free  can  combine  with  the  alkalies  of  the  intestinal  and  pancreatic  juices 
and  the  bile,  producing  soaps  which  are  of  great  importance  in  the 
emulsification  of  the  fats. 

If  to  a  soda  solution  of  about  1-3  p.  m.  pure,  perfectly  neutral 
olive-oil  is  added  in  not  too  large  a  quantity,  a  transient  emulsion  is 
obtained  after  vigorous  shaking.  If,  on  the  contrary,  one  adds  to  the 
same  quantity  of  soda  solution  an  equal  amount  of  commercial  olive- 
oil  (which  always  contains  free  fatty  acids),  the  vessel  need  only  be 
turned  over  for  the  two  liquids  to  mix,  and  immediately  there  appears 
a  very  finely  divided  and  permanent  emulsion,  making  the  liquid  appear 
like  milk.  The  free  fatty  acids  of  the  commercial  oil,  which  is  always 
somewhat  rancid,  combine  with  the  alkali  to  form  soaps  which  act  to 
emulsify  the  fats  (Brucke,  Gad,  Loewenthal  5).  This  emulsifying 
action  of  the  fatty  acids  split  off  by  the  pancreatic  juice  is  undoubtedly 
assisted  by  the  habitual  occurrence  of  free  fatty  acids  in  the  food,  as 
well  as  by  the  splitting  off  of  fatty  acids  from  the  neutral  fats  in  the 
stomach  (see  page  476). 

Bile    completely    prevents    peptic    zymolysis    in    artificial    digestion, 


lOn  the  digestion  of  cellulose  see  Henneberg  and  Stohmann,  Zeitschr,  f.  Biologie, 
21,  613;  v.  Knieriem,  ibid.,  67;  Hofmeister,  Arch.  f.  wiss.  u.  prakt.  Thierheilkunde, 
11;  Weiske,  Zeitschr.  f.  Biologie,  22,  373;  Tappeiner,  ibid.,  20  and  24;  Mallevre, 
Pfliiger's  Arch.,  49;  Omeliansky,  Arch.  d.  scienc.  biol.  de  St.  Petersbourg,  7;  E.  Muller, 
Pfliiger's  Arch.,  83;  Lohrisch,  Zeitschr.  f.  physiol.  Chem.,  47  (literature);  Pringsheim, 
ibid.  78,  266  (1912). 

2  Proceedings  of  Roy.  Soc,  60,  and  Journ.  of  Physiol.,  21.  In  regard  to  Pfliiger's 
work  see  Absorption. 

3  Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  20;  Rachford,  Journal  of  Phvsiol.,  12. 

4  Centralbl.  f.  Physiol.,  20. 

*  Brucke,  Wien,  Sitzungsber.,  61,  Abt.  2;  Gad,  Arch.  f.  (Anat.  u.)  Physiol.,  1878; 
Loewenthal,  ibid.,  1897. 


512  DIGESTION. 

because  it  retards  the  swelling  up  of  the  proteins.  The  passage  of  bile 
into  the  stomach  during  digestion  on  the  contrary,  seems,  according, 
to  several  investigators,  especially  Oddi  and  Dastre,1  to  have  no  dis- 
turbing action  on  gastric  digestion.  According  to  Boldyreff,2  after 
continuous  starvation,  on  feeding  fat  and  food  rich  in  fat,  as  well  as  after 
large  amounts  of  acid,  a  mixture  of  bile,  pancreatic  juice,  and  intestinal 
juice  pass  readily  into  the  stomach.  After  food  rich  in  fat,  which 
retards  the  secretion  of  gastric  juice  and  the  motility  of  the  stomach, 
a  digestion  due  to  this  alkaline  mixture  may  take  place  in  the  stomach. 

Bile  itself  has  no  solvent  action  on  proteins  in  neutral  or  alkaline 
reaction,  but  still'  it  may  exert  an  influence  on  protein  digestion  in  the 
intestine.  The  acid  contents  of  the  stomach,  containing  an  abundance 
of  proteins,  give  with  the  bile  a  precipitate  of  proteins  and  bile-acids. 
This  precipitate  carries  a  part  of  the  pepsin  with  it,  and  for  this  reason, 
and  also  on  account  of  the  partial  or  complete  neutralization  of  the 
acid  of  the  gastric  juice  by  the  alkali  of  the  bile  and  the  pancreatic 
juice,  the  pepsin  digestion  cannot  proceed  further  in  the  intestine. 
According  to  Baumstark  and  Cohnheim  3  connective  tissue  is  digested 
on  the  other  side  of  the  pylorus  in  the  intestine  by  the  pepsin-hydrochloric 
acid.  On  the  contrary,  the  bile  does  not  disturb  the  digestion  of  pro- 
teins by  the  pancreatic  juice  in  the  intestine.  The  action  of  these  diges- 
tive secretions,  as  above  stated,  is  not  disturbed  by  the  bile,  not  even 
by  the  faintly  acid  reaction  due  to  organic  acids;  but,  on  the  contrary, 
the  action  of  trypsin  is  accelerated  by  the  bile.  In  a  dog  killed  while 
digestion  is  going  on,  the  faintly  acid,  bile-containing  material  of  the 
intestine  shows  regularly  a  strong  digestive  action  on  proteins. 

The  precipitate  of  protein  and  bile-salts  formed  on  the  meeting  of  the 
acid  contents  of  the  stomach  with  the  bile  easily  redissolves  in  an  excess 
of  bile,  and  also  in  the  NaCl  formed  in  the  neutralization  of  the  hydro- 
chloric acid  of  the  gastric  juice.  This  may  take  place  even  in  faintly 
acid  reaction.  Since  in  man  the  excretory  ducts  of  the  bile  and  the 
pancreatic  juice  open  near  one  another,  in  consequence  of  which  the 
acid  contents  of  the  stomach  are  probably  immediately  in  great  part 
neutralized  by  the  bile  as  soon  as  it  enters,  it  is  doubtful  whether  a  pre- 
cipitation of  proteins  by  the  bile  occurs  in  the  intestine. 

Besides  the  previously  mentioned  processes  caused  by  enzymes, 
there  are  others  of  a  different  nature  going  on  in  the  intestine,  namely, 
the  fermentation  and  putrefaction  processes  caused  by  micro-organ- 
isms.     These  are   less  intense  in  the  upper  parts  of  the  intestine,  but 


» Oddi,  in  Centralbl.  f.  Physiol.,  1,  312;  Dastre,  Arch,  de  Physiol.  (5),  2,  316. 
1  Centralbl.  f.  Physiol.,  18,  457,  and  Pfluger's  Arch.,  121. 
3  Zeitschr.  f.  physio).  Chem.,  65,  477  (1910). 


PUTREFACTION  IN  THE  INTESTINE.  513 

increase  in  intensity  toward  the  lower  part,  and  decrease  in  the  large 
intestine  because  of  the  consumption  of  fermentable  material  and  by 
the  removal  of  water  by  absorption.  Fermentation  processes,  but  only 
very  slight  putrefaction,  occur  in  the  small  intestine  of  man.  Mac- 
fadyen,  M.  Nencki,  and  N.  Seeber  *  have  investigated  a  case  of 
human  anus  praeternaturlis,  in  which  the  fistula  occurred  at  the  lower 
end  of  the  ileum,  and  they  were  able  to  investigate  the  contents  of  the 
intestine  after  it  had  been  exposed  to  the  action  of  the  mucous  mem- 
brane of  the  entire  small  intestine.  The  mass  was  yellow  or  yellowish- 
brown,  due  to  bilirubin,  and  had  an  acid  reaction  which,  on  a  mixed 
but  principally  animal  diet,  calculated  as  acetic  acid,  amounted  to  1  p.  m. 
The  contents  were  nearly  odorless,  having  an  empyreumatic  odor  recall- 
ing that  of  volatile  fatty  acids,  and  infrequently  had  a  putrid  odor 
resembling  that  of  indol.  The  essential  acid  present  was  acetic  acid, 
accompanied  by  fermentation  and  paralactic  acid,  volatile  fatty  acids, 
succinic  acid,  and  bile-acids.  Coagulable  proteins,  peptone,  mucin, 
dextrin,  sugar,  and  alcohol  were  present.  Leucine  and  tyrosine  could 
not  be  detected. 

According  to  the  above-mentioned  investigators,  the  proteins  are 
only  to  a  very  slight  extent,  if  at  all,  decomposed  by  the  microbes  in 
the  small  intestine  of  man.  The  organisms  present  in  the  small  intestine 
preferably  decompose  the  carbohydrates,  forming  ethyl  alcohol  and  the 
above-mentioned  organic  acids. 

Further  investigations  of  Jakowsky  and  of  Ad.  Schmidt2  lead  to 
the  same  result,  namely,  that  in  man  the  putrefaction  of  the  proteins 
takes  place  chiefly  in  the  large  intestine,  and  the  conditions  are  the 
same  in  carnivora.  In  these  latter  it  has  been  possible  to  follow  the 
intestinal  digestion  by  investigating  the  contents  of  the  various  parts 
of  the  intestine  as  well  as  by  forming  fistulas  along  the  intestine.  Again 
Pawlow  and  his  pupils,  especially  London  3  and  his  collaborators,  have 
essentially  advanced  our  knowledge  on  this  subject. 

In  regard  to  the  digestion  of  protein,  it  has  been  found  that  after 
feeding  meat,  bread,  or  certain  protein  bodies,  the  digestion  in  the 
stomach  and  small  intestine  is  so  complete  that  on  the  passage  of  the 
contents  into  the  caecum  all  the  protein  is  digested  and  absorbed. 
Unboiled  white  of  egg  is  an  exception  and  is  digested  with  difficulty. 
In  experiments  with  unboiled  white  of  egg,  London  and  Suleima  reob- 


1  Arch.  f.  exp.  Path.  u.  Pharm.,  28. 

2  Jakowsky,  Arch,  des  scienc.  biol.  de  St.  Petersbourg,  1;   Ad.   Schmidt,  Arch.  f. 
Verdauunskr.,  4. 

'  The  works  of  London  and  collaborators  cannot  be  cited  in  detail,  but  may  be 
found  in  Zeitschr.  f.  physiol.  Chem.,  46-57. 


514  DIGESTION. 

tained  73  per  cent  of  the  coagulable  protein  from  a  fistula  in  the  ileum 
(2-3  cm.  in  front  of  the  caecum).  Elastin  is,  according  to  London,  * 
more  slowly  digested  in  the  small  intestine  than  other  proteins.  Kut- 
scher  and  Seemann,  Abderhalden,  London  and  collaborators  2  have 
also  found  that  non-biuret  giving  products  and  amino-acids  are  regularly 
split  off,  probably  by  the  combined  action  of  trypsin  and  erepsin.  These 
amino-acids  occur  to  a  slight  extent  only,  but  from  this  no  conclusion 
can  be  drawn  as  to  the  extent  of  amino-acid  formation,  because  we  do 
not  know  the  extent  of  their  absorption.  The  digestion  of  protein 
in  the  intestine,  it  seems,  according  to  Abderhalden,  London,  Oppler 
and  Reemlin,3  is  similar  to  the  artificial  digestion  with  trypsin,  nainely, 
that  the  destruction  takes  place  step-wise,  that  certain  amino-acids, 
such  as  tyrosine,  are  split  off  earlier  than  others.  Zunz  4  found  the  same 
end  result  in  the  protein  cleavage  in  the  small  intestine,  with  bread  as 
with  meat  feeding.  London,  Schittenhelm  and  Wiener  5  found 
that  a  cleavage  of  nucleic  acids  with  the  formation  of  nucleosides 
occurred  in  the  lower  part  of  the  jejunum  and  ileum. 

The  decomposition  products  of  the  proteins  formed  by  the  action  of 
gastric  juice  can,  according  to  London,  6  be  absorbed  without  further 
cleavage  by  the  pancreatic  juice,  and  a  further  cleavage  in  the  intestine 
seems  to  be  more  necessary  for  assimilation  than  for  absorption. 

The  carbohydrates  and  the  fats  (Levites  7)  may  be  so  completely 
split  in  the  stomach  and  small  intestine  that  their  absorption  is  com- 
plete before  the  contents  pass  into  the  caecum.  According  to  London 
and  Polowzowa  8  a  strong  cleavage  of  starch,  dextrins  and  disaccharides 
takes  place,  especially  in  the  duodenum,  while  the  absorption  is  less 
strong  here.  The  carbohydrates  are  here  prepared  for  the  absorption 
taking  place  in  the  lower  parts  of  the  intestine,  though  the  cleavage  also 
goes  on  in  the  other  parts,  namely  in  the  jejunum  and  the  upper  part 
of  the  ileum. 

As  above  remarked,  ordinarily  no  putrefaction  takes  place  in  the 
small  intestine,  but  occurs  generally  only  in  the  large  intestine.     This 


1  London  and  Suleima,  Zeitschr.  f.  physiol.  Chem.,  46;  London,  ibid.,  60. 

2  Kutscher   and   Seemann.   ibid.,   34;  Abderhalden   and   London,   with   Kautzsch, 
ibid.,  with  L.  Baumann,  ibid.,  51,  with  v.  Korosy,  ibid.,  53. 

3  Zeitschr.  f.  physiol.  Chem.,  55  and  58. 

4  Intern.  Beitr.  z.  Pathol,  u.  Ther.  d.  Ernahrungsstorungen,  2,  195,  459  (1910  and 
1911). 

'■>  Zeitschr.  f.  physiol.  Chem.,  72,  459  (1911). 
•  Ibid.,  49. 
7  Ibid.,  49  and  53. 
yIbid.,  56. 


PUTREFACTION  IN  THE  INTESTINE.  515 

putrefaction  of  the  proteins  is  not  the  same  as  the  pancreatic  digestion. 
In  putrefaction  the  decomposition  goes  much  further,  and  a  mixture  of 
products  is  obtained  which  have  become  known  through  the  labors 
of  numerous  investigators,  especially  Nbncki,  Baimanx,  Bkieger,  H. 
and  E.  Salkowski,  and  their  pupils.  The  products  which  are  formed 
in  the  putrefaetion  of  proteins  are  (in  addition  to  proteoses,  peptones, 
amino-acids,  and  ammonia)  indol,  skatol,  paracresol,  phenol,  phenylpro- 
pioitic  acid,  and  phenylacetic  acid,  also  paraoxyphenylacetic  acid  and 
hydroparacoumaric  acid  (besides  paracresol,  produced  in  the  putrefaction 
of  tyrosin),  volatile  fatty  acids,  carbon  dioxide,  hydrogen,  marsh-gas, 
■mcthylmercaptan,  and  sulphureted  hydrogen.  In  the  putrefaction  of 
gelatin  neither  tyrosine  nor  indol  is  formed,  while  glycocoll  is  produced 
instead. 

Among  these  products  of  decomposition  a  few  are  of  special  interest 
because  of  their  behavior  within  the  organism  and  because  after  their 
absorption  they  pass  into  the  urine.  A  few,  such  as  the  oxyacids,  pass 
unchanged  into  the  urine.  Others,  such  as  phenols,  are  directly  trans- 
formed into  ethereal  sulphuric  acids  by  synthesis,  and  are  eliminated  as 
such  by  the  urine;  on  the  contrary,  others,  such  as  indol  and  skatol,  are 
converted  into  ethereal  sulphuric  acids  after  oxidation  only  (for  details 
see  Chapter  XIV).  The  quantity  of  these  bodies  in  the  urine  also  varies 
with  the  extent  of  the  putrefactive  processes  in  the  intestine;  at  least 
this  is  true  for  the  ethereal  sulphuric  acids.  Their  quantity  increases 
in  the  urine  with  a  stronger  putrefaction,  and  the  reverse  takes  place, 
namely,  a  disappearance  from  the  urine,  or  a  great  reduction  in  quantity, 
as  Baumann,  Harley  and  Goodbody  1  have  shown  by  experiments 
on  dogs,  when  the  intestine  is  disinfected  by  various  agents. 

The  gases  which  are  produced  by  the  decomposition  processes  are 
mixed  in  the  intestinal  tract  with  the  atmospheric  air  swallowed  with 
the  saliva  and  food,  and  as  the  gas  developed  in  the  decomposition  of 
different  foods  varies,  so  the  mixture  of  gases  after  various  foods  should 
have  a  dissimilar  composition.  This  is  found  to  be  true.  Oxygen  is 
found  only  in  very  faint  traces  in  the  intestine;  this  may  be  accounted 
for  in  part  by  the  formation  of  reducing  substances  in  the  fermenta- 
tion processes  which  combine  with  the  oxygen,  and  partly,  perhaps 
chiefly,  to  a  diffusion  of  the  oxygen  through  the  tissues  of  the  walls  of 
the  intestine.  To  show  that  these  processes  take  place  mainly  in  the 
stomach,  the  reader  is  referred  to  page  486,  on  the  composition  of  the 
gases  of  the  stomach.  Xitrogen  is  invariably  found  in  the  intestine, 
and  it  is  probably  clue  chiefly  to  the  swallowed  air.     The  carbon  dioxide 

1  Baumann,  Zeitschr.  f.  physiol.  Chem.,  10;  Harley  and  Goodbody,  Brit.  Med. 
Journ.,  1899. 


516  DIGESTION. 

originates  partly  from  the  contents  of  the  stomach,  partly  from  the 
putrefaction  of  the  proteins,  partly  from  the  lactic-acid  and  butyric- 
acid  fermentation  of  carbohydrates,  and  partly  from  the  setting  free  of 
carbon  dioxide  from  the  alkali  carbonates  of  the  pancreatic  and  intes- 
tinal juices  by  their  neutralization  through  the  hydrochloric  acid  of 
the  gastric  juice  and  by  organic  acids  formed  in  the  fermentation. 
Hydrogen  occurs  in  largest  quantities  after  a  milk  diet,  and  in  smallest 
quantities  after  a  purely  meat  diet.  This  gas  seems  to  be  formed 
chiefly  in  the  butyric-acid  fermentation  of  carbohydrates,  although  it 
may  occur  in  large  quantities  in  the  putrefaction  of  proteins  under  certain 
circumstances.  There  is  no  doubt  that  the  methylmercaptan  and  sul- 
phureted  hydrogen  which  occur  normally  in  the  intestine  originate  from 
the  proteins.  The  marsh-gas  undoubtedly  originates  in  the  putrefac- 
tion of  proteins.  As  proof  of  this  Ruge  *■  found  26.45  per  cent  marsh- 
gas  in  the  human  intestine  after  a  meat  diet.  He  found  a  still  greater 
quantity  of  this  gas  after  a  vegetable  (leguminous)  diet;  this  coincides 
with  the  observation  that  marsh-gas  may  be  produced  by  a  fermentation 
of  carbohydrates,  but  especially  of  cellulose  (Tappeiner  2) .  Such  an 
origin  of  marsh-gas,  especially  in  herbivora,  is  to  be  expected.  A  small 
part  of  the  marsh-gas  and  carbon  dioxide  may  also  arise  from  the  decom- 
position of  lecithin  (Hasebroek3). 

Putrefaction  in  the  intestine  not  only  depends  upon  the  composi- 
tion of  the  food,  but  also  upon  the  albuminous  secretions  and  the  bile. 
Among  the  constituents  of  bile  which  are  changed  or  decomposed, , there 
are  not  only  the  pigments — the  bilirubin  yields  urobilin  and  a  brown 
pigment — but  also  the  bile-acids,  especially  taurocholic  acid.  Glyco- 
cholic  acid  is  more  stable,  and  a  part  is  found  unchanged  in  the  excre- 
ment of  certain  animals,  while  taurocholic  acid  is  so  completely  decom- 
posed that  it  is  entirely  absent  in  the  feces.  In  the  fetus,  on  the  con- 
trary, in  whose  intestinal  tract  no  putrefaction  processes  occur,  undecom- 
posed  bile-acids  and  bile-pigments  are  found  in  the  contents  of  the 
intestine.  The  transformation  of  bilirubin  into  urobilin  does  not  occur, 
as  previously  stated,  in  the  small,  but  in  the  large  intestine  in  man. 

As  under  normal  conditions  no  putrefaction,  or  a,t  least  none  worth 
mentioning,  occurs  in  the  small  intestine,  and  as  often  nearly  all  the  pro- 
tein of  the  food  is  absorbed,  it  follows  that  ordinarily  it  is  the  secretions 
and  cells  rich  in  proteins  which  undergo  putrefaction.  That  the  secre- 
tions  rich   in  proteins   are   destroyed   in  putrefaction  in  the   intestine 


1  Wien.  Sitzungsber.,  44. 

2  Zeitschr.  f.  Biologie.,  20  and  24. 

3  Zeitschr.  f.  phyaiol.  Chem.,  12. 


PUTREFACTION    IN  THE  INTESTINE.  517 

follows  from  the  fact  that  putrefaction  may  also  continue  during  com- 
plete fasting.  From  the  observations  of  Muller  l  upon  Cetti  it  was 
found  that  the  elimination  of  indican  during  starvation  rapidly  de- 
creased  and  after  the  third  day  of  starvation  it  had  entirely  disappeared, 
while  the  phenol  elimination,  which  at  first  decreased  so  that  it  was 
nearly  minimum,  increased  again  from  the  fifth  day  of  starvation,  and 
on  the  eighth  or  ninth  day  it  was  three  to  seven  times  as  much  as  in  man 
under  ordinary  circumstances.  In  dogs,  on  the  contrary,  the  elimina- 
tion of  indican  during  starvation  is  considerable,  but  the  phenol  elimina- 
tion is  slight.  Among  the  secretions  which  undergo  putrefaction  in  the 
intestine,  the  pancreatic  juice,  which  putrefies  most  readily,  takes  first 
place. 

From  the  foregoing  facts  it  must  be  concluded  that  the  products 
formed  by  the  putrefaction  in  the  intestine  are  in  part  the  same  as 
those  formed  in  digestion.  The  putrefaction  may  be  of  benefit  to  the 
organism  in  so  far  as  the  formation  of  such  products  as  proteoses,  pep- 
tones, polypeptides  and  amino-acids  is  concerned.  The  question  has 
indeed  been  asked  (Pasteur),  Is  digestion  possible  without  micro-organ- 
isms? Nuttal  and  Thierfelder  have  shown  that  guinea-pigs,  removed 
from  the  uterus  of  the  mother  by  Caesarian  section,  could  with  sterile 
air  digest  well  and  assimilate  sterile  food  (milk  and  crackers)  in  the 
complete  absence  of  bacteria  in  the  intestine,  and  developed  normally 
and  increased  in  weight.  Schottelius  2  has  arrived  at  other  results 
by  experiments  with  hens.  The  chickens,  hatched  under  sterile  con- 
ditions, kept  in  sterile  rooms  and  fed  with  sterile  food,  had  continuous 
hunger  and  ate  abundantly,  but  soon  died,  in  about  the  same  time  as  a 
starving  chicken.  On  mixing  with  the  food,  at  the  proper  time,  a  vari- 
ety of  bacteria  from  hen  feces,  they  gained  weight   again  and  recovered. 

The  bacterial  action  in  the  intestinal  canal  is,  at  least  in  certain  cases, 
as  with  food  rich  in  cellulose,  necessary,  and  it  acts  in  the  interest  of  the 
organism.  This  action  may,  by  the  formation  of  further  cleavage  prod- 
ucts, involve  a  loss  of  valuable  material  to  the  organism,  and  it  is  there- 
fore important  that  putrefaction  in  the  intestine  be  kept  within  certain 
limits.  If  an  animal  is  killed  while  digestion  in  the  intestine  is  going 
on,  the  contents  of  the  small  intestine  give  out  a  peculiar  but  not  putres- 
cent odor.  Also  the  odor  of  the  contents  of  the  large  intestine  is  far  less 
offensive  than  a  putrefying  pancreas  infusion  or  a  putrefying  mixture 
rich  in  protein.  From  this  one  may  conclude  that  putrefaction  in  the 
intestine  is  ordinarily  not  nearly  so  intense  as  outside  of  the  organism. 

1  Berlin,  klin.  Wochenschr.,  1887. 

2  Nuttal  and  Thierfelder,  Zeitschr.  f.  physiol.  Chem.,  21  and  22;  Schottelius,  Arch, 
f.  Hygiene,  34,  42,  and  67. 


518  DIGESTION. 

It  seems  thus  to  be  provided,  under  physiological  conditions,  that 
putrefaction  shall  not  proceed  too  far,  and  the  factors  which  here  come 
into  consideration  are  probably  of  different  kinds.  Absorption  is 
undoubtedly  one  of  the  most  important  of  them,  and  it  has  been  proved 
by  actual  observation  that  the  putrefaction  increases,  as  a  rule,  as  the 
absorption  is  checked  and  fluid  masses  accumulate  in  the  intestine.  The 
character  of  the  food  also  has  an  unmistakable  influence,  and  it  seems 
as  if  a  large  quantity  of  carbohydrates  in  the  food  acts  against  putre- 
faction (Hirschler  1).  It  has  been  shown  by  Pohl,  Biernacki, 
Rovighi,  Winternitz,  Schmitz,  and  others 2  that  milk  and  kephir 
have  a  specially  strong  preventive  action  on  putrefaction.  This  action 
is  not  due  to  the  casein,  but  chiefly  to  the  lactose  and  also  in  part  to  the 
lactic  acid. 

A  specially  strong  preventive  action  on  putrefaction  has  been 
ascribed  for  a  long  time  to  the  bile.  This  anti-putrid  action  does  not 
exist  in  neutral  or  faintly  alkaline  bile,  which  itself  easily  putrefies,  but 
to  the  free  bile-acids,  especially  taurocholic  acid  (Maly  and  Emich, 
Lindberger3).  There  is  no  question  that  the  free  bile-acids  have  a 
strong  preventive  action  on  putrefaction  outside  of  the  organism,  and 
it  is  therefore  difficult  to  deny  such  an  action  in  the  acid  reacting  con- 
tents of  the  intestine.  Notwithstanding  this,  the  anti-putrid  action 
of  the  bile  in  the  intestine  is  not  considered  by  certain  investigators 
(Voit,  Rohmann,  Hirschler  and  Terray,  Landauer  and  Rosen- 
berg 4)   as  of  great  importance. 

Biliary  fistulas  have  been  established  so  as  to  study  the  importance 
of  the  bile. in  digestion  (Schwann,  Blondlot,  Bidder  and  Schmidt,5 
and  others).  As  a  result  it  has  been  observed  that  with  fatty  foods  an 
imperfect  absorption  of  fat  regularly  takes  place  and  the  excrement 
contains,  therefore,  an  excess  of  fat  and  has  a  light-gray  or  pale  color. 
The  extent  of  deviation  from  the  normal  after  the  operation  is  essen- 
tially dependent  upon  the  character  of  the  food.  If  an  animal  is  fed 
on  meat  and  fat,  then  the  quantity  of  food  must  be  considerably  increased 
after  the  operation,  otherwise  the  animal  will  become  very  thin,  and 


1  Hirschler,  Zeitschr.  f.  physiol.  Chem.,  10;   Zimnitzki,  ibid.,  39  (literature). 

2  Schmitz,  ibid.,  17,  401,  which  gives  references  to  the  older  literature,  and  19.  See 
also  Salkowski,  Centralbl.  f.  d.  med.  Wiss.,  1893,  467,  and  Seelig,  Virchow's  Arch., 
126  (literature). 

3  Maly  and  Emich,  Monatshefte,  f.  Chem.,  4;  Lindberger,  footnote  4,  p.  506. 

4  Voit,  Beitr.  zur  Biologie,  Jubilaumschrift,  Stuttgart,  1882;  Rohmann,  Pfltiger's 
Arch.  29;  Hirschler  and  Terray,  Maly's  Jahresber.,  26;  Landauer,  Math,  u.  Naturw. 
Ber.  aus  Ungarn,  15;  Rosenberg,  Arch.  f.  (Anat.  u.)  Physiol.,  1901. 

'Schwann,  Miiller's  Arch.  f.  Anat.  u.  Physiol.,  1844;  Blondlot,  cited  from  Bidder 
and  Schmidt,  Verdauungssafte,  etc.,  98. 


PUTREFACTION  IN  THE   INTESTINE.  519 

indeed  die  with  symptoms  of  starvation.  In  these  cases  the  excrement 
has  the  odor  of  carrion,  and  this  was  considered  a  proof  of  the  action 
of  the  bile  in  checking  putrefaction.  The  emaciation  and  the  increased 
want  of  food  depend,  naturally,  upon  the  imperfect  absorption  of  the 
fats,  whose  high  calorific  value  is  reduced  and  must  be  replaced  by  the 
taking  up  of  larger  quantities  of  other  nutritive  bodies.  If  the  quan- 
tity of  proteins  and  fats  be  increased,  then  the  latter,  which  can  be  only 
incompletely  absorbed,  accumulate  in  the  intestine.  This  accumulation 
of  the  fats  in  the  intestine  only  renders  the  action  of  the  digestive  juices 
on  proteins  more  difficult,  and  thus  increases  the  amount  of  putrefac- 
tion. This  explains  th'e  appearance  of  fetid  feces,  whose  pale  color  is 
not  due  to  a  lack  of  bile-pigments,  but  to  a  surplus  of  fat  (Rohmann, 
Voit).  If  the  animal  is,  on  the  contrary,  fed  on  meat  and  carbohy- 
drates, it  may  remain  quite  normal,  and  the  leading  off  of  the  bile  does 
not  cause  any  increased  putrefaction.  The  carbohydrates  may  be 
uninterruptedly  absorbed  in  such  large  quantities  that  they  replace 
the  fat  of  the  food,  and  this  is  the  reason  why  the  animal  on  such  a  diet 
dots  not  become  emaciated.  As  with  this  diet  the  putrefaction  in  the 
intestine  is  no  greater  than  under  normal  conditions  even  though  the  bile 
is  absent,  it  would  seem  that  the  bile  in  the  intestine  exercises  no  pre- 
ventive action  on  putrefaction. 

To  this  conclusion  the  objection  may  be  made  that  the  carbohy- 
drates, which  are  capable  of  checking  putrefaction,  can,  so  to  speak, 
undertake  the  anti-putrid  action  of  the  bile.  But  as  there  are  also  cases 
(in  dogs  with  biliary  fistula)  where  the  intestinal  putrefaction  is  not 
increased  with  exclusive  meat  diet,1  it  is  maintained  that  the  absence 
of  bile  in  the  intestine,  even  by  exclusive  carbohydrate  food,  does  not 
always  cause  an  increased  putrefaction. 

Although  the  question  as  to  the  manner  in  which  the  putrefactive 
processes  in  the  intestine  under  physiological  conditions  are  kept  within 
certain  limits  cannot  le  answered  positively,  still  it  may  be  asserted 
that  the  faint  acid  reaction,  and  the  absorption  of  water,  and  the  rela- 
tively rapid  movement,  of  the  contents  of  the  small  intestine  and  their 
absorption,  are  important  factors. 

That  the  acid  reaction  in  the  intestine  has  a  preventive  influence  on 
the  putrefactive  processes  follows  from  the  existing  relation  between 
the  degree  of  acidity  of  the  gastric  juice  and  the  putrefaction  in  the 
intestine.  Since  the  investigations  and  observations  of  Kast,  Stadel- 
mann,  Wasbutzki,  Biernacki  and  Mester  had  proved  that  an 
increased  putrefaction  in  the  intestine  occurred  when  the  quantity  of 
hydrochloric    acid    in    the    gastric    juice    was    diminished    or  deficient, 

1  See  Hirschler  and  Terray,  1.  c. 


520  DIGESTION. 

Schmitz  1  has  shown  in  man  that  on  the  administration  of  hydro- 
chloric acid,  producing  a  hyperacidity  of  the  gastric  juice,  the  putrefac- 
tion in  the  intestine  may  be  checked.  The  question  arises  whether  the 
reaction  in  the  small  intestine  is  always  acid  and  whether  the  acidity 
is  strong  enough  to  prevent  putrefaction.  In  this  connection  it  must 
be  recalled  that  the  acidity  of  the  contents  of  the  small  intestine  is  not 
due  to  hydrochloric  acid,  but  chiefly  to  organic  acids,  acid  salts,  and 
free  carbon  dioxide.  There  are  several  observations  as  to  the  reaction 
of  the  intestinal  contents,  by  Moore  and  Rockwood,  Moore  and 
Bergin,  Matthes  and  Maequardsen,  I.  Munk,  Nencki  and  Zaleski, 
Hemmeter,2  although  they  are  somewhat  contradictory.  From  these 
reports  one  can  conclude  that  the  reaction  may  vary  not  only  among 
different  animals,  but  also  in  the  same  animals  under  varying  conditions. 
There  is  no  doubt  that  the  acid  reaction  in  many  cases  is  due  to  the  pres- 
ence cf  organic  acids.  On  testing  with  various  indicators  it  has  been 
shown  that  sometimes  the  upper  parts,  and  often  the  lower  parts,  are 
acid,  due  to  acid  salts  such  as  NaHCC>3  and  free  C02,  and  finally  that 
in  certain  animals  the  intestinal  contents  are  alkaline  throughout.  The 
question  how,  under  these  conditions,  putrefaction  is  excluded,  and  how 
the  acidity  of  the  gastric  contents  influences  the  intestinal  putrefaction, 
cannot  be  explained.  It  is  very  probable  that  the  bacterial  flora  of  the 
intestine  is  of  very  great  importance  and  it  is  possible,  as  Bienstock 
admits,  that  the  explanation  lies  in  an  antagonistic  bacterial  action  and 
that  the  carbohydrates,  especially  lactose,  which  retard  putrefaction, 
form  a  good  nutritive  media  for  those  bacteria  which  destroy  the  putre- 
factive producers  or  retard  their  development.  According  to  Horo- 
witz an  unequal  division  of  the  various  bacteria  occurs  in  dogs  in  the 
different  parts  of  the  intestine  and  certain  varieties  of  bacteria  occur 
in  greater  quantities  than  others,  according  to  the  kind  of  food  taken. 
The  influence  of  the  kind  of  food  upon  the  intestinal  flora  has  also  been 
studied  by  Kendall.  Perhaps,  also,  agreeing  with  the  experience  of 
Conradi  and  Kurpjuweit,3  the  toxins  produced  by  the  intestinal  bacteria 
may,  by  their  antiseptic  action,  keep  the  putrefactive  processes  in  the 
intestine  within  bounds. 


1  Zeitschr.  f.  physiol.  Chem.,  19,  401,  which  includes  all  the  pertinent  literature. 

2  Moore  and  Rockwood,  Journ.  of  Physiol.,  21;  Moore  and  Bergin,  Amer.  Journ. 
of  Physiol.,  3;  Matthes  and  Marquardsen,  Maly's  Jahresber.,  28;  Munk,  Centralbl.  f. 
Physiol.,  16;  Nencki  and  Zaleski,  Zeitschr.  f.  physiol.  Chem.,  27;  Hemmeter,  Pfluger's 

Arch.,  81. 

3  Bienstock,  Arch.  f.  Hygiene,  39;  Horowitz.  Zeitschr.  f.  physiol.  Chem.,  52;  Ken- 
dall, Journ.  of  biol.  Chem.  6,  499  (1909);  Conradi  and  Kurpjuweit,  Munch,  med. 
Wochenschr.,  1905. 


feci;-.  521 

Feces.  It  is  evident  that  the  residue  which  remains  after  complete 
digestion  and  absorption  in  the  intestine  must  be  different,  both  quali- 
tatively and  quantitatively,  according  to  the  variety  and  quantity  of 
the  food.  In  man  the  quantity  of  excrement  from  a  mixed  diet  is 
120-150  grams,  with  30-37  grams  of  solids,  per  twenty-four  hours,  while 
the  quantity  from  a  vegetable  diet,  according  to  Voit,1  was  333 
grams,  with  75  grams  of  solids.  With  a  strictly  meat  diet  the  excre- 
ment is  scanty,  pitch-like,  and  black.  The  scanty  feces  in  starva- 
tion have  a  similar  appearance.  A  large  quantity  of  coarse  bread  yields 
a  great  amount  of  light-colored  excrement.  In  these  cases  the  feces 
are  also  habitually  poorer  in  nitrogen  than  after  food  rich  in  protein. 
The  individuality  also  plays  an  important  role  in  the  utility  of  the  food 
and  the  formation  of  feces  (Schierbeck  2).  If  there  is  a  large  propor- 
tion of  fat,  it  takes  a  lighter  clay-like  appearance.  The  decomposi- 
tion products  of  the  bile-pigments  seem  to  play  only  a  small  part  in  the 
normal  color  of  the  feces. 

The  constituents  of  the  feces  are  of  different  kinds.  In  the  excre- 
ment are  found  digestible  or  absorbable  constituents  of  the  food,  such 
as  muscle  fibers,  connective  tissues,  lumps  of  casein,  grains  of  starch, 
and  fat,  which  have  not  had  sufficient  time  to  be  completely  digested 
or  absorbed  in  the  intestinal  tract.  In  addition  the  excrement  con- 
tains indigestible  bodies,  such  as  the  remains  of  plants,  keratin  sub- 
stances, and  others;  also  form-elements  originating  from  the  mucous  coat 
and  the  glands;  constituents  of  the  different  secretions,  such  as  mucin, 
cholic  acid,  dyslysine,  and  cholesterin  (koprosterin  or  stercorin),  purine 
bases,3  and  enzyro.es;  mineral  bodies  of  the  food  and  the  secretions; 
and,  lastly,  products  of  putrefaction  or  of  digestion,  such  as  skatol,  indol, 
volatile  fatty  acids,  purine  bases,  lime,  and  magnesia  soaps.  Occasion- 
ally, also,  parasites  of  different  kinds  occur;  and  lastly,  the  excrement 
contains  micro-organisms  of  various  species. 

That  the  mucous  membrane  of  the  intestine  by  its  secretion  and  by 
the  abundant  quantity  of  detached  epithelium  contributes  essentially 
to  the  formation  of  feces  follows  from  the  discovery  first  made  by  L. 
Hermann   and  substantiated   by  others,4    that   a  clean,   isolated  loop 


1  Zeitschr.  f .  Biologie,  25,  264. 

2  Arch,  f .  Hygiene,  51. 

s  In  regard  to  the  purine  bases  in  feces,  see  Hall,  Journ.  of  Path,  and  Bacteriol., 
9;  Schittenhelm,  Arch.  f.  klin.  Med.,  81.  Schittenhelm  and  Kriiger,  Zeitschr.  f.  physiol. 
Chem.,  45. 

4  Hermann,  Pfliiger's  Arch.,  46.  See  also  Ehrenthal,  ibid.,  48;  Berenstein,  ibid., 
53;  Klecki,  Centralbl.  f.  Physiol.,  7;  736,  and  F.  Voit,  Zeitschr.  f.  Biologie,  29;  v. 
Moraczewski,  Zeitschr.  f.  physiol.  Chem.,  25;  F.  Lippich,  Prager  med.  Wochenschr., 
32. 


522  DIGESTION. 

of  intestine  collects  material  similar  to  feces.  These  masses  are  rich 
in  mineral  substances  and  especially  rich  in  bodies  soluble  in  alcohol- 
ether,  among  which  cholesterin  occurs,  as  previously  mentioned  (Chap- 
ter VII).  With  a  mixed  diet  with  an  excess  of  meat,  the  human  feces 
consist  only  in  small  part  of  food  residues  and  consist  in  great  part, 
or  after  meat  or  milk  diet,  almost  entirely,  of  intestinal  secretions.  Many 
foods,  therefore,  produce  a  large  quantity  of  feces  chiefly  by  causing  an 
abundant  secretion.1 

The  reaction  of  the  feces  is  very  variable,  but  in  man  with  a  mixed 
diet  it  is  neutral  or  faintly  alkaline.  It  is  often  acid  in  the  inner  part, 
while  the  outer  layers  in  contact  with  the  mucous  coat  have  an  alka- 
line reaction.  In  nursing  infants  it  is  habitually  acid.  The  odor  is 
perhaps  chiefly  due  to  skatol,  which  was  first  found  in  the  feces  by 
Brieger,  and  so  named  by  him.  Indol  and  other  substances  also  take 
part  in  the  production  of  odor.  The  color  is  ordinarily  light  or  dark 
brown,  and  depends  above  all  upon  the  nature  of  the  food.  Medicinal 
bodies  may  give  the  feces  an  abnormal  color.  The  excrement  is  col- 
ored black  by  bismuth,  yellow  by  rhubarb,  and  green  by  calomel.  This 
last-mentioned  color  was  formerly  accounted  for  by  the  formation  of  a 
little  mercury  sulphide,  but  now  it  is  said  that  calomel  checks  the  putre- 
faction and  the  decomposition  of  the  bile-pigments,  so  that  a  part  of  the 
bile-pigments  passes  into  the  feces  as  biliverdin.  In  the  yolk-yellow  or 
greenish-yellow  excrement  of  nursing  infants  one  can  detect  bilirubin. 
Neither  bilirubin  nor  biliverdin  seems  to  exist  in  the  excrement  of  mature 
persons  under  normal  conditions.  In  adults  under  normal  conditions 
the  feces  contain  neither  bilirubin  nor  biliverdin.  On  the  contrary,  there 
is  found  stercobilin  (Masitjs  and  Vanlair),  which  is  identical  with  uro- 
bilin (Jaff£2).  Bilirubin  may  occur  in  pathological  cases  in  the  feces 
of  mature  persons.  It  has  been  observed  in  a  crystallized  state  (as 
hsematoidin)  in  the  feces  of  children  as  well  as  of  grown  persons. 

The  absence  of  bile  (acholic  feces)  causes  the  feces  to  have,  as  above 
stated,  a  gray  color,  due  to  large  quantities  of  fat;  this  may,  however, 
be  partly  attributed  to  the  absence  of  bile-pigments.  In  these  cases 
a  large  quantity  of  crystals  has  been  observed  which  consist  principally 
of  magnesium  soaps  or  sodium  soaps.  Hemorrhage  in  the  upper  parts 
of  the  digestive  tract  yields,  when  it  is  not  very  abundant,  a  dark-brown 
excrement,  due  to  haematin. 


1  In  regard  to  the  constitution  of  feces  with  various  foods,  see  Hammerl,  Kermauner, 
Moeller,  and  Prausnitz,  Zeitschr.  f.  Biologie.  35,  and  Poda,  Micko,  Prausnitz  and 
Mtiller,  ibid.,  39. 

2  See  bile-pigments,  Chapter  VII,  and  urobilin,  Chapter  XIV. 


MECONIUM.     INTESTINAL  CONCREMENT8.  523 

Excretin,  so  named  by  Marcet,1  is  a  crystalline  body  occurring  in  human 
excrement,  but  which,  according  to  Hopi'e-Seylek,  is  perhaps  only  impure  choles- 
terin  (koprosterin  or  stercorin?).  Excretolic  acid  is  the  name  given  by  Marcet 
to  an  oily  body  with  an  excrementitious  odor. 

In  consideration  of  the  very  variable  composition  of  feces,  quanti- 
tative analyses  are  of  little  value  and  therefore  will  be  omitted.2 

Meconium  is  a  dark  brownish-green,  pitchy,  mostly  acid  mass  without  any 
strong  odor.  It  contains  greenish-colored  epithelium  cells,  cell-detritus,  numer- 
ous fat-globules,  and  cholesterin  plates.  The  amount  of  water  is  720-800, 
and  solids  2S0-200  p.  m.  Among  the  solids  there  are  mucin,  bile-pigments, 
and  bile-acids,  cholesterin,  fat,  soaps,  traces  of  enzymes,  calcium  and  magnesium 
phosphates.  Sugar  and  lactic  acid,  soluble  protein  bodies  and  peptones,  also 
leucine  and  tyrosine  and  the  other  products  of  putrefaction  occurring  in  the 
intestine,  are  absent.  Meconium  may  contain  undecomposed  taurocholic  acid, 
bilirubin  and  biliverdin,  but  it  does  not  contain  any  stercobilin.  which  is  con- 
sidered as  proof  of  the  non-existence  of  putrefactive  processes  in  the  digestive 
tract  of  the  fetus. 

The  coyitents  of  the  intestine  under  abnormal  conditions  are  perhaps  less  the 
subject  of  chemical  analysis  than  of  an  inspection  and  microscopical  investiga- 
tion or  bacteriological  examination.  On  this  account  the  question  as  to  the 
properties  of  the  contents  of  the  intestine  in  different  diseases  cannot  be  thor- 
oughly treated  here.3 

Appendix. 

INTESTINAL   CONCREMENTS. 

Calculi  occur  very  seldom  in  the  human  intestine  or  in  the  intestine 
of  carnivora,  but  they  are  quite  common  in  herbivora.  Foreign  bodies 
or  undigested  residues  of  food  may,  when  for  some  reason  or  other  they 
are  retained  in  the  intestine  for  some  time,  become  incrusted  with  salts, 
especially  ammonium-magnesium  phosphate  or  magnesium  phosphate, 
and  these  salts  usually  form  the  chief  constituent  of  the  concrements. 
In  man  they  are  sometimes  oval  or  round,  yellow,  yellowish-gray,  or 
brownish-gray,  of  variable  size,  consisting  of  concentric  layers  and 
containing  chiefly  ammonium-magnesium  phosphate  and  calcium  phos- 
phate, besides  a  small  quantity  of  fat  or  pigment.  The  nucleus  ordi- 
narily consists  of  some  foreign  body,  such  as  the  stone  of  a  fruit,  a 
fragment  of  bone,  or  something  similar.  Sjoqvist4  has  recorded  an  ex- 
traordinary concrement  consisting  principally  of  fatty  acids  and  a  bile-acid. 
In  those  countries  where  bread  made  from  oat-bran  is  an  important  food, 

1  Annal.  de  chim.  et  de  phys.,  59. 

1  In  regard  to  these  analyses  as  well  as  to  the  feces  under  abnormal  conditions 
and  to  the  pertinent  literature,  see  Ad.  Schmidt  and  J.  Strassburger,  Die  Faeces  des 
Menschen,  etc.,  Berlin,  1901  and  1902. 

*  See  Schmidt  and  Strassburger,  1.  c. 

4  Hygiea,  Festband,  1908. 


524  DIGESTION. 

we  often  find  in  the  large  intestine,  balls  similar  to  the  so-called  hair- 
balls  (see  below).  Such  calculi  contain  calcium  and  magnesium  phos- 
phate (about  70  per  cent),  oat-bran  (15-18  per  cent),  soaps  and  fat  (about 
10  per  cent).  Concretions  which  contain  very  much  fat  (about  74  per 
cent)  occasionally  occur,  and  those  consisting  of  fibrin  clots,  sinews, 
or  pieces  of  meat  incrusted  with  phosphates  are  also  rare. 

Intestinal  calculi  often  occur  in  animals,  especially  in  horses  fed  on  bran. 
These  calculi,  which  attain  a  very  large  size,  are  hard  and  heavy  (as  much  as  8 
kilos)  and  consist  in  great  part  of  concentric  layers  of  ammonium-magnesium 
phosphate.  Another  variety  of  concrements  which  occur  in  horses  and  cattle 
consists  of  gray-colored,  often  very  large,  but  relatively  light  stones  which  contain 
plant  residues  and  earthy  phosphates.  Stones  of  a  third  variety  are  sometimes 
cylindrical,  sometimes  spherical,  smooth,  shining,  brownish  on  the  surface,  con- 
sisting of  matted  hairs  and  plant-fibers,  and  termed  hair-balls.  The  so-called 
"  ^egagropil.e,"  which  occur  in  the  antilope  rupicapra,  belong  to  this  group, 
and  are  generally  considered  as  nothing  else  than  the  hair-balls  of  cattle. 

The  so-called  oriental  bezoar-stone  also  belongs  to  the  intestinal  concrements, 
and  probably  originates  from  the  intestinal  tract'  of  the  capra  .egagrus  and  ante- 
lope dorcas.  There  may  exist  two  varieties  of  bezoar-stones.  One  is  olive- 
green,  faintly  shining  and  formed  of  concentric  layers.  On  heating  it  melts  with 
the  development  of  an  aromatic  odor.  It  contains  as  chief  constituent  lithofellic 
acid,  C20H36O4,  which  is  related  to  cholic  acid,  and  besides  this  a  bile-acid,  litho- 
bilic  acid.  The  others  are  nearly  blackish  brown  or  dark  green,  very  glossy, 
consisting  of  concentric  layers,  and  do  not  melt  on  heating.  They  contain  as 
chief  constituent  ellagic  acid,  a  derivative  of  gallic  acid,  of  the  formula  CuHeOs, 
which,  according  to  Graebe,1  is  the  dilactone  of  hexaoxydiphenyldicarboxylic 
acid,  and  which  gives  a  deep-blue  color  with  an  alcoholic  solution  of  ferric  chlo- 
ride. The  last-mentioned  bezoar-stone  originates,  to  all  appearances,  from  the 
food  of  the  animal. 

Ambergris  is  generally  considered  an  intestinal  concrement  of  the  sperm  whale. 
Its  chief  constituent  is  ambrain,  which  is  a  non-nitrogenous  substance  perhaps 
related  to  cholesterin.  Ambrain  is  insoluble  in  water  and  is  not  changed  by  boil- 
ing alkalies.     It  dissolves  in  alcohol,  ether,  and  oils. 

VI.     ABSORPTION. 

The  contents  of  the  intestine  are  gradually  pushed  onward  by  the 
peristalsis  or  rhythmical  movement  of  the  intestinal  musculature,  but 
the  mechanism  is  not  well  known.2  By  these  processes  the  intestinal 
contents  are  intimately  mixed  and  the  constituents  of  the  food  which 
are  valuable  to  the  organism  are  transformed,  in  the  manner  previously 
mentioned,  so  that  they  are  adaptable  for  the  processes  of  absorption. 
In  discussing  the  absorption  processes  we  must  treat  of  the  form  into 
which  the  different  foods  are  changed  before  absorption,  of  the  man- 
ner in  which  this  is  accomplished,  and  lastly,  of  the  forces  which  act 
in  these  processes. 

1  Ber.  d.  d.  chem.  Gesellsch.,  36. 

2  See  Cannon,  Amer.  Journ.  of  Physiol.,  6,  12,  29;  Magnus,  Pfluger's  Arch.,  102, 
103,  108,  111;  Baumstark  and  Cohnheim,  Zeitschr.  f.  physiol.  Chem.,  65. 


ABSORPTION  OF  PROTEINS.  525 

Before  we  can  answer  the  question  as  to  the  form  in  which  the  pro- 
teins are  absorbed  from  the  intestinal  canal,  it  is  of  interest  to  learn 
whether  the  animal  body  can,  perhaps,  also  utilize  such  proteins  as  are 
introduced  intravenously,  Bubcutaneously,  or  into  a  body-cavity,  i.e., 
evading  the  intestinal  canal,  or,  as  it  is  called  parenteral. 

Since  the  first  investigations  of  Zuntz  and  v.  Mering  on  this  sub- 
ject, several  experimenters  x  have  shown,  without  any  doubt,  that  the 
animal  body  can  more  or  less  completely  utilize  different,  parenterally 
introduced  proteins,  although  different  varieties  of  animals  show  a  differ- 
ence in  this  regard.  Still  we  do  not  know  where  and  how  these  foreign 
proteins  are  changed  and  assimilated,  but  Cramer  ascribes  great  impor- 
tance to  the  leucocytes  in  this  regard.  See  Abderhalden's  experiment 
given  on  page  54. 

That  the  animal  body  can  also  assimilate  not  previously  digested 
or  split  proteins  introduced  directly  into  the  intestine  has  been  shown 
by  Brucke,  Bauer  and  Yoit,  Eichhorst,  Czerny  and  Latschen- 
berger,  Yoit  and  Friedlaxder,  and  others.2  In  the  experiments 
of  the  two  last-mentioned  investigators  neither  casein  (as  milk)  nor 
hydrochloric-acid  myosin  or  acid  albuminate  (in  acid  solution)  was 
absorbed,  while,  on  the  contrary,  about  21  per  cent  of  ovalbumin  or 
seralbumin  and  69  per  cent  of  alkali  albuminate  (dissolved  in  alkali) 
were  absorbed.  Mendel  and  Rockwood,  on  the  contrary,  in  experi- 
ments with  casein  and  edestin  in  the  living  intestinal  loop,  could  prove 
only  the  slightest  absorption  en  excluding  digestion  as  completely  as 
possible,  while  the  corresponding  proteoses  were  abundantly  absorbed. 

It  is  difficult  to  decide  in  these  experiments  as  to  how  far  the  pro- 
teins were  taken  up  in  an  actually  unchanged  or  partly  modified  form. 
The  alimentary  albuminaria,  observed  repeatedly  after  the  introduction 
of  large  quantities  of  protein  into  the  intestinal  canal,  indicates  an 
absorption  of  undigested  protein  under  certain  circumstances.  To  decide 
this  question  the  biological  method,  using  the  precipitine  reaction,  has 
been  made  use  of,  and  Ascoli  and  Vigno,3    using  this  method,  claim  to 

1  Zuntz  and  v.  Mering,  Pfliiger's  Arch.,  32;  Xeumeister,  Verh.  d.  phys.-med. 
Gesellsch.  zu  Wiirzburg,  18S9,  and  Zeitschr.,  f.  Biologie,  27;  Friedenthal  and  Lewan- 
dowsky,  Arch.  f.  (Anat.  u.)  Physiol.,  1S99;  Munk  and  Lewandowsky,  ibid.,  1S99, 
Supp.;  Oppenheimer,  Hofmeisters  Beitrage,  4;  Mendel  and  Rockwood,  Amer.  Journ. 
of  Physiol.,  12;  Heilner,  Zeitschr.  f.  Biol.,  50,  and  Munch,  med.  Wochenschr.,  49; 
Cramer,  Journ.  of  Physiol.,  3",  with  Pringle,  ibid.;  Rona  and  Michaelis.  Pfluuer's 
Arch.,  123  and  124;  v.  Korosy,  Zeitschr.  f.  physiol.  Chem.  62,  68  (1909),  69,  313  (1910). 

2  Brucke,  Wien.  Sitzungsber.,  59;  Bauer  and  Voit,  Zeitschr.  f.  Biologie,  5;  Eich- 
horst, Pfliiger's  Arch.,  4;  Czerny  and  Latschenberger,  Yirchow's  Arch.,  59;  Voit  and 
Friedlander,  Zeitschr.  f.  Biologie,  33.  Contradictory  observations  can  be  found  in 
Keller,   Beitr.   z.   Frage  d.   Resorption  im  Dickdarm.   I naug. -Dissert.  Breslau,   1909. 

3  Zeitschr.  f.  Physiol.  Chem.,  39. 


526  DIGESTION. 

have  shown  the  passage  of  non-modified  protein  into  the  blood  and 
lymph  (page  66).  Based  upon  many  investigations  on  this  subject 
we  can  consider  it  possible  that  under  certain  circumstances,  as  on  flood- 
ing the  intestinal  canal  with  protein,  with  a  greater  permeability  of  the 
intestinal  wall,  as  in  new-born  and  sucking  animals,  and  with  a  dimin- 
ished modification  by  the  gastric  juice,  a  passage  of  non-modified  pro- 
tein may  take  place  in  the  blood-vessels,  but  that  under  normal  con- 
ditions this  is  not  the  case,  or  at  least  does  not  take  place  to  any  men- 
tionable  degree.  As  a  rule,  the  absorption  of  protein  follows  a  modi- 
fication of  it.  In  this  connection  the  experiments  of  Orni  1  are  of  interest 
which  show  that  the  dog's  intestine  takes  up  the  serum  of  the  dog  but 
not  that  of  the  ox  or  horse.  In  regard  to  the  previously  split  proteins 
the  question  arises,  whether  the  proteins  are  chiefly  absorbed  as  pro- 
teoses or  peptones  or  as  simpler  atomic  complexes. 

According  to  the  earlier  investigations  of  Ludwig  and  Schmidt- 
Mulheim,  as  well  as  those  of  Munk  and  Rosenstein,2  it  is  generally 
agreed  that  the  products  of  protein  digestion  do  not  pass  into  the 
blood  through  the  lymph  vessels,  but  through  the  intestinal  capillaries. 
The  question  of  the  absorption  of  these  products  resolves  itself  into 
the  form  in  which  they  are  taken  up  by  the  intestine  and  the  form  in 
which  they  pass  into  the  blood. 

It  was  mentioned  above  that  proteoses  and  peptones  as  well  as  non- 
biuret-giving  products  and  amino-acids  have  been  found  in  the  con- 
tents of  the  intestine.  The  amino-acids  occur  to  a  less  extent  than  the 
proteoses  and  peptones.  This  may  indicate  that  the  amino-acids  are 
more  abundantly  formed,  but  also  more  quickly  absorbed,  but  it  may 
also  indicate  that  the  amino-acids  are  produced  to  a  slight  extent  only, 
in  the  intestinal  contents.  There  is  no  doubt  that  the  amino-acids  can 
be  absorbed  as  such,  but  there  is  still  another  question,  namely,  whether 
the  proteoses  and  peptones  are  absorbed  as  such  or  only  after  a  pre- 
vious cleavage  into  amino-acids. 

Nolf  and  Honof.e  found,  what  was  later  substantiated  by  Zunz,3 
that  the  proteoses  and  peptones  disappear  more  quickly  from  the 
intestine  than  the  non-biuret-giving  products.  This  does  not  prove 
that  the  proteoses  are  absorbed  as  such,  but  rather  against  such  a  view. 
A  more  direct  proof  for  the  absorption  of  the  non-split  proteoses  lies 
in  the  fact,  as  shown  by  Nolf,  that  the  proteoses  when  introduced  in 


1  Pfltiger's  Arch.  126,  428  (1909). 

*  Schmidt-Mulheim,  Arch.  f.  (Anat.  u.)  Physiol.,  1877;  Munk  and  Rosenstein. 
Virchow'e  Arch.,  128. 

3  Nolf  and  Honored  Arch,  internat.  de  Physiol.,  1905;  Nolf,  Journ.  de  Physiol,  et 
Pathol,  gdn.,  1907;  Zunz,  Memoires  cour.,  etc.,  Acad.  Roy.  Med.,  Belg.,  20,  Fasc.  1. 


ABSORPTION  OF  PROTEINS.  521 

large  quantities  in  the  intestine  pass  in  small  amounts  into  the  blood. 
Another  proof  is  the  findings  of  Borchakdt,1  that  after  feeding  dogs 
with  not  too  large  amounts  of  elastin,  the  passage  of  a  proteose,  the 
hemielastose,  could  be  detected  in  the  blood.  Attention  must  also  be 
called  to  the  fact  that  according  to  Hofmeistek  -  the  walls  of  the 
stomach  and  intestine  are  the  only  parts  of  the  body  in  which  pro- 
teoses and  peptones  occur  during  digestion. 

We  have  reason  for  believing  that  the  proteoses,  as  well  as  their 
cleavage  products,  are  taken  up  by  the  intestine,  and  if  this  is  the  case 
the  next  question  to  be  answered  is,  in  what  form  do  these  bodies  leave 
the  intestine  and  pass  into  the  blood? 

In  order  to  decide  this  question  the  blood  has  been  repeatedly 
tested  in  regard  to  the  quantity  of  proteoses.  As  seen  on  page  264 
this  has  led  to  very  contradictory  results,  and  if  we  exclude  those 
exceptional  cases  where  a  large  quantity  of  proteose  was  introduced 
into  the  intestine  at  once,  then  we  can  say  that  the  occurrence  of  pro- 
teoses in  the  blood,  or  at  least  in  the  blood-plasma,  has  not  been  posi- 
tively shown  under  physiological  conditions.3  It  can  also  be  said  that 
such  investigations  do  not  prove  much  because  of  the  large  quantity 
of  blood  passing  through  the  intestine  for  a  given  time,  and  the  quan- 
tity of  proteose  must  be  so  small,  so  that  when  divided  in  the  entire  blood 
it  can  hardly  be  detected.  It  is  therefore  of  interest  that  neither  amino- 
acids  nor  proteoses  were  found  in  the  blood  after  cutting  out  several 
organs  or  groups  of  organs  so  that  the  blood  circulated  only  through 
the  intestinal  canal,  heart,  lungs,  pancreas  and  intercostal  muscles 
(Kutscher  and  Seemann,  v.  Korosy4). 

We  are  therefore  obliged  to  consider  that  the  proteoses  and  amino- 
acids  are  transformed  in  the  intestinal  walls  in  some  manner  or  other. 
Such  a  belief,  especially  applied  to  the  proteoses,  coincides  with  the 
observations  of  Hofmeister,  that  the  proteoses  occurring  in  the  mucous 
membrane  during  digestion  disappear  at  the  temperature  of  the  room 
from  the  removed,  but  still  apparently  living,  mucous  membrane  after  a 
certain  time.  This  also  coincides  well  with  the  observations  of  Ludwig 
and  Salvioli.5  These  investigators  introduced  a  peptone  solution  into 
a  double-ligatured,  isolated  piece  of  the  small  intestine,  which  was  kept 
alive  by  passing  defibrinated  blood  through  it,  and  observed  that  the 

1  In  regard  to  the  literature  on  proteoses  in  the  blood  see  Chapter  V,  footnotes- 
1,  2  and  3,  p.  264. 

2  Zeitschr  f.  physiol.  Chem.,  6,  and  Arch.  f.  exp.  Path.  u.  Pharm.,  19,  20,  22. 

*  See  footnote  1. 

*  Kutscher  and  Seemann,  Zeitschr.  f.  physiol.  Chem.,  34;  v.  Korosy,  ibid.,  57. 

s  Arch.  f.  (Anat.  u.)  Physiol.,  1880,  Supplbd.  See  also  Cathcart  and  Leatheer 
.'ourn.  of  Physiol.,  33. 


528  DIGESTION. 

peptone  disappeared  from  the  intestine,  but  that  the  blood  passing 
through  did  not  contain  any  peptone. 

What  becomes  of  the  amino-acids  in  the  intestinal  wall?  Ktjtscher 
and  Seemann  have  shown  that  the  crystalline  cleavage  products  are 
so  transformed  in  the  intestinal  wall  that  they  cannot  be  detected. 
We  have  here  to  think  of  two  possibilities:  The  amino-acids  are  either 
further  split  or  they  are  used  in  synthesis  (of  proteins?). 

It  is  a  long-known  fact  that  with  the  digestion  and  absorption  an 
increased  elimination  of  nitrogen  in  the  urine  goes  hand  in  hand.  The 
•quantity  of  nitrogen  eliminated  in  the  urine  after  partaking  of  protein 
corresponded,  according  to  Asher  and  Haas,1  to  65  per  cent  of  the 
nitrogen  introduced.  It  is  hardly  credible  that  this  elimination  of 
nitrogen  depends  upon  an  increased  destruction  of  body  protein,  and 
it  is  more  probable  that  this  represents  decomposed  food-protein. 
But  according  to  Nencki  and  Zaleski  2  an  abundant  formation  of 
ammonia  occurs  in  the  cells  of  the  digestive  apparatus  after  a  rich 
protein  diet,  so  we  must  consider  the  possibility  that  a  considerable  part, 
perhaps  the  very  greatest  part,  of  the  amino-acids  are  deamidized  in 
the  intestinal  wall.  The  other  part  of  the  amino-acids  may  be  used 
in  the  syntheses  to  be  mentioned  below.  Such  a  partial  deamidization 
of  the  digestive  products  has  been  shown  by  Cohnheim  3  in  his  absorp- 
tion experiments  with  the  fish  intestine. 

The  proteoses  taken  up  by  the  intestinal  mucosa,  if  this  does  take 
place,  can  naturally  undergo  a  further  conversion  into  amino-acids  in 
the  walls  of  the  intestine.  Still  there  are  other  possibilities.  A  direct 
utilization  of  the  proteoses  in  the  synthesis  of  the  proteins  in  the  intes- 
tine is  not  very  probable,  but  on  the  contrary  it  is  more  probable  that 
the  proteoses,  in  order  to  undergo  further  cleavage  or  further  utiliza- 
tion, are  taken  up  by  the  leucocytes  and  carried  off.  Hofmeister 
has  advocated  such  a  possibility  for  a  long  time.  Heidenhain  raised 
objections  to  this  suggestion  in  which  he  called  attention  to  the  dis- 
proportion between  the  number  of  leucocytes  and  the  large  quantity  of 
peptones  (proteoses)  to  be  absorbed,  but  at  that  time  the  deep  cleavage 
of  a  great  part  of  the  protein  into  amino-acids  was  not  known.  Recently 
Pringle  .  and  Cramer  4  urged  the  theory  of  the  importance  of  the 
leucocytes,  and  the  observations  of  Inagaki  5    also  show  the  possibility 

1  Bioch.  Zeitschr.,  12. 

2  Arch,  des  scienc.  biol.  de  St.  P6tersbourg,  4;  Arch.  f.  exp.  Path.  u.  Pharm.,  37; 
see  also  Salaskin,  Zeitschr.  f.  Physiol.  Chem.,  25. 

1  Zeitschr.  f.  physiol.  Chem.,  59. 

♦Hofmeister,  1.  c;  Heidenhain,  Pfluger's  Arch.,  43;  Pringle  and  Cramer,  Journ. 
of  Physiol.,  37. 

6  Zeitschr.  f.  physiol.  Chem.,  50. 


ABSORPTION   OF  PROTEINS.  529 

of  the  leucocytes  taking  up  the  proteoses  and  fixing  them,  it  seems,  in 
the  cell  substance. 

It  is  for  the  present  impossible  to  say  with  certainty  whether  or 
not  and  to  what  extent  the  proteoses,  as  such,  are  absorbed  and  to  give 
their  further  fate  thereafter  in  the  intestine.  The  present  view  is  prob- 
ably as  follows:  That  they  do  not  pass  as  such  into  the  blood,  and 
that  they  are  transformed  into  amino-acids  in  part  in  the  intestinal 
-contents  and  in  part  in  the  intestinal  mucosa,  and  then  from  these  amino- 
acids  the  coagulable  proteins  are  constructed  by  synthesis.  In  sup- 
port of  the  theory  of  a  protein  synthesis  from  amino-acids  we  have  a 
series  of  experiments  where  deeply  split  or  completely  split  proteins 
were  fed.  In  these  experiments  by  Loewi,  Henderson  and  Dean, 
Henriques  and  Hansen,  and  especially  by  Abderhalden  and  his 
co-workers  l  on  dogs,  mice  and  rats,  it  was  possible  to  keep  the  animals 
in  nitrogenous  equilibrium  or  indeed  nitrogen  retention  for  a  long  time 
with  the  cleavage  products  of  proteins  besides  non-nitrogenous  food- 
stuffs and  salts.  According  to  the  recent  experiments  of  Abderhalden 
the  organism  can  build  up  proteins  from  amino-acids  when  the  indi- 
vidual amino-acids  are  supplied  in  proportions  as  they  exist  in  the  cell 
proteins.  Certain,  sometimes  absent  amino-acids  seem  to  be  capable 
of  being  produced  within  the  organism  (for  example,  glycocoll,  proline) 
while  other  (tryptophane)  cannot  be  produced.  This  explains  why 
gelatine  which  does  not  contain  any  tryptophane  cannot  replace  protein 
in  the  food. 

The  results  of  the  experiments  are  generally  considered  as  proof  of 
the  ability  of  the  animal  body  to  construct  proteins  from  amino-acids 
by  synthesis,  and  in  the  present  state  of  our  knowledge  we  can  hardly 
draw  other  conclusions  from  them  or  advance  any  simpler  theory. 

Where  does  the  protein  synthesis  take  place?  If  it  were  positively 
sure  that  the  amino-acids  did  not  pass  into  the  blood  then  we  would 
have  transferred  this  synthesis  to  the  intestinal  walls.  Otherwise  we 
must  think  in  the  first  place  of  the  liver;  but  this  organ  does  not  seem 
to  play  an  important  role  in  this  synthesis.  Abderhalden  and  London  2 
made  an  experiment  on  a  dog  with  an  Eck  fistula  (see  page  397),  feeding 
the  dog  with  decomposed  protein,  and  they  found  that  this  animal 
behaved  exactly  like  a  normal  animal,  as  it  was  kept  for  eight  days  not 
only   in   nitrogenous   equilibrium   but   also   in   nitrogen   retention.     On 

1  Loewi,  Arch.  f.  exp.  Path.  u.  Pharm.,  48.  See  also  Henderson  and  Dean,  Amer. 
Journ.  of  Physiol.,  9;  Abderhalden  and  Rona,  Zeitschr.  f.  physiol.  Chem.,  42,  44,  47,  and 
52;  Henriques  and  Hansen,  ibid.,  43,  49;  Henriques,  ibid.,  54;  Abderhalden  with 
Olinger,  ibid.,  57,  with  Messner  and  Windrath,  ibid.,  59;  Abderhalden,  ibid.,  77,  22, 
78,  1  (1912). 

*  Zeitschr.  f.  physiol.  Chem.,  54. 


530  DIGESTION. 

the  other  hand  it  is  not  possible  to  deny  the  importance  of  the  liver  for 
the  protein  syntheses.  As  Embden  and  his  collaborators  have  shown  on 
perfusing  the  liver  containing  a  large  amount  of  glycogen,  that  d-alanine 
was  formed  and  Embden  explains  this  formation  by  a  destruction  of 
glucose  or  lactic  acid  and  pyroracemic  acid.  With  experiments  with 
blood  perfusion  of  the  liver,  a-amino-acids  are  formed  from  the  am- 
monium salts  of  the  corresponding  a-keto-acids.  The  combination 
NH4.O.CO.CO.R  passes  into  HO.CO.CH(NH2).R.  The  cleavage 
products  of  the  carbohydrates  can  be  converted  in  the  liver  into  char- 
acteristic constituents  of  the  protein  molecule.1  In  this  connection 
we  must  here  mention  the  experiments  of  Luthje  2  in  which  he  found 
a  nitrogen  retention  after  feeding  only  one  amino-acid  with  abundance 
of  carbohydrate. 

What  kind  of  protein  is  formed  in  the  synthesis?  This  we  do  not 
know.  Abderhalden's  belief  is  that  it  is  plasma  protein,  which,  as 
is  well  known,  is  the  same  in  each  animal  independent  of  the  kind  of 
protein  introduced  with  the  food  and  from  which  the  cells  of  the  body 
then  create  the  further  protein  material.  Objections  can  be  raised 
against  this  hypothesis,  but  still  it  is  worth  consideration.  In  favor 
of  this  we  can  also  add  that  according  to  the  investigations  of  Freund 
and  v.  Korosy  3  the  blood  coming  from  the  intestine  during  digestion 
is  richer  in  coagulable  protein  than  other  blood,  and  also  that  this 
protein,  Freund  asserts,  belongs  to  the  globulin  group.  This  globulin, 
according  to  Freund  and  Toepfer,  is  not  identical  with  the  ordinary 
serglobulin  mixture,  but  is  a  pseudoglobulin  formed  in  the.  intestine 
from  the  food  protein  by  synthesis,  and  which  is  more  easily  decom- 
posed or  further  utilized  in  the  liver  and  other  organs.  Further  research 
in  this  direction  is  necessary,  as  we  have  other  investigations  which 
are  essentially  different.  If  a  re-formation  of  coagulable  proteins  takes 
place  from  amino-acids  during  digestion,  it  is  to  be  expected  that  a 
relatively  greater  quantity  of  coagulable  protein  should  occur  in  the 
mucosa  of  the  digesting  intestine  as  compared  with  the  non-digesting 
intestine.  Pringle  and  Cramer,  by  a  method  which  requires  con- 
firmation, claim  that  in  the  digesting  animal  (cat),  the  blood,  and  to  a 
still  higher  degree  the  intestinal  mucosa,  and  especially  the  lymph  nodes 
of  the  intestine,  are  richer  in  non-coagulable  protein  than  the  starving 
animal,  a  condition  which  is   related  to  the  r61e  of  the  leucocytes  in  the 

iBioch.  Zeitschr.  29,  423  (1910);  38, '393,  407,  414  (1911);  45,  1-207  (1912); 
summary,  45,  201. 

2  Pfliiger's  Arch.  113,  547  (1906). 

a  v.  Korosy,  Zeitschr.  f.  physiol.  Chem.,  57;  Freund,  Zeitschr.  f.  exp.  Path.  u. 
Therap.,  4;  G.  Toepfer  and  Freund,  and  Toepfer,  ibid.,  3;  Pringle  and  Cramer,  Journ. 
of  Physiol.,  37. 


ABSORPTION   OF  PROTEINS.  531 

protein   assimilation.     This   question  of  the   absorption  of  proteins   in 
the  intestine  is  still  unexplained  in  many  directions. 

The  extent  of  the  protein  absorption  is  dependent  essentially  upon 
the  kind  of  food  introduced,  since  as  a  rule  the  protein  substances  from 
an  animal  source  are  much  more  completely  absorbed  than  from  a 
vegetable  source.  As  proof  of  this  the  following  observations  are 
given:  In  his  experiments  on  the  utilization  of  certain  foods  in  the  intes- 
tinal canal  of  man,  Rubner  found  that  with  an  exclusively  animal  diet, 
on  partaking  of  an  average  of  738-884  grams  of  fried  meat,  or  948  grams 
of  eggs  per  day,  the  nitrogen  deficit  with  the  excrement  was  only  2.5-2.8 
per  cent  of  the  total  nitrogen  introduced.  With  a  strictly  milk  diet 
the  results  were  somewhat  unfavorable,  since  after  partaking  of  4100 
grams  of  milk  the  nitrogen  deficit  increased  to  12  per  cent.  The  con- 
ditions are  quite  different  with  vegetable  food,  as  shown  by  the  re- 
searches of  Meyer,  Rubner,  Hultgren  and  Landergren,  who  made 
experiments  with  various  kinds  of  rye  bread  and  found  that  the  loss  of 
nitrogen  through  the  feces  amounted  to  22-48  per  cent.  Experiments 
with  other  vegetable  foods,  and  also  the  investigations  of  Schuster, 
Cramer,  Meinert,  Mori,1  and  others  on  the  utilization  of  foods  with 
mixed  diets,  have  led  to  similar  results.  With  the  exception  of  rice, 
wheat  bread,  and  certain  very  finely  divided  vegetable  foods,  it  is  found 
in  general  that  the  nitrogen  deficit  by  the  feces  increases  with  a  larger 
quantity  of  vegetable  material  in  the  food. 

The  reason  for  this  is  manifold.  The  large  quantity  of  cellulose 
frequently  present  in  vegetable  foods  impedes  the  absorption  of  pro- 
teins. The  greater  irritation  produced  by  the  vegetable  food  itself  or 
by  the  organic  acids  formed  in  the  fermentation  in  the  intestinal  canal 
causes  a  more  violent  peristalsis,  which  drives  the  contents  of  the  intes- 
tine faster  than  otherwise  along  the  intestinal  canal.  Another  and  most 
important  reason  is  the  fact  that  a  part  of  the  vegetable  protein  sub- 
stances seems  to  be  indigestible,  and  because  of  the  difficultly  digestible 
vegetable  food,  a  large  quantity  of  digestion  fluids  containing  nitrogen 
is  secreted. 

In  speaking  of  the  functions  of  the  stomach  we  stated  that  after 
the  removal  or  excision  of  this  organ,  an  abundant  digestion  and  absorp- 
tion of  proteins  may  take  place.  It  is  therefore  of  interest  to  learn  how 
the  digestion  and  absorption  of  proteins  go  on  after  the  extirpation  of 
the   second  protein-digesting  organ,   the  pancreas.     In   this   connection 


1  Rubner,  Zeitschr.  f.  Biologie,  15;  Meyer,  ibid.,  7;  Hultgren  and  Landergren, 
Nord.  med.  Arch.,  21;  Schuster,  in  Voit's  "  Untersuch.  d.  Kost,"  etc.,  142;  Cramer, 
Zeitschr.  f.  physiol.  Chem.,  6;  Meinert,  "  Ueber  Massennahrung,"  Berlin,  1SS5;  Kell- 
ner  and  Mori,  Zeistchr.  f.  Biologie,  25. 


532  DIGESTION. 

there  are  the  observations  on  animals  after  complete  or  partial  extirpa- 
tion of  the  gland  by  Minkowski  and  Abelmann,  Sandmeyer,  V.  Har- 
lbt,  after  destroying  the  gland  by  Rosenberg,  and  also  in  man  after 
closing  the  pancreatic  duct  by  Harley  and  Deucher.  In  all  these 
cases  such  discrepancy  of  figures  has  resulted  for  the  utilization  of  the 
proteins — between  80  per  cent  after  the  apparently  complete  exclusion 
of  pancreatic  juice  in  man  (Deucher)  and  18  per  cent  after  extirpa- 
tion of  the  gland  in  dogs  (Harley) — that  one  can  hardly  draw  any 
clear  conception  as  to  the  extent  and  importance  of  the  trypsin  diges- 
tion in  the  intestine.  That  on  completely  preventing  the  entrance  of 
pancreatic  juice  only  a  slight  diminution  in  the  protein  absorption  takes 
place  follows  from  the  researches  of  Lombroso  and  Niemann.1  In 
order  to  understand,  in  these  cases,  why  the  digestion  and  absorption 
took  place  so  abundantly  it  would  be  of  interest  to  know  how  other 
digestion  fluids  act  substitutingly.  In  this  regard  Zunz  and  Mayer2 
found  that  in  dogs  (meat  digestion)  the  tying  of  the  pancreatic  passages 
is  essentially  compensated  for  by  an  increased  secretion  of  pepsin  and 
other  proteolytic  enzymes,  and  that  in  this  case  the  demolition  of  the 
protein  in  the  stomach  goes  further  than  in  a  normal  animal. 

The  carbohydrates  are,  it  seems,  chiefly  absorbed  as  monosaccharides. 
Glucose,  fructose,  and  galactose  are  probably  absorbed  as  such.  The 
two  disaccharides,  saccharose  and  maltose,  ordinarily  undergo  an  inver- 
sion in  the  intestinal  tract  and  are  converted  into  glucose  and  fructose. 
Lactose  is  also,  at  least  in  certain  animals,  inverted  in  the  intestine. 
In  other  mature  animals,  on  the  contrary,  if  the  lactase  formation  is  not 
excited  by  milk  food,  the  sugar  is  not  inverted  or  only  to  a  slight 
extent  (Voit  and  Lusk,  Weinland,  Portier,  Rohmann  and  Nagano), 
and  it  probably  is  absorbed  as  such  in  these  animals  if  it  does  not  under- 
go fermentation,  or,  as  Rohmann  and  Nagano3  assumed,  if  it  is  not 
transformed  in  the  intestinal  mucosa  in  some  unknown  way.  An 
absorption  of  non-inverted  carbohydrates  is  not  improbable,  and  accord- 
ing to  Otto  and  v.  Mering  the  portal  blood  contains,  after  a  carbo- 
hydrate diet,  besides  glucose,  a   dextrin-like  carbohydrate.      Moscati4 

1  Abelmann,  "  Ueber  die  Ausniitzung  der  Nahrungsstoffe  nach  Pankreasexstirpa- 
tion"  (Inaug.-Dissert.  Dorpat,  1890),  cited  from  Maly's  Jahresber.,  20;  Sandmeyer, 
Zeitschr.  f.  Biologie,  31;  Rosenberg,  Pfliiger's  Arch.,  70;  Harley,  Journ.  of  Pathol, 
and  Bacterid.,  1895;  Deucher,  Correspond.  Blatt.  f.  Schweiz.  Aerzte,  28;  Lombroso, 
Arch.  f.  exp.  Path.  u.  Pharm.,  60;  Niemann,  Zeitschr.  f.  exp.  Path.  u.  Therap.,  5; 
See  also  Brugsch  and  Pletnew,  Zeitschr.  f.  exp.  Path.  u.  Therap.  6,  326. 

2  Mem.  de  l'acad.  roy.  de  m6dic.  de  Belg.,  18. 

J  Voit  and  Lusk,  Zeitschr.  f.  Biologie,  28;  Rohmann  and  Nagano,  Pfliiger's  Arch., 
95,  which  contains  the  references  to  the  literature. 

4  Otto,  see  Maly's  Jahresber.,  17;  v.  Mering,  Arch.  f.  (Anat.,  4.)  Physiol.,  1877; 
Moscati,  Zeitschr.  f.  physiol.  Chem.,  50 


ABSORPTION   OF  CARBOHYDRATES.  533 

• 

believes  that  when  homogeneous  starch  solutions  are  injected  intra- 
venously or  subcutaneously,  the  starch  is  taken  up  by  the  organs,  namely 
the  spleen,  liver  and  lungs,  and  is  utilized  as  the  starch  can  be  changed 
into  glycogen.  A  part  of  the  carbohydrates  is  destroyed  by  fermenta- 
tion in  the  intestine,  with  the  formation  of  lactic  and  acetic  acids  and 
other  bodies. 

The  different  varieties  of  sugars  arc  absorbed  with  varying  degrees 
of  rapidity,  but  as  a  general  thing  absorption  occurs  very  quickly.  This 
absorption  takes  place  more  quickly  in  the  upper  part  of  the  intestine 
than  in  the  lower  part  (Rohmann,  Lannois  and  Lepine,  Rohmann 
and  Nagano  1).  It  is  generally  admitted  that  the  simpler  sugars  are 
more  quickly  absorbed  than  the  disaccharides,  while  the  reports  as  to 
the  absorption  of  the  disaccharides  differ  somewhat  (Hedon,  Alber- 
toni,  Waymouth  Reid,  Rohmann  and  Nagano).  There  seems  to  be 
no  doubt  that  lactose  is  absorbed  more  slowly  than  the  two  other  disac- 
charides. According  to  the  extensive  experiments  of  Rohmann  and 
Nagano,  saccharose  is  absorbed  more  quickly  than  maltose.  Nagano  2 
contends  that  the  pentoses  are  absorbed  more  slowly  than  hexoses. 

On  the  introduction  of  starch  even  in  very  considerable  quantities 
into  the  intestinal  tract  no  glucose  passes  into  the  urine,  a  condition 
which  probably  depends  in  this  case  upon  the  absorption  and  assimila- 
tion and  the  slow  saccharification  taking  place  simultaneously.  If, 
on  the  contrary,  large  quantities  of  sugar  are  introduced  at  one  time, 
then  an  elimination  of  sugar  by  the  urine  takes  place,  and  this  elimina- 
tion of  sugar  is  called  alimentary  glycosuria.  In  these  cases  the  assimila- 
tion of  the  sugar  and  the  absorption  do  not  take  place  together. 

That  quantity  of  sugar  to  which  we  must  raise  the  ingested  sub- 
stance in  order  to  produce  an  alimentary  glycosuria  gives,  according 
to  Hofmeister,3  the  assimilation  limit  for  that  same  sugar.  This  limit- 
is  different  for  various  kinds  of  sugar;  and  it  also  varies  for  the  same 
sugar  not  only  in  different  animals,  but  also  in  different  members  of  the 
same  species,  as  also  in  the  same  individual  under  varying  circum- 
stances. In  general  it  can  be  said  that  in  regard  to  the  ordinary  varie- 
ties of  sugar,  such  as  glucose,  fructose,  galactose,  saccharose,  maltose, 
and  lactose,  the  assimilation  limit  is  highest  for  glucose  and  lowest  for 
lactose.  It  must  be  admitted  that  with  an  overabundant  quantity  of 
sugars  in  the  intestinal  tract  the  disaccharides  do  not  have  sufficient 
time  for  their  complete  inversion,  and  this  has  been  directly  shown  by 


'Lannois  and  Lupine,  Arch,  de  physiol.  (3),  1;  Rohmann,  Pfluger's  Arch.,  41;  see 
also  footnote  3,  p.  532. 

2  In  regard  to  the  literature  on  the  absorption  of  sugars,  see  footnote  3,  p.  532. 
1  Arch.  f.  exp.  Path.  u.  Pharm.,  25  and  26. 


534  DIGESTION. 

• 
Rohmann  and  Nagano.     It  is,   therefore,   not  remarkable  that  disac- 
eharides,  as  well,  have  been  found  in  the  urine  in  cases  of  alimentary 
glycosuria.1 

The  investigations  of  Ludwig  and  v.  Mering  and  others  have 
explained  how  the  sugars  enter  into  the  blood-stream,  namely,  that  they 
as  well  as  other  bodies  soluble  in  water  do  not  ordinarily  pass  over  into 
the  chylous  vessels  in  measurable  quantities,  but  are  chiefly  taken  up  by 
the  blood  in  the  capillaries  of  the  villi,  and  in  this  way  pass  into  the  mass 
of  the  blood.  These  investigations  have  been  confirmed  by  observa- 
tions of  I.  Munk  and  Rosenstein  2  on  human  beings. 

The  reason  why  the  sugars  and  other  soluble  bodies  do  not  pass 
over  into  the  chylous  vessels  in  appreciable  quantity  is,  according  to 
Heidenhain,3  to  be  found  in  the  anatomical  conditions,  in  the  arrange- 
ment of  the  capillaries  close  under  the  layer  of  epithelium.  Ordinarily 
these  capillaries  find  the  necessary  time  for  the  removal  of  the  water 
and  the  solids  dissolved  in  it.  But  when  a  large  quantity  of  liquid, 
such  as  a  sugar  solution,  is  introduced  into  the  intestine  at  once,  this  is 
not  possible,  and  in  these  cases  a  part  of  the  dissolved  bodies  passes  into 
the  chylous  vessels  and  the  thoracic  duct  (Ginsberg  and  Rohmann4). 

The  passage  of  sugar  into  the  urine,  when  at  one  time  large  quanti- 
ties of  sugar  are  taken  and  the  assimilation  limit  is  exceeded,  can  be 
best  explained  by  the  assumption  that  a  part  of  the  sugar  escaped  the 
liver  and  passed  into  the  large  circulation,  or  that  the  liver  did  not  have 
time  to  retain  the  sugar  and  transform  it  into  glycogen.  According  to 
the  observations  of  de  Filippi5  upon  dogs  with  Eck  fistula,  it  seems  as  if 
the  role  of  the  liver  in  these  cases  is  too  highly  estimated.  An  animal 
with  Eck  fistula  could  take  an  unlimited  quantity  of  starch  without 
glycosuria  occurring.  The  assimilation  limit  was  in  these  cases  some- 
what lower,  but  qualitatively  they  behave  like  normal  animals  and  with 
increasing  sugar  supply  they  could  also  retain  increasing  quantities  of 
sugar. 

The  introduction  of  larger  quantities  of  sugar  into  the  intestine  at 
one  time  can  readily  cause  a  disturbance  with  diarrheal  evacuations 
of  the  intestine.     If  the  carbohydrate  is  introduced  in  the  form  of  starch, 


1  For  the  literature  in  regard  to  the  passage  of  various  kinds  of  sugars  into  the 
urine,  see  C.  Voit.  Ueber  die  Glykogenbildung,  Zeitschr.  f.  Biologie,  28,  and  F.  Voit, 
footnote  1,  p.  396.  See  also  Blumenthal,  Zur  Lehre  von  der  Assimilationsgrenze 
der  Zuckerarten,  Inaug.-Dissert.  1903,  Strassburg  and  Brasch,  Zeitschr.  f.  Biol.,  50. 

2v.  Mering.  Arch.  f.  (Anat.  u).  Physiol.,  1877;  Munk  and  Rosenstein,  Virchow's 
Arch.  123. 

3  Pfluger's  Arch.,  43,  Suppl. 

4  Ginsberg,  Pfluger's  Arch.,  44;  Rohmann,  ibid.,  41. 
'•  Zeitschr.  f.  Biol.,  4!)  and  50. 


ABSORPTION  OF  FATS.  535 

then  very  large  quantities  may  be  absorbed  without  causing  any  dis- 
turbance, and  the  absorption  may  be  very  complete.  Rubner  found 
the  following:  On  partaking  508-G70  grams  of  carbohydrates,  as  wheat 
bread,  per  day,  the  part  not  absorbed  amounted  to  only  0.8-2.6  per  cent. 
For  peas,  where  357-588  grams  were  eaten,  the  loss  was  3.6-7  per  cent, 
and  for  potatoes  (718  grams)  7.6  per  cent.  Constantinidi  found  on 
partaking  367-380  grams  of  carbohydrates,  chiefly  as  potatoes,  a  loss 
of  only  0.4-0.7  per  cent.  In  the  experiments  of  Rubner,  as  also  of 
Hultgren  and  Landergren,1  with  rye  bread  the  utilization  of  car- 
bohydrates was  less  complete,  and  the  loss  in  a  few  cases  rose  even  to 
10.4-10.9  per  cent.  It  at  least  follows  from  the  experiments  made  thus 
far  that  man  can  absorb  more  than  500  grams  of  carbohydrates  per  diem 
without  difficulty. 

We  generally  consider  the  pancreas  as  the  most  important  organ 
in  the  digestion  and  absorption  of  amylaceous  bodies,  and  it  is  a  ques- 
tion how  these  bodies  are  absorbed  after  the  extirpation  of  the  pan- 
creas. As  on  the  absorption  of  proteins,  so  also  on  the  absorption  of 
starch,  the  observations  have  given  variable  results.  In  certain  cases 
the  absorption  was  not  impaired,  while  in  others  it  was,  on  the  contrary, 
rather  diminished,  and  with  dogs  devoid  of  pancreas  it  has  been  found 
that  the  absorption  was  decreased  to  50  per  cent  of  the  starch  partaken 
(Rosenberg,  Cavazzani2). 

Em  unification  used  to  be  considered  as  of  the  greatest  importance 
in  the  absorption  of  fats,  and  this  emulsion  occurs  in  the  chyle  on  the 
introduction  into  the  intestine  of  not  only  neutral  fats,  but  also  of  fatty 
acids.  The  fatty  acids  do  not  exist  as  such  in  the  emulsified  fat  of  the 
chyle.  The  investigations  of  I.  Munk,  later  confirmed  by  others,  have 
shown  that  the  fatty  acids  undergo  in  great  part  a  synthesis  into  neutral 
fats  in  the  walls  of  the  intestine,  and  are  carried  as  such  by  the  stream 
of  chyle  into  the  blood.  This  synthesis  seems  to  take  place  in  the 
mucous  membrane  (Moore  and  others3). 

The  assumption  that  the  fat  is  absorbed  chiefly  as  an  emulsion  is 
partly  based  on  the  abundance  of  emulsified  fat  in  the  chyle  after  feed- 
ing with  fat,  and  partly  on  the  fact  that  a  fat  emulsion  is  often  found 
in  the  intestine  after  such  food.     As  an  abundant  cleavage  of  neutral 

1  Rubner,  Zeitschr.  f.  Biologie,  15  and  19;  Constantinidi,  ibid.,  23;  Hultgren  and 
Landergren,  Nord.  med.  Arch.  21. 

2  Cavazzani,  Centralbl.  f.  Physiol.,  7.  See  footnote  1,  p.  532;  also  Lombroso, 
Hofmeister's  Beitriige,  8. 

3  Munk,  Virchow's  Arch.,  80.  See  also  v.  Walther,  Arch.  f.  (Anat,  u.)  Physiol., 
1890;  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  21;  Frank,  Zeitschr.  f.  Biologie, 
36;  Moore,  see  Biochem.  Centralbl.,  1,  741;  Frank  and  Ritter,  Zeitschr.  f.  Biologie, 
47;  Noll,  Pfl tiger's  Arch.  136. 


536  DIGESTION. 

fats  occurs  in  the  intestinal  canal,  and  also  as  the  fatty  acids  do  not 
occur  in  the  chyle  as  such,  but  as  emulsified  fat  after  a  synthesis  with 
glycerin  into  neutral  fats,  it  is  to  be  doubted  whether  the  emulsified 
fat  of  the  chyle  originates  from  an  absorption  of  emulsified  fat  in  the 
intestine  or  from  a  subsequent  emulsification  of  neutral  fats  formed 
synthetically.  This  doubt  has  greater  warrant  in  the  observation  of 
Frank  l  that  the  fatty-acid  ethyl  ester  is  extensively  taken  up  from  the 
intestine,  not  as  such,  but  as  split-off  fatty  acids  from  which  then  the 
neutral  emulsified  fats  of  the  chyle  are  formed. 

The  assumption  of  an  absorption  of  fats  as  an  emulsion  is  inconsist- 
ent with  the  fact  that  an  emulsion  produced  by  means  of  soaps  is  not 
permanent  in  an  acid  liquid;  hence  we  cannot  consider  as  possible  the 
presence  of  an  emulsion  in  the  intestine  so  long  as  it  is  acid.  This 
difficulty  is  not  too  serious,  as  the  reaction  is  often  only  due  to  carbonic 
acid  and  bicarbonates,  and  besides  as  found  by  Kuhne  and  recently 
shown  by  Moore  and  Krumbholz,2  the  proteins  have  a  preserving 
action  upon  fat  emulsions. 

The  earlier  opinions  as  to  fat  absorption  were,  that  fat  was  absorbed 
as  soaps,  soluble  in  water,  as  well  as  finely  emulsified  fat,  and  this  last 
form  was  considered  as  of  the  greatest  importance.  This  view  has 
recently  undergone  essential  modifications,  due  to  the  work  of  Moore 
and  Rockwood,  and  especially  to  the  extensive  work  of  Pfluger.3 

Moore  and  Rockwood  have  shown  the  great  solvent  action  of  the 
bile  for  fatty  acids,  and  on  continuing  these  investigations  further, 
Moore  and  Parker  have  found  that  the  bile  increases  the  solubility 
of  soaps  in  water,  and  can  prevent  their  gelatinization,  a  fact  which  is 
of  greater  importance  for  the  absorption  of  fats  than  the  solubility  of 
the  fatty  acids  in  bile.  The  quantity  of  lecithin  in  the  bile  is  of  great 
importance  for  the  solubility  therein  of  the  fatty  acids  as  well  as  the 
soaps.  According  to  the  above-mentioned  investigators,  the  absorption 
of  fat  from  the  intestine  is  essentially  dependent  upon  the  solubility  of 
the  soaps  and  free  fatty  acids  in  the  bile.  The  neutral  fats  are  split 
and  the  free  fatty  acids  are  in  part  absorbed,  dissolved  as  such  by  the 
bile,  and  in  part  combined  with  alkalies,  forming  soaps.  Neutral  fats 
are  regenerated  from  the  fatty  acids,  and  the  alkali  set  free  from  the 
soaps  is  secreted  again  into  the  intestine  and  used  for  the  re-formation 


1  Zeitschr.  f.  Biologie,  36. 

1  Kuhne,  Lehr.  der  physiol.  Chem.,  122;  Moore  and  Krumbholz,  Journ.  of  Physiol., 
22. 

3  In  regard  to  the  recent  literature  on  fat  absorption,  see  the  works  of  Pfluger, 
Pfluger 's  Arch.,  80,  81,  82,  85,  88,  89,  and  90,  where  the  work  of  other  investigators  is 
cited  and  discussed.     See  also  Croner,  Bioch.  Zeitschr.  23;  Lombroso,  Arch,  di  Fisiql.  5. 


ABSORPTION  OF  FATS.  537 

of  soaps.  According  to  Croner  the  absorption  of  soaps  occurs  only 
in  the  lower  parts  of  the  small  intestine. 

The  importance  of  the  bile,  the  soaps,  and  the  alkali  carbonates  has 
been  closely  studied,  principally  in  the  very  thorough  investigations  of 
Pfluger.  He  has  quantitatively  determined  the  solvent  power  of 
the  above-mentioned  bodies — each  alone  as  well  as  different  mixtures 
of  these — for  the  various  fatty  acids,  and  has  closely  studied  the  mode 
of  action  of  the  bile.  From  his  investigations  he  has  arrived  at  the 
conclusion  that  no  unsplit  fat  is  absorbed,  that  all  fats,  before  their 
absorption,  must  first  be  split  into  glycerin  and  fatty  acids,  and  that  the 
bile,  on  account  of  its  solvent  power  for  soaps  and  fatty  acids,  is  sufficient 
for  the  absorption  of  large  quantities  of  fat  eaten.  The  object  of  the 
formation  of  an  emulsion  is,  according  to  this  view,  that  the  fat  in  this 
condition  forms  such  a  large  surface  for  the  action  of  the  steapsin  or 
the  fat-splitting  agents.  The  possibility  that  all  the  fat  must  be  first 
split  and  that  no  unsplit  fat  is  absorbed  is,  according  to  these  researches, 
not  to  be  denied. 

The  next  question  is  whether  all  the  fat  or  the  greater  part  of  it 
passes  into  the  .blood  through  the  lymphatics  and  the  thoracic  duct. 
According  to  the  researches  of  Walther  and  Frank  x  on  dogs,  it  seems 
that  only  a  small  part  of  the  fats,  or  at  least  of  the  fatty  acids  fed, 
passes  into  the  chylous  vessels;  but  these  observations  can  hardly  be 
applied  to  the  absorption  of  neutral  fats,  or  to  the  absorption  in  man 
under  normal  circumstances.  Munk  and  Rosenstein,2  in  their  inves- 
tigations on  a  girl  with  a  lymph  fistula,  found  60  per  cent  of  the  fat 
ingested  in  the  chyle,  and  of  the  total  quantity  of  fat  in  the  chyle  only 
4-5  per  cent  existed  as  soaps.  On  feeding  with  a  foreign  fatty  acid, 
such  as  erucic  acid,  they  found  37  per  cent  of  the  introduced  body  as 
neutral  fat  in  the  chyle.  Not  all  the  fat  introduced  is  found  in  the 
chyle,  and  there  is  always  a  not  inconsiderable  part  of  the  absorbed 
fat  whose  fate  we  are  not  able  to  follow. 

The  completeness  with  which  fats  are  absorbed  depends,  under  nor- 
mal conditions,  essentially  upon  the  kind  of  fat.  In  this  regard  it  is 
known,  especially  from  the  investigations  of  Munk  and  Arnschixk,3 
that  the  varieties  of  fat  with  high  melting-points,  such  as  mutton-tallow, 
and  especially  stearin,  are  not  so  completely  absorbed  as  the  fats  with 
low  melting-points,  such  as  hog-  and  goose-fat,  olive-oil,  etc.  The  kind 
of  fat  also  has  an  influence  on  the  rapidity  of  absorption,  as  Munk  and 
Rosenstein   found   that   solid   mutton-fat    was   absorbed   more   slowly 


1  Walther,  Arch.  f.  (Anat.  u.)  Physiol.,  1S90;  Frank.  Qrid.,  1S92. 

2  Virchow's  Arch.,  123. 

s  Munk,  Virchow's  Arch.,  80  and  95;  Arnschink,  Zeitschr.  f.  Biologie.  26. 


538  DIGESTION. 

than  fluid  lipanin.  The  extent  of  absorption  in  the  intestinal  tract  is, 
under  physiological  conditions,  very  considerable.  In  the  case  of  a 
dog  investigated  by  Voit  it  was  found  that  out  of  350  grams  of  fat 
(butter)  partaken,  346  grams  were  absorbed  from  the  intestinal  canal, 
and  according  to  the  investigations  of  Rubner  x  the  human  intestine 
can  absorb  over  300  grams  of  fat  per  diem.  The  fats  are,  according 
to  Rubner,  much  more  completely  absorbed  when  free,  in  the  form  of 
butter  or  lard,  than  when  inclosed  in  cell-membranes,  as  in  bacon. 

Claude  Bernard  showed  long  ago  with  experiments  on  rabbits  in 
which  the  ductus  choledochus  was  made  to  open  into  the  small  intestine 
above  the  pancreatic  duct,  that  after  food  rich  in  fats  the  chylous  vessels 
of  the  intestine  above  the  pancreas  passages  were  transparent,  while 
below  they  were  milk-white,  and  also  that  the  bile  alone  cannot  pro- 
duce an  absorption  of  the  emulsified  fat  without  the  pancreatic  juice. 
Dastre  2  has  performed  the  reverse  experiment  on  dogs.  He  tied  the 
ductus  choledochus  and  adjusted  a  biliary  fistula  so  that  the  bile  flowed 
into  the  intestine  below  the  mouth  of  the  pancreatic  passages.  On 
killing  the  animal  after  a  meal  rich  in  fat  the  chylous  vessels  were  first 
found  milk-white  below  the  discharge  of  the  biliary  fistula.  From  this 
Dastre  draws  the  conclusion  that  a  combined  action  of  the  bile  and  pan- 
creatic juice  is  important  in  the  absorption  of  fats — a  conclusion  which 
stands  in  accord  with  the  experience  of  many  others. 

Through  numerous  observations  of  many  investigators,  such  as 
Bidder  and  Schmidt,  Voit,  Rohmann,  Fr.  Muller,  I.  Munk,3  and 
others,  it  has  been  shown  that  the  exclusion  of  the  bile  from  the  intes- 
tinal tract  diminishes  the  absorption  of  fat  to  such  an  extent  that  only 
one-seventh  to  about  one-half  of  the  quantity  of  fat  ordinarily  absorbed 
undergoes  absorption.  In  icterus  with  entire  exclusion  of  the  bile,  a 
considerable  decrease  in  the  absorption  of  fat  is  noticed.  As  under 
normal  conditions,  so  also  in  the  absence  of  bile  in  the  intestine,  the  lower 
melting  parts  of  the  fat  are  more  completely  absorbed  than  those  which 
have  a  high  melting-point.  I.  Munk  found  in  his  experiments  on  dogs 
with  lard  and  mutton-tallow  that  the  absorption  of  the  high-melting 
tallow  was  reduced  twice  as  much  as  the  lard  on  the  exclusion  of  the 
bile  from  the  intestine. 

We  also  learn  from  the  investigations  of  Rohmann  and  I.  Munk 
that  in  the  absence  of  bile  the  relation  between  fatty  acids  and  neutral 
fats  is  changed,  namely,  about   80-90  per  cent  of  the  fat  existing  in  the 


1  Voit,  Zeitschr.  f.  Biologie,  9;  Rubner,  tirid.,  lo. 

2  Arch,  de  Physiol.  (5),  2. 

a  F.  Muller,  Sitzungsber.  der  phys.-med.  Gesellsch.  zu  Wiirzburg,  1SS5;    I.  Munk, 
Virchow's  Arch.,  122.     See  also  footnotes  4  and  5,  p.  518. 


ABSORPTION   OF   FATS.  539 

feces  consists  of  fatty  acid,  while  under  normal  conditions  the  feces 
contain  1  part  neutral  fat  to  about  2-2£  parts  free  fatty  acids.  It  ia 
not  possible  to  state  how  this  increased  quantity  of  fatty  acids  in  the  fat 
of  the  feces  is  produced  upon  the  exclusion  of  the  bile  from  the  intestine. 

There  is  no  doubt  that  the  bile  is  of  great  importance  in  the  absorp- 
tion of  fats.  Still  there  is  also  no  doubt  that  rather  considerable  quan- 
tities of  fat  may  be  absorbed  from  the  intestine  in  the  absence  of  bile. 
What  relation  does  the  pancreatic  juice  bear  to  this  fact? 

Upon  this  point  a  rather  large  number  of  observations  on  animals 
have  been  made  by  Abelmann  and  Minkowski,  Sandmeyer,  Harley, 
Rosenberg,  Hedon  and  Ville,  and  also  on  man  by  Fr.  Muller  and 
Deucher.1  In  all  of  these  investigations  a  more  or  less  diminished 
absorption  of  fat  was  observed  after  the  extirpation  or  destruction  of 
the  gland,  or  the  exclusion  of  the  juice  from  the  intestine.  The  results 
are  very  diverse  as  to  the  extent  of  this  diminution,  as  in  certain  cases 
no  absorption  of  fat  was  observed,  while,  in  other  cases,  a  considerable 
absorption  was  noted  in  the  same  class  of  animal  (dog)  and  even  in  the 
same  animal.  According  to  Minkowski  and  Abelmann,  after  the  total 
extirpation  of  the  pancreas,  the  fat  of  the  food  introduced  is  not  absorbed 
at  all,  with  the  exception  of  milk,  of  which  28-53  per  cent  of  the  fat  is 
absorbed.  Other  investigators  have  obtained  different  results,  and  Har- 
ley has  observed  a  case  where  in  a  dog  an  absorption  of  only  4  per  cent 
of  the  milk  fat,  or,  on  the  complete  exclusion  of  intestinal  bacteria,  even 
no  absorption,  took  place.  The  conditions  may  vary  in  the  different 
cases,  and  the  behavior  is  not  the  same  in  different  varieties  of  animals. 

As  shown  by  Lombroso,  there  exists  an  essential  difference  between 
the  action  of  the  extirpation  of  the  gland,  or  a  prevented  flow  of  the 
secretion  into  the  intestine.  In  the  last  case,  as  the  experiments  reported 
by  Niemann  show,  no  essential  disturbance  of  the  absorption  takes  place, 
while  the  total  extirpation  of  the  gland  is  followed  by  a  marked  dis- 
turbance (Lombroso2).  This  investigator  is  also  of  the  opinion  that 
the  pancreas,  independent  of  the  external  secretion  in  any  way  (by 
endocrinic  bodies),  influences  the  absorption  of  the  foodstuffs  and  the 
activity  of  the  pancreas  enzymes  in  the  intestine.  In  order  to  judge 
this  view  it  would  be  of  the  greatest  interest  to  know  how  the  exclusion 
of  the  pancreatic  juice  from  the  intestine  acts  upon  the  other  factors 

1  Muller,  "  Unters.  liber  den  Icterus,"  Zeitschr.  f.  klin.  Med.,  12;  H6don  and 
Ville,  Arch,  de  Physiol.  (5),  9;  Harley,  Journ.  of  Physiol.,  18,  Journ.  of  Pathol,  and 
Bacteriol.,  1895,  and  Proceed.  Roy.  Soc,  61.  In  regard  to  the  other  authors  see  foot- 
note 1,  p.  532. 

2  Lombroso,  see  Bioch.  Centralbl.,  3,  67  and  566,  and  4,  73S;  also  Compt.  rend, 
soc.  biol.,  57;  Hofmeister's  Beitriige,  8,  11;  Pfluger's^Arch.,  112;  and  Arch.  f.  exp. 
Path.  u.  Pharm.,  56  and  60;  Niemann,  1.  c. 


540  DIGESTION. 

of  the  digestion,  such  as  upon  the  formation  of  the  secretions  and  their 
activity.  As  to  this  we  know  at  present  very  little,  but  the  work  of 
Zunz  and  Mayer  (see  page  532),  indicates  that  such  a  reverse  action  is 
possible.  Under  these  circumstances  it  is  not  possible  to  give  Lombroso's 
views  too  great  a  prominence. 

Lombroso  has  also  found  that  after  the  extirpation  of  the  pancreas 
in  the  dog,  sometimes  more  fat  is  eliminated  than  was  contained  in  the 
food;  that  this  eliminated  fat,  which  depends  upon  a  fat  secretion  into 
the  intestinal  canal,  has  a  different  composition  from  the  introduced  fat, 
and  that  in  these  cases  an  absorption  of  fat  also  takes  place.  That  some 
fat  can  be  absorbed  in  animals  even  in  the  absence  of  the  bile  as  well 
as  pancreatic  juice  has  been  shown  by  the  investigations  of  Hedon  and 
Ville  and  Cunningham.1 

The  reason  for  the  fact  that  the  fat  absorption  is  diminished  in  the 
absence  of  bile  from  the  intestine  must  be  sought  for  in  the  above-men- 
tioned rdle  of  this  fluid.  It  is  more  difficult  to  state  why  the  absence 
of  pancreatic  juice  causes  a  reduction  in  the  absorption  of  fat.  The  most 
natural  view  is  that  the  neutral  fats  are  here  less  completely  split,  but 
this  does  not  seem  to  be  the  case,  because  the  non-absorbed  fat  of  the 
feces  consists,  on  the  exclusion  of  bile  and  pancreatic  juice  (Minkowski 
and  Abelmann,  Harley,  Hedon  and  Ville,  Deucher),  principally  of 
free  fatty  acids.  A  still  unknown  change  caused  by  gastric  or  intestinal 
lipase  or  by  micrororganisms  may  produce  a  cleavage  of  the  fat  in  these 
cases.  The  imperfect  fat  absorption  after  the  extirpation  of  the  pan- 
creas can  possibly  be  explained  by  the  removal  of  a  considerable  part 
of  the  alkalies  necessary  for  the  formation  of  the  emulsion  and  for  the 
solution  of  the  fatty  acids,  but  as  Sandmeyer  found  in  dogs  deprived  of 
their  pancreas,  that  the  fat  absorption  was  raised  by  giving  chopped 
pancreas  with  the  fat,  this  can  hardly  be  a  sufficient  explanation.  The 
reason  for  this  is  perhaps  that  after  the  extirpation  of  the  pancreas  the 
splitting  of  the  fat  is  chiefly  brought  about  by  bacteria  in  those  parts  of 
the  intestinal  canal  where  the  conditions  for  absorption  are  not  favor- 
able. 

The  soluble  salts  are  also  absorbed  with  the  water.  The  proteins, 
which  can  dissolve  a  considerable  quantity  of  salts,  such  as  earthy  phos- 
phates which  are  otherwise  insoluble  in  alkaline  water,  are  of  great 
'  importance  in  the  absorption  of  such  salts. 

The  soluble  constituents  of  the  digestive  secretions  can  be  absorbed 
like  the  other  soluble  substances  and  toxines,  and  ferments  may  also  be 
absorbed,  especially  by  a  diseased  change  in  the  intestinal  walls. 

The  occurrence  of  urobilin  in  urine  attests  the  absorption  of  the  bile- 

1  Hedon  and  Ville,  1.  c.;  Cunningham,  Journ.  of  Physiol.,  32. 


ABSORPTION  OF  BILE.  541 

constituents  under  physiological  conditions  despite  the  fact  that  the 
occurrence  of  very  small  traces  of  bile-acids  in  the  urine  is  disputed. 
The  absorption  of  bile-acids  by  the  intestine  seems  to  be  positively  proved 
by  other  observations.  Tappeineh  '  introduced  a  solution  of  bile- 
salts  of  a  known  concentration  into  an  intestinal  knot  and  after  a  time 
investigated  the  contents.  He  found  that  in  the  jejunum  and  the  ileum, 
but  not  in  the  duodenum,  an  absorption  of  bile-acids  took  place,  and 
further  that  of  the  two  bile-acids  only  the  glycocholic  acid  was  absorbed 
in  the  jejunum.  Further,  Schiff  long  ago  expressed  the  opinion  that 
bile  undergoes  an  intermediate  circulation,  in  such  wise  that  it  is 
absorbed  from  the  intestine,  then  carried  to  the  liver  by  the  blood,  and 
lastly  eliminated  from  the  blood  by  this  organ.  Although  this  view  has 
met  with  seme  opposition,  still  its  correctness  seems  to  be  established  by 
the  researches  of  various  investigators,  and  more  recently  by  Prevost  and 
Binet,  and  specially  by  Stadelmann  and  his  pupils.2  After  the  intro- 
duction of  foreign  bile  into  the  intestine  of  an  animal,  the  foreign  bile- 
acids  appear  again  in  the  secreted  bile. 

How  does  the  removal  of  large  portions  of  the  various  parts  of  the 
intestine  affect  absorption?  Harley3  has  been  able  to  perform  a  par- 
tial extirpation  of  the  large  intestine  and  in  another  instance  a  com- 
plete extirpation.  This  last  condition  increased  the  feces  considerably, 
especially  because  of  the  large  increase  in  the  water  (five-fold).  Fats 
and  carbohydrates  were  absorbed  just  as  completely  as  in  the  normal. 
The  absorption  of  the  proteins,  on  the  contrary,  was  reduced  to  only 
84  per  cent  as  compared  to  93-98  per  cent  in  normal  dogs.  After  extir- 
pation, the  feces  sometimes  did  not  contain  any  urobilin,  or  only  traces 
thereof,  while  bile-pigments  existed  in  large  amounts. 

Erlanger  and  Hewlett  found  that  dogs  from  which  70-83  per 
cent  of  the  total  length  of  the  jejunum  and  ileum  had  been  removed, 
could  be  kept  alive,  like  other  animals,  if  only  the  food  was  not  too  rich 
in  fat.  When  the  food  contained  large  amounts  of  fat  then  25  per 
cent  was  evacuated  by  the  feces  as  compared  to  4-5  per  cent  in  the 
normal  animal.  Under  these  same  conditions  the  amount  of  nitrogen 
in  the  feces  was  increased  to  twice  the  normal  amount.  London  and 
Stassow4  found  on  resection  of  the  ileum  that  the  eliminated  diges- 
tion and  absorption  were  performed  by  the  parts  of  the  intestine  higher 


1  Wien.  Sitzungsber.,  77. 

2  Schiff,  Pfliiger's  Arch.,  3;  Prevost  and  Binet,  Compt.  Rend.,  106;  Stadelmann, 
see  footnote  1,  p.  416. 

3  Proceed.  Roy.  Soc,  64. 

4  Erlanger  and  Hewlett,  Amer.  Journ.  of  Physiol.  6;  London  and  Stassow.  Zeitschr. 
f.physiol.  Chem.  74,  349  (1911). 


542  DIGESTION. 

up;  after  resection  of  the  jejunum  the  large  intestine  seems  to  have  a 
compensating  action. 

After  the  exclusion  of  the  colon  in  rabbits,  Bergmann  and  Hult- 
gren  x  could  find  no  definite  action  upon  the  availability  of  the  cellu- 
lose nor  could  any  diminution  in  the  utility  of  the  other  constituents 
of  the  food  be  observed.  Zuntz  and  Ustjanzew  2  also  found  that  the 
removal  of  the  caecum  had  no  influence  on  the  utilization  of  nitrogen; 
but  in  respect  of  other  factors  they  arrive  at  different  results.  They 
found,  namely,  that  the  caecum  of  the  rodent  is  of  great  importance  for 
the  digestion  of  crude  fiber  and  the  pentosans.  On  feeding  hay  and 
wheat  to  rabbits  after  the  removal  of  the  csecum,  the  digestion  coefficient 
for  crude  fiber  fell  from  42.8  to  23.4-18.7  per  cent,  and  for  pentosans 
from  50  to  40-28.7  per  cent. 

The  question  as  to  the  forces  which  are  active  in  the  intestine  during 
absorption  has  not  been  satisfactorily  answered.  Attempts  have  been 
made  to  explain  absorption  as  a  filtration,  due  to  a  certain  difference 
in  the  hydrostatic  pressure  between  the  intestinal  contents  and  the 
blood.  A  sufficiently  great  difference  in  pressure  does  not  seem  to 
exist  and  besides  this  the  absorbed  solution  on  account  of  its  composi- 
tion cannot  be  considered  as  a  filtrate  from  the  intestinal  contents. 
Diffusion  processes  without  doubt  play  a  much  more  important  role. 
These  attempt  to  keep  the  same  concentration  of  all  dissolved  sub- 
stances on  both  sides  of  the  intestinal  epithelium  (in  intestinal  contents 
and  in  the  blood).  Such  processes  must  be  influenced,  as  mentioned 
in  Chapter  I  on  the  osmotic  pressure,  to  a  high  degree  upon  the  perme- 
ability of  the  intestinal  membrane  for  dissolved  solids  and  for  water. 
Nevertheless  the  diffusion  stream  does  not  give  sufficient  explanation 
for  the  absorption,  as,  according  to  Cohnheim,  3  the  result  is  different 
according  to  whether  the  intestine  is  alive  or  is  dead  and  in  general  a 
streaming  from  the  lumen  of  the  intestine  into  the  outside  fluid  is 
noticeable  in  the  living  intestine  quite  independent  of  the  differences 
in  concentration.  How  this  streaming  is  brought  about  has  not  been 
explained. 

Other  investigators  have  suggested  the  question  whether  surface- 
tension  forces  (adsorption  phenomenon)  are  active  in  absorption.4 
Still  it  has  not  been  possible  to  bring  the  absorbability  of  a  substance 
in  simple  relation  to  its  influence  on  the  surface-tension  of  the  water. 


1  Skand.  Arch.  f.  Physiol.,  14. 

2  Verhandl.  d.  physiol.  Gesellsch.  zu  Berlin,  1904-1905. 

3  Zeitschr.  f.  physiol.  Chem.  36-39. 

4  J.  Traube,  Bioch.  Zeitschr.  24,  324  (1910)  which  also  contains  literature. 


THEORIES  OF  ABSORPTION.  543 

Under  these  circumstances  and  as  it  is  not  within  the  scope  of  this 
book  to  enter  into  details  upon  the  numerous  investigations  as  to  the 
theory  of  absorption,  we  must  refer  to  larger  works  l  and  to  text-books 
on  physiology  for  further  information. 


1  See  Hober,  Physikalische  Chemie  der  Zelle,  Leipzig,  190G,  Koranyi  and  Richter, 
Phyeikalische  Chemie  u.  Medizin.  Leipzig  1907,  Bd.  1,  295.  I.  Munk,  Ergebnirae 
der  Physiologie,  I,  Abt.  1;  Hamburger,  Osmotisher  Druck  und  Ionealehre,  Bd.  2, 
Wiesbaden,  1904. 


CHAPTER  IX. 
TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 
I.     THE   CONNECTIVE  TISSUES. 

The  form-elements  of  the  typical  connective  tissues  are  cells  of 
various  kinds,  of  a  not  very  well-known  chemical  composition,  and 
gelatin-yielding  fibrils,  which,  like  the  cells,  are  imbedded  in  an  interstitial 
or  intercellular  substance.  The  fibrils  consist  of  collagen,  the  interstitial 
substance  contains  chiefly  mucoid  (tendon-mucoid) ,  besides  serglobulin 
and  seralbumin,  which,  occur  in  the  parenchymatous  fluid  (Loebisch1). 

The  connective  tissue  also  often  contains  fibers  or  formations  con- 
sisting of  elastin,  sometimes  in  such  great  quantities  that  the  connective 
tissue  is  transformed  into  elastic  tissue.  A  third  variety  of  fibers,  the 
reticular  fibers,  also  occurs,  and  according  to  Siegfried  these  consist 
of  reticulin. 

If  finely  divided  tendons  are  extracted  in  cold  water  or  NaCl  solu- 
tions, the  protein  bodies  soluble  in  the  nutritive  fluid  in  addition  to  a 
little  mucoid  are  dissolved.  If  the  residue  is  extracted  with  half- 
saturated  lime-water,  then  the  mucoid  is  dissolved  and  may  be  precipi- 
tated from  the  filtered  extract  by  adding  an  excess  of  acetic  acid.  The 
extracted  residue  contains  the  fibrils  of  the  connective  tissue  together 
with  the  cells  and  the  elastic  substance. 

The  so-called  tendon  mucin  is  not  true  mucin,  but  a  mucoid,  which, 
as  first  shown  by  Levene  and  then  by  Cutter  and  Gies,  contains  a. 
part  of  its  sulphur  as  an  acid  related  to  chondroitin-sulphuric  acid. 
These  mucoids,  which,  according  to  Cutter  and  Gies,  are  mixtures  of 
several  glycoproteins,  contain  2.2-2.33  per  cent  sulphur,  as  shown  by 
the  analyses  of  Chittenden  and  Gies,  as  well  as  those  of  Cutter  and 
Gies.  The  quantity  of  sulphur  split  off  as  sulphuric  acid  was  1.33-1.62 
per  cent  (Cutter  and  Gies).  van  Lier  2  has  prepared  a  substance  at  least 
closely  related  to  tendon  mucoid  from  the  hard  skin  of  man  and  certain 
animals. '  This  mucoid  yielded  an  ethereal  sulphuric  acid,  a  glucothionic 
acid  with  1.58-3.03  per  cent  sulphur  in  the  barium  salt,  and  was  variable 
in  different  animals.     It  gives  the  orcin  reaction  for  glucuronic  acid. 


1  Zeitschr.  f.  physiol.  Chem.,  10. 

2  Levene,  ibid.,  31  and  39;  Cutter  and  Gies.  Amer.  Journ.  of  Physiol.,  6;  Chitten- 
den and  Gies,  Journ.  of  Exp.  Med.,  1;  van  Lier,  Zeitschr.  f.  physiol.  Chem.  61. 

544 


CONNECTIVE  TISSUES.  545 

The  fibrils  of  the  connective  tissue  are  elastic  and  swell  slightly  in 
water,  somewhat  more  in  dilute  alkalies  or  in  acetic  acid.  On  the  other 
hand,  they  shrink  by  the  action  of  certain  metallic  salts,  such  as  ferrous 
sulphate  or  mercuric  chloride,  and  tannic  acid,  which  form  insoluble 
compounds  with  the  collagen.  Among  these  compounds,  which  prevent 
putrefaction  of  the  collagen,  that  with  tannic  acid  has  been  found  of 
the  greatest  technical  importance  in  the  preparation  of  leather.  In 
regard  to  the  collagens,  gelatins,  elastins,  and  reticulins,  see  pages  116 
to  121. 

The  tissues  described  under  the  names  mucous  or  gelatinous  tissues 
are  characterized  more  by  their  physical  than  by  their  chemical  prop- 
erties, and  have  been  but  little  studied.  This  much,  however,  is 
known,  that  the  mucous  or  gelatinous  tissues  contain,  at  least  in  certain 
cases,  as  in  the  Acalephse,  no  mucin. 

The  umbilical  cord  is  the  most  accessible  material  for  the  investiga- 
tion of  the  chemical  constituents  of  the  gelatinous  tissues.  The  mucin 
occurring  therein  yields,  according  to  van  Lier,  an  ethereal  sulphuric 
acid  (glucothionic  acid)  like  the  tendon  mucoid.  C.  Th.  Morner  1 
has  found  a  mucoid  in  the  vitreous  humor  which  contains  12.27  per 
cent  nitrogen  and  1.19  per  cent  sulphur. 

Young  connective  tissue  is  richer  in  mucoid  than  old.  Halliburton  2 
found  an  average  of  7.66  p.  m.  mucoid  in  the  skin  of  very  young  children 
and  only  3.85  p.  m.  in  the  skin  of  adults.  In  so-called  myxcedema, 
in  which  a  re-formation  of  the  connective  tissue  of  the  skin  takes  place, 
the  quantity  of  mucoid  is  also  increased. 

The  connective  tissue  and  also  the  elastic  tissue  are  richer  in  water 
and  poorer  in  solids  in  young  animals  as  compared  with  full-grown 
animals.  This  may  be  seen  from  the  following  analyses  of  the  Achilles 
tendon  (Buerger  and  Gies)  and  of  the  ligamentum  nuchse  (Vande- 
grift  and  Gies3): 

Achilles  tendon.  Ligament. 

Calf.  Ox.  Calf.  Ox. 

Water 675.1p.m.        628.7    p.m.  651.0p.m.        575.7    p.m. 

Solids 324.9  "             371.3  "  394.0  "            424.3 

<  )r«anic  bodies 318.4  "             366.6  "  342.4  "             419.6 

Inorganic  bodies 6.1  "                 4.7  "  6.6  "                4.7 

Fat 10.4  "                  11.2 

Proteid 2.2  "                 6.16 

Mucoid 12.83  "                 5.25 

Elastin 16.33  "                 316.70 

Collagen 315.88  "                 72.30 

Extractives,  etc 8.96  "                 7.99 

1  Zeitschr.  f.  physiol.  Chem.,  IS,  250. 

2  Mucin  in  Myxcedema:  Further  Analyses.  King's  College  Collected  Papers 
No.  1,  1893. 

1  Buerger  and  Gies,  Amer.  Journ.  of  Physiol.,  6;  Vandegrift  and  Gies,  ibid,  5. 


546  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

In  regard  to  the  mineral  bodies  it  must  be  remarked  that  according 
to  the  determinations  of  H.  Schulz  the  connective  tissue  is  rich  in  silicic 
acid.  The  greatest  amount  was  found  by  him,  in  the  crystalline  lens 
of  the  ox,  namely,  0.5814  gram  per  kilo  of  dried  substance.  In  man  he 
found  0.0637  gram  in  the  tendons,  0.1064  gram  in  the  fascia,  and  0.244 
gram  in  Wharton's  jelly  for  every  kilo  of  dried  substance.  The  quantity 
of  silicic  acid  is  higher  in  the  young  than  in  the  old;  in  man  it  is  highest 
in  the  embryonic  connective  tissue  of  the  umbilical  cord.  In  the  last- 
named  substance  Schulz  also  found  0.403  gram  FeoOs,  0.693  gram 
MgO,  3.297  grams  CaO,  and  3.794  grams  P2O5  for  every  kilo  of  dried 
substance.  The  report  of  Schulz  on  the  quantity  of  silicic  acid  does 
not  correspond  with  the  investigations  of  Frauenberger  1  who  found, 
in  Wharton's  jelly,  only  a  fraction  of  the  quantity  of  silicic  acid  that 
Schulz  gives. 

H.     CARTILAGE. 

Cartilaginous  tissues  consist  of  cells  and  an  original  hyaline  matrix, 
which,  however,  may  become  changed  in  such  wise  that. there  appears 
in  it  a  network  of  elastic  fibers  or  connective-tissue  fibrils. 

Those  cells  that  offer  great  resistance  to  the  action  of  alkalies  and 
acids  have  not  been  carefully  studied.  According  to  earlier  opinions 
the  matrix  was  considered  as  consisting  of  a  body  analogous  to  colla- 
gen, so-called  chondrigen.  The  investigations  of  Morochowetz  and 
others,  but  especialty  those  of  C.  Morner,2  have  shown  that  the 
matrix  of  the  cartilage  consists  of  a  mixture  of  collagen  with  other 
bodies. 

The  tracheal,  thyroideal,  cricoidal,  and  arytenoidal  cartilages  of 
full-grown  cattle  contain,  according  to  Morner,  four  constituents  in 
the  matrix,  namely,  chondromucoid,  chondroitin-sulphuric  acid,  collagen, 
and  the  albumoid. 

Chondromucoid.  This  body,  according  to  C.  Morner,  has  the  com- 
position C  47.30,  H  6.42,  N  12.58,  S  2.42,  O  31.28  per  cent.  Sulphur  is 
in  part  loosely  combined  and  may  be  split  off  by  the  action  of  alkalies, 
and  a  part  separates  as  sulphuric  acid  when  boiled  with  hydrochloric 
acid.  Chondromucoid  is  decomposed  fay  dilute  alkalies  and  yields  alkali 
albuminate,  peptone  substances,  chondroitin-sulphuric  acid,  alkali  sul- 
phides, and  some  alkali  sulphates.  On  boiling  with  acids  it  yields  acid 
albuminate,    peptone    substances,    chondroitin-sulphuric    acid,    and    on 

1  Schulz,  Pfluger's  Arch,  84  and  89,  131  and  144;  Frauenberger,  Zeitschr.  f.  physiol. 
Chem.,  57. 

1  Morochowetz,  Verhandl.,  d.  naturh.  med.  Vereins  zu  Heidelberg,  1,  Heft  5;  Morner, 
Skand.  Arch.  f.  Physiol.,  1. 


(  1 1  ON  DROITI N -SULPHURIC  ACID.  547 

account  of  the  further  decomposition  of  this  last  body,  sulphuric  acid 
and  a  reducing  substance  are  forme  1. 

Chondromucoid  is  a  white,  amorphous,  acid-reacting  powder  which 
is  insoluble  in  water,  but  dissolves  easily  on  the  addition  of  a  little 
alkali.  This  solution  is  precipitated  by  acetic  acid  in  great  excess  and 
by  small  quantities  erf  mineral  acids.  The  precipitation  may  be  retarded 
by  neutral  sails  or  by  ehondroitin-sulphuric  acid.  The  solution  con- 
taining NaCl  and  acidified  with  HC1  is  not  precipitated  by  potassium 
ferrocyanide.  Precipitants  for  chondromucoid  are  alum,  ferric  chloride, 
sugar  of  lead,  or  basic  lead  acetate.  Chondromucoid  is  not  precipitated 
by  tannic  acid,  and  it  may  by  its  presence  prevent  the  precipitation 
of  gelatin  by  this  acid.  It  gives  the  usual  color  reactions  for  proteins, 
namely,  with  nitric  acid,  with  copper  sulphate  and  alkali,  with  Mil- 
lion's and  Adamkiewicz-Hopkins'  reagents. 

Chcaidroitin-sulphuric  Acid,  chondroitic  acid.  This  acid,  which 
was  first  prepared  pure,  from  cartilage,  by  C.  Morner  and  identified 
by  him  as  an  ethereal  sulphuric  acid,  occurs,  according  to  Morner,  in 
all  varieties  of  cartilage  and  also  in  the  tunica  intima  of  the  aorta  and 
as  traces  in  the  bone  substance. '  K.  Morner  x  has  also  found  it  in  the 
ox-kidney  and  in  human  urine  as  a  regular  constituent.  Its  occurrence 
in  amyloid,  as  mentioned  on  page  173,  has  been  disputed  by  Hanssen. 
In  the  opinion  of  Levene,2  the  glucothionic  acid  which  is  prepared  from 
tendon  mucoid,  and  which  gives  the  orcin  reaction  for  glucuronic  acid, 
and  yields  furfurol  on  distillation  with  hydrochloric  acid,  is  not  identical 
with  the  ehondroitin-sulphuric  acid,  but  is  probably  related  thereto. 

Chondroitin-sulphuric  acid  has  the  formula  C18H27NSO17,  accord- 
ing to  Schmiedeberg.3  As  primary  products  this  acid  yields,  on 
cleavage,  sulphuric  acid  and  a  nitrogenous  substance,  chondroitin,  accord- 
ing to  the  following  equation: 

C18H27NSO17+H2O  =  H2S04+Ci8H27NOi4. 

Chondroitin,  which  is  similar  to  gum  arabic,  and  which  is  a  monobasic 
acid,  yields  acetic  acid  and  a  new  nitrogenous  substance,  chondrosin, 
as  cleavage  products,  on  decomposition  with  dilute  mineral  acids: 

C18H27NOi4+3H20  =  3C2H402+Ci2H2iN01i. 

Chondrosin,  which  is  also  a  gummy  substance  soluble  in  water,  is  a 
monobasic   acid   and   reduces   copper   oxide   in   alkaline   solutions   even 

1  C.  Morner,  1.  c,  and  Zeitschr.  f.  physiol.  Chem.,  20  and  23;  K.  Morner,  Skand. 
Arch.  f.  Physiol.,  6. 

2  Zeitschr.  f.  physiol.  Chem.,  39. 

*  Arch.  f.  exp.  Path.  u.  Pharm.,  28. 


548  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

more  strongly  than  glucose.  It  is  dextrogyrate,  and  represents  the 
reducing  substance  obtained  by  previous  investigators  in  an  impure 
form  on  boiling  cartilage  with  an  acid.  The  products  obtained  on  decom- 
posing chondrosin  with  barium  hydroxide  tend  to  show,  according  to 
Schmiedeberg,  that  chondrosin  contains  the  atomic  groups  of  glucuronic 
acid  and  glucosamine.  This  assumption  does  not  seem  to  have  sufficient 
foundation.  According  to  Orgler  and  Neuberg,  chondrosin  does  not 
give  the  orcin  test  nor  does  it  yield  furfurol.  They  claim  that  on 
cleavage  with  baryta  it  yields,  besides  a  carbohydrate  complex  which 
has  not  been  studied,  an  oxyamino-acid  having  the  formula  C6H13O6N, 
a  hexosamine  acid  or  tetraoxyaminocaproic  acid.  In  opposition  to  this 
S.  Frankel  has  found  that  the  chondrosin  gives  the  orcin  as  well  as  the 
phloroglucin  test  with  hydrochloric  acid,  and  he  has  prepared  an  acid 
with  the  formula  CeHnNOe,  which  he  calls  aminoglucuronic  acid, 
which  gives  the  above  tests  and  also  reduces.  Among  other  investi- 
gators, Pons  and  Kondo  l  have  also  found  that  chondroitin-sulphuric 
acid  gives  the  orcin  test  and  yields  furfurol,  according  to  Pons  6.6-6.9 
per  cent.  The  chondrosin  obtained  after  boiling  with  acid  and  distilling 
off  the  furfurol  does  not,  according  to  Pons,  give  furfurol,  which  agrees 
with  Orgler  and  Neuberg's  statement.  From  the  hydrolytic  products 
of  chondroitin-sulphuric  acid  with  hydrochloric  acid,  Pons  obtained 
with  phenylhydrazin.  a  crystalline  substance  melting  at  143°  C. 

Chondroitin-sulphuric  acid  appears  as  a  white  amorphous  powder 
which  dissolves  very  easily  in  water,  forming  an  acid  solution  and,  when 
sufficiently  concentrated,  a  sticky  liquid  similar  to  a  solution  of  gum 
arabic.  Nearly  all  of  its  salts  are  soluble  in  water.  The  neutralized 
solution  is  precipitated  by  stannous  chloride,  basic  lead  acetate,  neutral 
ferric  chloride,  and  by  alcohol  in  the  presence  of  a  little  neutral  salt. 
The  solution,  on  the  other  hand,  is  not  precipitated  by  acetic  acid, 
tannic  acid,  potassium  ferrocyanide  and  acetic  acid,  sugar  of  lead,  mer- 
curic chloride,  or  silver  nitrate.  Acidified  solutions  of  alkali  chondroitin- 
sulphates  cause  a  precipitation  when  added  to  solutions  of  gelatin  or 
proteid. 

The  preparation  of  chondromucoid,  and  its  separation  from  chondroitin- 
sulphuric  acid  can  be  accomplished  after  the  method  of  C.  Morner,  but  for 
details  we  refer  to  the  original  work. 

The  pre-existing  chondroitin-sulphuric  acid,  or  that  formed  by  the 
decomposition  of  chondromucoid,  is  obtained  by  lixiviating  the  cartilage 
with  a  5-per  cent  caustic-alkali  solution.  The  alkali  albuminate  formed 
by  the  decomposition  of  the  chondromucoid  can  be  removed  from  the 
solution  by  neutralization,  then  the  peptone  precipitated  by  tannic  acid, 

1  Orgler  and  Neuberg,  Zeitschr.  f.  physiol.  Chem.,  37;  Frankel,  Annal.  d.  Chem.  u. 
Pharm.,  351;  Pons.  Arch,  intern,  de  Physiol.,  8  (1909);  Kondo,  Bioch.  Zeitschr.,  26. 


CARTILAGENEOUS  TISSUE.  549 

the  excess  of  this  acid  removed  with  sugar  of  lead,  and  the  lead  removed 
from  the  filtrate  by  H2S.  If  further  purification  is  necessary,  the  acid  is 
precipitated  with  alcohol,  the  precipitate  dissolved  in  water,  this  solu- 
tion dialyzed  and  precipitated  again  with  alcohol — this  solution  in  water 
and  precipitation  with  alcohol  being  repeated  a  few  times — and  lastly 
the  acid  is  treated  with  alcohol  and  ether.  Other  methods  for  the  prepara- 
tion of  the  acid  (from  the  septum  narium  of  the  pig)  have  been  suggested 
by  Schmiedeberg  and  Kondo. 

The  collagen  of  the  cartilage  gives,  according  to  C.  Morner,  a  gelatin 
which  contains  only  16.4  per  cent  N,  and  which  can  hardly  be  considered 
identical  with  ordinary  gelatin. 

In  the  above-mentioned  cartilages  of  full-grown  animals  the  chon- 
droitin-sulphuric acid  and  chondromucoid,  perhaps  also  the  collagen, 
are  found  surrounding  the  cells  as  round  balls  or  lumps.  These  balls 
(Morner's  chondrin-balls) ,  which  give  a  blue  color  with  methyl-violet, 
lie  in  the  meshes  of  a  trabecular  structure,  which  is  colored  when 
brought  in  contact  with  tropseolin. 

The  albumoid  is  a  nitrogenized  body  which  contains  loosely  com- 
bined sulphur.  It  is  soluble  with  difficulty  in  acids  and  alkalies  and 
resembles  keratin  in  many  respects,  but  differs  from  it  by  being  soluble 
in  gastric  juice.  In  other  respects  it  resembles  elastin,  but  differs  from 
this  substance  in  containing  sulphur.  This  albumoid  gives  the  color 
reactions  of  the  protein  bodies. 

Cartilage  gelatin  and  the  albumoid  may  be  prepared  according  to 
the  folowing  method  of  Morner:  First  remove  the  chondromucoid 
and  chondroitin-sulphuric  acid  by  extraction  with  dilute  caustic  potash 
(0.2-0.5  per  cent),  remove  the  alkali  from  the  remaining  cartilage  by 
water,  and  then  boil  with  water  in  a  Papin's  digester.  The  collagen 
passes  into  solution  as  gelatin,  while  the  albumoid  remains  undissolved 
(contaminated  by  the  cartilage-cells).  The  gelatin  may  be  purified  by 
precipitating  with  sodium  sulphate,  which  must  be  added  to  saturation 
in  the  faintly  acidified  solution,  redissolving  the  precipitate  in  water, 
dialyzing  well,  and  precipitating  with  alcohol. 

In  Morner's  experience  no  albumoid  is  found  in  young  cartilage, 
but  only  the  three  first-mentioned  constituents.  Nevertheless,  the  young 
cartilage  contains  about  the  same  amounts  of  nitrogen  and  mineral 
substances  as  the  old.  The  cartilage  of  the  ray  (Raja  batis  Lin.),  which 
has  been  investigated  by  Lonnberg,1  contains  no  albumoid  and  only 
a  little  chondromucoid,  but  a  large  proportion  of  chondroitin-sulphuric 
acid  and  collagen. 

According  to  Pfluger  and  Handel,2    glycogen  occurs  to  a  slight 

1  Maly's  Jahresber.,  19,  325. 

1  Pfluger  in  Pfliiger's  Arch.,  92;  Handel,  ibid. 


550  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

extent  in  all  matrices,  and  of  these  it  is  richest  in  the  cartilage.  Ten- 
dons, ligamentum  nucha?,  and  cartilage  of  the  ox  contained  0.06,  0.07, 
and  2.17  p.  m.  glycogen  respectively  (Handel). 

Hoppe-Seyler  found  in  fresh  human  rib-cartilage  676.7  p.  m.  water, 
301.3  p.  m.  organic,  and  22  p.  m.  inorganic  substance,  and  in  the  cartilage 
of  the  knee-joint  735.9  p.  m.  water,  248.7  p.  m.  organic,  and  15.4  p.  m. 
inorganic  substance.  Pickardt  found  402-574  p.  m.  water  and  72.86 
p.  m.  ash  (no  iron)  in  the  laryngeal  cartilage  of  oxen.  The  ash  of  car- 
tilage contains  considerable  amounts  (even  800  p.  m.)  of  alkali  sulphate, 
which  probably  does  not  exist  originally  as  such,  but  is  produced  in  great 
part  by  the  incineration  of  the  chondroitin-sulphuric  acid  and  the  chon- 
dromucoid.  The  analyses  of  the  ash  of  cartilage  therefore  cannot 
give  a  correct  idea  of  the  quantity  of  mineral  bodies  existing  in  this  sub- 
stance. The  cartilage  is  richest  in  sodium  of  all  the  tissues  of  the  body, 
and  according  to  Bunge  1  the  amount  of  Na  and  CI  is  greatest  in  young 
animals.  In  1000  parts  of  cartilage  dried  at  120°  C,  Bunge  found  91.26 
parts  Na^O  in  the  shark,  33.98  in  the  ox  embryo,  32.45  in  a  fourteen-day- 
old  calf,  and  26.4  in  a  ten-weeks-old  calf. 

Ochronose  is  the  brown  to  black  coloration  of  the  cartilage  which 
sometimes  occurs,  and  which  has  also  been  observed  in  several  cases  of 
alcaptonuria  (see  Chapter  XIV)  or  after  lengthy  treatment  with  carbolic 
acid  bandages  (Poulsen,  Adler2).  The  nature  of  these  melanine- 
like  pigments  is  unknown. 

The  Cornea.  The  corneal  tissue,  which,  in  a  chemical  sense,  is  con- 
sidered by  many  investigators  to  be  related  to  cartilage,  contains  traces 
of  proteid  and  a  collagen  as  chief  constituent,  which  C.  Morner3  claims 
contains  16.95  per  cent  N.  According  to  him  it  also  contains  a  mucoid 
which  has  the  composition  C  50.16,  H  6.97,  N  12.79,  and  S  2.07  per 
cent.  On  boiling  with  dilute  mineral  acid  this  mucoid  yields  a  reducing 
substance.  The  globulins  found  by  other  investigators  in  the  cornea 
are  not  derived  from  the  matrix,  according  to  Morner,  but  from  the 
layer  of  epithelium.  Morner  believes  that  Descemet's  membrane 
consists  of  membranin  (page  171),  which  contains  14.77  per  cent  N  and 
0.90  per  cent  S. 

In  the  cornea  of  oxen  His4  found  758.3  p.  m.  water,  203.8  p.  m. 
gelatin-forming  substance,  28.4  p.  m.  other  organic  substance,  besides 
8.1  p.  m.  soluble  and  1.1  p.  m.  insoluble  salts. 


1  Hoppe-Seyler,  cited  from  Kiihne's  Lehrbuch  d.  physiol.  Chem.,  387;  Pickardt,, 
Centralbl.  f.  Physiol.,  6,  735;  Bunge,  Zeitschr.  f.  physiol.  Chem.,  28. 

2  See  Maly's  Jahresb.,  40,  424,  Adler,  Zeitschr.  f.  Krebsforschung,  11. 
'  Zeitschr.  f.  physiol.  Chem.,  18. 

4  Cited  from  Camgee,  Physiol.  Chem.,  1880,  451. 


BONE.  551 


IU.     BONE. 

The  bony  structure  proper,  when  free  from  other  format  inns  occurring 
in  bones,  such  a-  marrow,  nerves,  and  blood-vessels,  consists  of  cella  and 

a  matrix. 

The  alls  have  not  been  closely  studied  in  regard  to  their  chemical 
constitution.  On  boiling  with  water  they  yield  no  gelatin.  They 
contain  no  keratin,  which  usually  should  not  be  present  in  the  bony 
structure  (Herbert  Smith  *). 

The  matrix  of  the  bony  structure  contains  two  chief  constituents, 
namely,  an  organic  substance,  and  the  so-called  bone-earths,  lime-salts, 
inclosed  in  or  combined  with  it.  If  bones  are  treated  with  dilute  hydro- 
chloric acid  at  the  ordinary  temperature,  the  lime-salts  are  dissolved 
and  the  organic  substance  remains  as  an  elastic  mass,  preserving  the 
shape  of  the  bone. 

The  organic  matrix  consists  chiefly  of  ossein,  which  is  generally 
considered  as  identical  with  the  collagen  of  the  connective  tissue.  It 
also  contains,  as  Hawk  and  Gies  2  have  shown,  mucoid  and  albuminoid. 
After  the  removal  of  the  lime-salts  by  hydrochloric  acid  of  2-5  p.  m. 
these  experimenters  were  able  to  extract  the  mucoid  by  one-half  sat- 
urated lime-water,  and  to  precipitate  it  with  2  p.  m.  hydrochloric  acid. 
After  the  removal  of  the  osseomucoid  and  collagen  (by  boiling  with 
water)  they  obtained  the  albuminoid  as  an  insoluble  residue. 

The  osseomucoid  on  boiling  writh  hydrochloric  acid  yielded  a  reduc- 
ing substance  and  sulphuric  acid;  1.11  per  cent  sulphur  appearing  in 
this  form.  The  osseomucoid  stands  close  to  the  chondro-  and  tendon 
mucoid  in  elementary  composition,  as  may  be  seen  from  the  follow- 
ing analyses: 

c  h  n          s            o 

Osseomucoid 47.43  6.63  12.22  2.32  31 .40  (Hawk  and  Gies) 

Chondromucoid. .  ..   47.30  6.42  12.58  2.42  31.28  (C.  Morner) 

Tendon  mucoid ...  .  48.76  6.53  11.75  2.33  30.60  (Chittenden  and  Gies) 

Corneal  mucoid..  ..   50.16  6.97  12.79  2.07  28.01   (C.  Morner) 

The  osseoalbuminoid  is  insoluble  in  2  p.  m.  hydrochloric  acid,  and 
in  5  p.  m.  Xa2CC>3,  but  dissolves  in  10  per  cent  KOH  with  the  formation 
of  albuminates.  The  composition  of  chondro-  and  osseoalbuminoid 
is  as  follows: 

c 

Osseoalbuminoid 50  16 

Chondroalbuminoid .  .  .   50  46 


1  Zeitschr.  f.  Biologie,  19. 

s  Amer.   Journ.   of   Physiol.,    5   and 


H 

N 

s 

0 

7.03 

16. 

17 

1.18 

25 

46\ 

Hawk  and 

7.05 

14. 

95 

1.86 

25 

.68J 

Gies 

552  TISSUES  OF  THE  CONNECTIVE   SUBSTANCE. 

The  inorganic  constituents  of  the  bony  structure,  the  so-called 
bone-earths,  which,  after  the  complete  calcination  of  the  organic  sub- 
stance, remain  as  a  white  brittle  mass,  consist  chiefly  of  calcium  and 
phosphoric  acid,  but  also  contain  carbonic  acid  and,  in  smaller  amounts, 
magnesium,  chlorine,  and  fluorine.  Iron,  which  has  been  found  in  bone- 
ash,  does  not  seem  to  belong  exactly  to  the  bony  substance,  but  to  the 
nutritive  fluids  or  to  the  other  constituents  of  bones.  The  traces  of 
sulphate  occurring  in  the  bone-ash  are  derived,  according  to  Morner, 
from  the  chondroitin-sulphuric  acid.  According  to  Gabriel,  potassium 
and  sodium  are  essential  constituents  of  bone-earth,  and  this  has  been 
substantiated  by  Aron  l. 

The  opinions  of  investigators  differ  slightly  as  to  the  manner  in 
which  the  mineral  bodies  of  the  bony  structure  are  combined  with 
each  other.  Chlorine  is  present  in  the  same  form  as  7m  apatite 
3(Ca3P208)CaCl2-  If  we  eliminate  the  magnesium,  the  chlorine,  and 
the  fluorine,  the  last,  Gabriel  claims,  occurring  only  as  traces,  the  remain- 
ing mineral  bodies  form  the  combination  3(Ca3P20s)CaC03.  In  his 
opinion  the  simplest  expression  for  the  composition  of  the  ash  of  bones 
and  teeth  is  (Ca3(P04)2+Ca5HP30,3+Aq),  in  which  2-3  per  cent  of  the 
lime  is  replaced  by  magnesia,  potash,  and  soda,  and  4-6  per  cent  of  the 
phosphoric  acid  by  carbonic  acid,  chlorine,  and  fluorine.  Recently,  on 
the  contrary,  Gassmann  has  given  important  reasons  for  the  follow- 
ing complex  combination  in  Werner's  2  sense. 


/OP03Ca\ 
Cf(      >  Ca  3 

.      VoPOsCa/  - 


C03 


Analyses  of  bone-earths  have  shown  that  the  mineral  constituents 
exist  in  rather  constant  proportions,  which  are  nearly  the  same  in  dif- 
ferent animals.  As  an  example  of  the  composition  of  bone-earth  we  here 
give  the  analyses  of  Zalesky.3     The  figures  represent  parts  per  thousand: 

Man.  Ox.  Tortoise.  Guinea-pig. 

Calcium  phosphate,  Ca3P208 838 . 9  860 . 9  859 . 8  873 . 8 

Magnesium  phosphate,  MgJPaOs 10.4  10.2  13.6  10.5 

Calcium  combined  with  C02,  Fl,  and  CI. .  .     76 . 5  73 . 6  63 . 2  70 . 3 

C02                   57.3  62.0  52.7  

Chlorine 1.8  2.0         1.3 

Fluorine* 2.3  3.0  2.0  


1  Morner,  Zeitschr.  f.  physiol.  Chem.,  23;  Gabriel,  ibid.,  18,  which. also  contains 
the  pertinent  literature;  Aron,  Pfliiger's  Arch.,  106. 

2  Gassmann,   Zeitschr.  f.   physiol.  Chem.    70  and  83;  Werner,   Ber.  d.  d.  Chem. 
Gesellsch.,  40. 

1  Hoppe-Seyler,  Med.-chem.  Untersuch.,  p.  19. 

4  The  reports  as  to  the  quantity  of  fluorine  disagree;  see  Harms,   Zeistchr.  f. 
Biologic,  38;  Jodlbauer,  ibid.,  41. 


Ox. 

Elephant. 

Femur 

Femur. 

857.2 

900.3 

15.3 

19.6 

4.5 

4.7 

3.0 

2.0 

119.6 

72.7 

BONK.  553 

Some  of  the  ( '( )•_>  is  always  lost  on  calcining,  so  that  the  bone-ash 
does  not  contain  the  entire  CO2  of  the  bony  substance. 

Gautier  and  Clausmann  1  have  determined  the  fluorine  in  various 
organs  and  tissues.  In  man  the  diaphysis  end  of  the  femur  had  0.495 
p.  m.  fluorine,  and  the  epiphysis  end  0.119  p.  m.  fluorine.  In  children 
the  diaphysis  end  of  the  long  bones  contained  0.156  p.  m.  fluorine 
and  the  epiphysis  end  0.037  p.  m.  A  similar  difference  also  occurs  in 
animals.  Cartilage  of  man  with  0.014  p.  in.  fluorine  and  tendons  (calf) 
with  0.0035  p.  m.  fluorine,  are  much  poorer  in  fluorine  than  the  bones. 
The  dentin  (dog)  contains  0.56  p.  m.  fluorine  and  the  enamel  (of  a 
young  dog)  contained  i.66.  p.  m.  fluorine,  all  results  obtained  from  the 
fresh  substance. 

Ad.  Carnot  2  found  the  following  composition  for  the  bone-ash  of 
man,  ox,  and  elephant: 

Man. 
Femur  Femur 

(body).  (head). 

Calcium  phosphate 874 . 5  878 . 7 

Magnesium  phosphate 15.7           17.5 

Calcium  fluoride 3.5             3.7 

Calcium  chloride 2.3             3.0 

Calcium  carbonate 101.8  92.3 

Iron  oxide 1.0            1.3            1.3            1.5 

The  quantity  of  organic  substance  in  the  bones,  calculated  from  the 
loss  of  weight  in  burning,  varies  between  300  and  520  p.  m.  This 
variation  may  in  part  be  explained  by  the  difficulty  in  obtaining  the 
bony  substance  entirely  free  from  water,  and  partly  by  the  very  variable 
amount  of  blood-vessels,  nerves,  marrow,  and  the  like  in  different  bones. 
The  unequal  amounts  of  organic  substance  found  in  the  compact  and 
in  the  spongy  parts  of  the  same  bone,  as  well  as  in  bones  at  different 
periods  of  development  in  the  same  animal,  probably  depend  upon 
the  varying  quantities  of  these  above-mentioned  tissues.  Dentin,  which 
is  comparatively  pure  bony  structure,  contains  only  260-280  p.  m. 
organic  substance,  and  Hoppe-Seyler  3  therefore  thinks  it  probable 
that  perfectly  pure  bony  substance  has  a  constant  composition  and 
contains  only  about  250  p.  m.  organic  substance.  The  question  whether 
these  substances  are  chemically  combined  with  the  bone-earths  or  only 
intimately  mixed  has  not  been  decided. 

The  nutritive  fluids  which  circulate  through  the  bones  have  not  been  isolated 
and  we  only  know  that  they  contain  some  protein  and  some  NaCl  and  alkali 
sulphate. 

1  Compt.  Rend.,  156. 

iIbid.,  114. 

» Physiol.  Chem.,  102-104 


554  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

Bone  Marrow.  We  differentiate  between  the  red  and  yellow  mar- 
row, to  which  also  belongs  the  gelatinous  marrow,  poor  in  fat,  found  in 
fat  atrophy  and  in  old  age.  The  difference  between  the  first  two-men- 
tioned kinds  of  marrow  lies,  essentially,  in  the  fact  that  the  red  marrow 
contains  a  greater  quantity  of  erythrocytes  besides  a  higher  content  of 
protein  and  less  fat.  The  fat  of  the  yellow  marrow  is,  according  to 
X erring,1  richer  in  oleic  acid  and  poorer  in  solid  fats  than  the  fat  of  the 
red  marrow.  Besides  the  fat,  lecithin  also  occurs  in  the  bone-marrow 
and  this  varies  in  amount  in  different  animals  and  at  various  ages,  as 
mentioned  on  page  244.  The  protein  consists  of  a  globulin  coagulating 
at  47-50°  C.  (Forrest)  and  a  nucleoprotein  with  1.6  per  cent  phos- 
phorus (Halliburton2)  besides  fibrinogen  (P.  Muller3),  traces  of 
albumin  and  proteose.  In  the  extractives  are  found  lactic  acid,  inosite, 
hypoxanthine,  cholesterine  and  bodies  of  an  unknown  kind.  The  quan- 
titative composition  of  both  kinds  of  marrow  varies  considerably  with 
the  fat  content,  and  the  reports  of  the  different  investigators  are  corre- 
spondingly discrepant   (Xerking,  Hutchinson  and  Macleod  4) . 

The  diverse  quantitative  composition  of  the  various  bones  of  the 
skeleton  depends  probably  on  the  varying  quantities  of  other  tissues, 
such  as  marrow,  blood-vessels,  etc.,  which  they  contain.  The  same 
reason  explains,  to  all  appearances,  the  larger  quantity  of  organic 
substance  in  the  spongy  part  of  the  bones  as  compared  with  the  more 
compact  parts.  Schrodt  5  has  made  comparative  analyses  of  different 
parts  of  the  skeleton  of  the  same  animal  (dog)  and  has  found  an  essen- 
tial difference.  The  quantity  of  water  in  the  fresh  bones  varies  between 
138  and  443  p.  m.  The  bones  of  the  extremities  and  the  skull  contain 
138-222,  the  vertebrae  168-443,  and  the  ribs  324-356  p.  m.  water.  The 
quantity  of  fat  varies  between  13  and  269  p.  m.  The  largest  amount 
of  fat,  256-269  p.  m.,  is  found  in  the  long  tubular  bones,  while  only 
13-175  p.  m.  fat  is  found  in  the  small  short  bones.  The  quantity  of 
organic  substance,  calculated  from  fresh  bones,  was  150-300  p.  m.,  and 
the  quantity  of  mineral  substances  290-563  p.  m.  Contrary  to  the 
general  supposition  the  greatest  amount  of  bone-earths  was  not  found 
in  the  femur,  but  in  the  first  three  cervical  vertebrae.  In  birds  the 
tubular  bones  are  richer  in  mineral  substances  than  the  flat  bones 
(During),  and  the  greatest  quantity  of  mineral  bodies  has  been  found 
in  the  humerus  (Hiller,  During  6). 

1  Bioch.  Zeitschr.,  10. 

2  Forrest,  Journ.  of  Physiol.,  17;  Halliburton,  ibid.,  18. 
'See  footnote  1,  p.  253. 

4  Nerking,  1.  c;    Hutchinflon  and  Macleod,  Journ.  of  Anat.  and  Physiol.,  36. 

'Cited  from  Maly's  Jahresber.,  6. 

6  Hiller,  eited  from  Maly's  Jahresber.,  14;  During,  Zeitschr.  f.  physiol.  Chem.,  23. 


DISEASES  OF  THE  BONES.  555 

We  do  not  possess  trustworthy  information  in  regard  to  the  compo- 
sition of  bones  at  different  ages.  The  analyses  by  E.  Voit  of  bones  of 
dogs,  and  by  Brubacher  of  bones  of  children,  apparently  indicate  that 
the  skeleton  becomes  poorer  in  water  and  richer  in  ash  with  increase 
in  age.  Graffenberger  1  has  found  in  rabbits,  6£-7£  years  old,  that 
the  bones  contained  only  140-170  p.  m.  water,  while  the  bones  of  the 
full-grown  rabbit  2-4  years  old  contained  200-240  p.  m.  The  bones  of 
old  rabbits  contain  more  carbon  dioxide  and  less  calcium  phosphate. 

The  composition  of  bones  of  animals  of  different  species  is  but  little  known. 
The  bones  of  birds  contain,  as  a  rule,  somewhat  more  water  than  those  of  mam- 
malia, and  the  bones  of  fishes  contain  the  largest  quantity  of  water.  The  bones 
of  fishes  and  amphibians  contain  a  greater  amount  of  organic  substance.  The 
bones  of  pachyderms  and  cetaceans  contain  a  large  proportion  of  calcium  carbo- 
nate; those  of  granivorous  birds  always  contain  silicic  acid.  The  bone-ash  of 
amphibians  and  fishes  contains  sodium  sulphate.  The  bones  of  fishes  seem  to 
contain  more  soluble  salts  than  the  bones  of  other  animals. 

A  great  many  experiments  have  been  made  to  determine  the  exchange 
of  material  in  the  bones — for  instance,  writh  food  rich  in  l'me  and  with 
food  deficient  in  lime — but  the  results  have  always  been  doubtful  or 
contradictory.  The  attempts  to  substitute  other  alkaline  earths  or 
alumina  for  the  lime  of  the  bones  have  also  given  conflicting  results.2 
On  feeding  sufficient  calcium  and  phosphorus  in  the  food  Aron  3  found, 
by  strongly  reducing  the  sodium  and  at  the  same  time  giving  a  large 
amount  of  potassium,  that  the  development  of  the  bones  was  below 
normal.  On  the  administration  of  madder,  the  bones  of  the  animal  are 
found  to  be  colored  red  after  a  few  days  or  weeks;  but  these  experiments 
have  not  led  to  any  positive  conclusion  in  regard  to  the  growth  or 
metabolism  in  the  bones. 

Under  pathological  conditions,  as  in  rachitis  and  softening  of  the 
bones,  an  ossein  has  been  found  which  does  not  give  any  typical  gelatin 
on  boiling  with  water.  This  finding  is  still  uncertain  as  otherwise  path- 
ological conditions  seem  to  affect  chiefly  the  quantitative  composition 
of  the  bones,  and  especially  the  relation  between  the  organic  and  the  inor- 
ganic substance.  In  rachitis  the  bones  are  poorer  in  solids  and  these 
are  poorer  in  mineral  substances  than  under  normal  conditions. 
Attempts  have  been  made  to  produce  rachitis  in  animals  by  the  use  of 
food  deficient  in  lime.  From  experiments  on  fully  developed  animals 
opposing  results  have  been  obtained.     In  young,  undeveloped  animals 


1  Voit,    Zeitschr.    f.    Biologie,    16;  Brubacher,    ibid.,    27;  Graffenberger   in    Maly's 
Jahresber.,  21. 

2  See  H.  Weiske,  Zeitschr.  f.  Biologie,  31,  and  W.  Stoeltzner,  Pfliiger'a  Arch.,  122, 
and  H.  Stoeltzner,  Bioch.  Zeitschr.,  12. 

1  Pfluger'a  Arch.,  106. 


556  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

Erwin  Voit,  Aron  and  Sebatjer  and  others x  produced,  by  lack  of 
lime-salts,  a  change  similar  to  rachitis.  In  full-grown  animals  the 
bones  were  changed  after  a  long  time  because  of  the  lack  of  lime-salts 
in  the  food,  but  did  not  become  soft,  only  thinner  (osteoporosis).  The 
attempts  to  remove  the  lime-salts  from  the  bones  by  the  addition  of 
lactic  acid  to  the  food  have  led  to  no  positive  results  (Heitzmann, 
Heiss,  Baginsky  2).  Weiske,  on  the  contrary,  has  shown,  by  admin- 
istering dilute  sulphuric  acid  or  monosodium  phosphate  with  the  food 
(presupposing  that  the  food  gave  no  alkaline  ash)  to  sheep  and  rab- 
bits, that  the  quantity  of  mineral  bodies  in  the  bones  might  be  dimin- 
ished. On  feeding  continuously  for  a  long  time  with  a  food  which  yielded 
an  acid  ash  (cereal  grains),  Weiske  observed  a  diminution  in  the  min- 
eral substances  of  the  bones  in  full-grown  herbivora.3  A  few  investi- 
gators are  of  the  opinion  that  in  rachitis,  as  in  osteomalacia,  in  which 
disease  the  calcium  content  of  the  bones  is  also  diminished,  a  solution 
of  the  lime-salts  by  means  of  lactic  acid  takes  place.  This  was  sug- 
gested by  the  fact  that  O.  Weber  and  C.  Schmidt4  found  lactic  acid, 
in  the  cyst-like,  altered  bony  substance  in  osteomalacia. 

Well-known  investigators  have  disputed  the  possibility  of  the  lime- 
salts  being  washed  from  the  bones  in  osteomalacia  by  means  of  lactic 
acid.  They  have  given  special  prominence  to  the  fact  that  the  lime- 
salts  held  in  solution  by  the  lactic  acid  must  be  deposited  on  neutraliza- 
tion of  the  acid  by  the  alkaline  blood.  This  objection  is  not  very  impor- 
tant, as  the  alkaline  blood-serum  has  the  property  to  a  high  degree  of 
holding  earthy  phosphates  in  solution,  which  fact  has  been  recently 
shown  by  Hofmeister.  The  investigations  of  Levy  contradict  the 
claim  as  to  the  solution  of  the  lime-salts  by  lactic  acid  in  osteomalacia. 
He  found  that  the  normal  relation  6PC>4:10Ca  is  retained  in  all  parts 
of  the  bones  in  osteomalacia,  which  would  not  be  the  case  if  the  bone- 
earths  were  dissolved  by  an  acid.  The  decrease  in  phosphate  occurs  in 
the  same  quantitative  relation  as  the  carbonate,  and  according  to  Levy, 
in  osteomalacia  the  exhaustion  of  the  bone  takes  place  by  a  decalcifica- 
tion in  which  one  molecule  of  phosphate-carbonate  after  the  other  is 
removed.     This  does  not  agree  with  the  findings  of  MoCrudden  5    who 


1  Zeitschr.  f.   Biologie,   16;  Aron  and  Sebauer,   Bioch.  Zeitsohr.,  8;  A.  Baginsky, 
Arch.  f.  (Anat.  u.)  Physiol.,  1881. 

2  Heitzmann,  Maly's  Jahresber.,  3,  229;  Heiss,  Zeitschr.  f.  Biolojrie,  12;  Basrinsky;, 
Virchow's  Arch.,  87. 

3  See  Maly's  Jahresber.,   22;  also  Weiske,   Zeitschr.   f.   physiol.   Chem.,   20,   and 
Zeitschr.  f.  Biologie,  31. 

*  Cited  from  v.  Corup-Besanez.  Lehrb.  de.  physiol.  Chem.,  4.  Aufl. 
6  Hofmeister,  Ergebn.  d.  Physiol.  10;  Levy.  Zeitschr.  f.  physiol.  Chem.  19;  McCrud- 
den,  Journ.  of  biol.  Chem.,  7. 


TOOTH-STRUCTURE.  557 

found  a  changed  relation  between  the  Ca  and  phosphoric  acid  in  osteo- 
malacia. 

Rachitic  bones  are  always  poorer  in  mineral  substances  than  normal  bones. 
The  relation  between  Ca,PO«  and  CO.,  was  found  by  GASSMANN  to  be  the  same 
as  in  normal  bones  while  he  found  a  pathological  increase  in  the  magnesium.  The 
organic  substance  was  found  in  rachitis  to  be  relatively  as  well  as  absolutely 
increased,  at  least  in  certain  cases  (Gassmann).  The  statements  differ  in 
regard  to  the  water  content.  According  to  Brubacher  this  is  larger  while  accord- 
ing to  Gassmann  it  is  10  p.  m.  smaller  than  in  normal  bones.  In  opposition  to 
rachitis,  osteomalacia  is  often  characterized  by  the  considerable  amount  of  fat 
in  the  bones,  230-290  p.  m.,  but  as  a  rule  the  composition  varies  so  much  that 
the  analysts  are  of  little  value.  In  a  case  of  osteomalacia,  Chabrie  l  found  a 
larger  quantity  of  magnesium  than  calcium  in  a  bone.  The  ash  contained  417 
p.  m.  phosphoric  acid,  222  p.  m.  lime,  269  p.  m.  magnesia,  and  86  p.  m.  carbon 
dioxide.  McCrudden  found  more  magnesium  than  calcium;  other  investigators 
have  on  the  contrary  found  more  calcium  than  magnesium. 

The  tooth-structure  is  closely  related,  from  a  chemical  standpoint, 
to  the  bony  structure. 

Of  the  three  chief  constituents  of  the  teeth — dentin,  enamel,  and 
cement — the  cement  is  to  be  considered  as  true  bony  structure,  and  as 
such  has  already  been  discussed  to  some  extent.  Dentin  has  the  same 
composition  as  the  bony  structure,  but  contains  somewhat  less  water. 
The  organic  substance  yields  gelatin  on  boiling;  but  the  dental  tubes 
are  not  dissolved,  therefore  they  cannot  consist  of  collagen.  In  dentin 
2G0-280  p.  m.  organic  substance  has  been  found.  Enamel  is  an  epithe- 
lium formation  containing  a  large  proportion  of  lime-salts.  Correspond- 
ing to  its  character  and  origin,  the  organic  substance  of  the  enamel 
does  not  yield  any  gelatin.  Completely  developed  enamel  contains 
the  least  water,  the  greatest  quantity  of  mineral  substances,  and  is  the 
hardest  of  all  the  tissues  of  the  body.  In  full-grown  animals  it  con- 
tains hardly  any  water,  and  the  quantity  of  organic  substance  amounts 
to  only  20-40-68  p.  m.  The  relative  amounts  of  calcium  and  phosphoric 
acid  are  shown  by  the  analyses  of  Hoppe-Seyler  to  be  about  the  same 
as  in  bone-earths.  The  quantity  of  chlorine  according  to  him  is  remark- 
ably high,  0.3-0.5  per  cent,  while  Bertz  2  found  that  the  ash  of  enamel 
was  free  from  chlorine  and  that  dentin  was  very  poor  in  chlorine. 

Carnot,3  who  has  investigated  the  dentin  from  elephants,  has  found  4.3  p.  m. 
calcium  fluoride  in  the  ash.  In  ivory  he  found  only  2  p.  m.  Dentin  from 
elephants  is  rich  in  magnesium  phosphate,  which  is  still  more  abundant  in  ivory. 


1  Gassmann,  Zeitschr.  f.  physiol.  Chem.  70;  Brubacher,  Zeitschr.  f.  biol.  27.  See 
also  Cappezzuoli,  Bioch.  Zeitschr.  16;  Chabrie,  Les  phenomenee  chim.  de  1' ossification, 
Paris,  1895,  65. 

2  See  Maly's  Jahresber.,  30. 
3Compt.  Rend.,  114. 


558  TISSUES  OF  THE  CONNECTIVE   SUBSTANCE. 

Gabriel  found  that  the  quantity  of  fluorine  is  very  small  and 
amounts  to  1  p.  m.  in  ox-teeth.  It  is  no  greater  in  the  teeth  and  enamel 
than  in  the  bones.1  The  same  investigator  found  that  the  amount  of 
phosphates  is  strikingly  small  in  the  enamel,  and  in  the  teeth  consider- 
able lime  is  replaced  by  magnesia.  This  coincides  with  Bertz's  find- 
ings, that  dentin  contains  twice  as  much  magnesia  as  the  enamel. 

According  to  Gassmann,2  the  teeth  among  themselves  have  dif- 
ferent composition,  and  in  man  the  wisdom  teeth  are  poorer  in  organic 
substance  and  richer  in  lime  than  the  canine  teeth.  The  great  tend- 
ency of  the  first  to  caries  is  probably  explained  by  this  fact.  The  reason 
for  the  degeneration  of  the  teeth  is  considered  by  C.  Rose  3  to  be  a  lack 
of  earthy  salts,  and  according  to  him  one  finds  the  best  teeth  in  localities 
where  the  drinking  water  has  high  permanent  hardness. 

IV.     THE   FATTY  TISSUE. 

The  membranes  of  the  fat-cells  withstand  the  action  of  alcohol  and 
ether.  They  are  not  dissolved  by  acetic  acid  or  by  dilute  mineral  acids, 
but  are  dissolved  by  artificial  gastric  juice.  They  may  possibly  con- 
sist of  a  substance  closely  related  to  elastin.  The  fat-cells  contain, 
besides  fat,  a  yellow  pigment  which  in  emaciation  does  not  disappear 
so  rapidly  as  the  fat;  and  this  is  the  reason  that  the  subcutaneous  cel- 
lular tissue  of  an  emaciated  corpse  has  a  dark  orange-red  color.  The 
cells  deficient  in,  or  nearly  free  from  fat,  which  remain  after  the  complete 
disappearance  of  the  latter,  seem  to  have  an  albuminous  protoplasm 
rich  in  water.  Adipose  tissue  is  rich  in  a  fat-splitting  enzyme  and  in 
catalases. 

The  less  water  the  fatty  tissue  contains  the  richer  it  is  in  fat. 
Schulze  and  Reinecke4  found  in  1000  parts: 

Water.  Membrane.  Fat. 

Fatty  tissue  of  oxen 99 . 7  16 . 6  883 . 7 

Fatty  tissue  of  sheep 104.8  16.4  878.8 

Fatty  tissue  of  pigs 64.4  13.6  922.0 

The  fat  contained  in  the  fat-cells  consists  mainly  of  triglycerides  of 
stearic,  palmitic,  and  oleic  acids.  Besides  these,  especially  in  the  less 
solid  kinds  of  fats,  there  are  glycerides  of  other  fatty  acids  (see  Chapter 
IV).     In  all  animal  fats  there  are  besides  these,  as  Fr.  Hofmann  5  has 

1  See  footnote  4,  p.  552. 

2  Zeitschr.  f.  physiol.  Chem.,  55. 

3  Deutech.  Monatsh.  f.  Zahnheilk.,  1908. 

4  Annal.  d.  Chem.  u.  Pharm.,  142. 

6  Ludwig-Festsohrift,  1874,  Leipzig. 


FATTY  TISSUE.  559 

shown,  also  free,  non-volatile  fatty  acids,  although  in  very  small 
amounts. 

Human  fat  is  relatively  rich  in  olein,  the  quantity  in  the  subcutaneous 
fatty  tissue  being  70-80  per  cent  or  more.1  In  new-born  infants  it  is 
poorer  in  oleic  acid  than  in  adults  (Knopfelmacher,  Siegert,  Jaeckle)  ; 
the  quantity  of  olein  increases  until  the  end  of  the  first  year,  when  it  is 
about  the  same  as  in  adults.  The  composition  of  the  fat  in  man  as  well 
as  in  different  individuals  of  the  same  species  of  animals  is  rather  variable, 
a  fact  which  is  probably  dependent  upon  the  food.  According  to  the 
researches  of  Henriques  and  Hansen  the  fat  of  the  subcutaneous  fatty 
tissue  is  richer  in  olein  than  that  of  the  internal  organs;  this  has  also  been 
observed  by  Leick  and  Winkler.2  In  animals  with  a  thick  subcutaneous 
fat  deposit  the  outer  layers,  according  to  Henriques  and  Hansen,  are 
richer  in  olein  than  the  inner  layers.  The  fat  of  cold-blooded  animals 
is  especially  rich  in  olein,  The  fat  of  domestic  animals  has,  according 
to  Amthor  and  Zink,  a  less  oily  consistency  and  a  lower  iodine  and 
acetyl  equivalent  than  the  corresponding  fat  of  wild  animals.  Under 
pathological  conditions  the  fat  may  have  a  markedly  pronounced  varia- 
tion. The  fat  of  lipoma  seems,  from  Jaeckle's  experience,  to  be  poorer 
in  lecithin  than  other  fats. 

The  fat  stored  up  in  the  organs  and  tissues  can  be  changed  somewhat 
by  the  composition  of  the  fat  of  the  food,  still,  according  to  Abderhalden 
and  Brahm,3  the  fat  actually  occurring  in  the  cells  (with  the  exception 
of  the  real  fat  cells)  is  not  dependent  in  its  composition  upon  the  kind 
of  food  fat  taken. 

The  properties  of  fats  in  general,  and  the  three  most  important  varieties 
of  fat  in  particular,  have  been  considered  in  a  previous  chapter,  hence 
the  formation  of  the  adipose  tissue  is  of  chief  interest  at  this  time. 

The  formation  of  fat  in  the  organism  may  occur  in  various  ways.  The 
fat  of  the  animal  body  may  consist  partly  of  fat  absorbed  from  the  food 
and  deposited  in  the  tissues,  and  partly  of  fat  formed  in  the  organism 
from  other  bodies,  such  as  proteins  (?)  or  carbohydrates. 

That  the  fat  from  the  food  which  is  absorbed  in  the  intestinal  canal 
may  be  retained  by  the  tissues  has  been  shown  in  several  ways.  Rad- 
ziejewski,  Lebedeff,  and  Munk  have  fed  dogs  with  various  fats,  such 
as    linseed-oil,    mutton-tallow,    and    rape-seed-oil,    and    have    afterward 


1  See  Jaeckle,  Zeitschr.  f.  physiol.  Chem.,  36  (literature). 

2  Knopfelmacher,  Jahrbuch  f.  Kinderheilkunde  (X.  F.),  45  (older  literature); 
Siegert,  Hofmeister's  Beitriige,  1;  Jaeckle,  Zeitschr.  f.  physiol.  Chem.,  36  (literature); 
Henriques  and  Hansen,  Skand.  Arch.  f.  Physiol.,  11;  Leick  and  Winkler,  Arch.  f.  Path, 
u.  Pharm.,  48. 

8  Zeitschr.  f.  physiol.  Chem.,  65. 


560  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

found  the  administered  fat  in  the  tissues.  Hofmann  starved  dogs 
until  they  appeared  to  have  lost  their  fat,  and  then  fed  them  upon  large 
quantities  of  fat  and  only  little  proteins.  When  the  animals  were  killed 
he  found  so  large  a  quantity  of  fat  that  it  could  not  have  been  formed 
from  the  administered  proteins  alone,  but  the  greater  part  must  have 
been  derived  from  the  fat  of  the  food.  Pettenkofer  and  Voit  arrived 
at  similar  results  in  regard  to  the  action  of  the  absorbed  fats  in  the  organ- 
ism, though  their  experiments  were  of  another  kind.  Munk  found 
that  on  feeding  with  free  fatty  acids,  these  are  deposited  in  the  tissues, 
not,  however,  as  such;  but  they  are  transformed  by  synthesis  with 
glycerin  into  neutral  fats  on  their  passage  from  the  intestine  into  the 
thoracic  duct.  The  connection  between  the  fat  of  the  food,  and  of  the 
body  has  also  been  shown  by  others,  especially  Rosenfeld.  Coro- 
nedi  and  Marchetti  and  in  particular  Winternitz  1  have  shown  that 
iodized  fat  is  taken  up  in  the  intestinal  tract  and  deposited  in  the  various 
organs. 

Proteins  and  carbohydrates  are  considered  as  the  mother-substances 
of  the  fats  formed  in  the  organism. 

The  formation  of  the  so-called  corpse-wax,  adipocere,  which  consists 
of  a  mixture  of  fatty  acids,  ammonia,  and  lime-soaps,  from  parts  of  the 
corpse  rich  in  proteins,  is  sometimes  given  as  a  proof  of  the  formation 
of  fats  from  proteins.  The  accuracy  of  this  view  has,  however,  been  dis- 
puted, and  many  other  explanations  of  the  formation  of  this  substance 
have  been  offered.  According  to  the  experiments  of  Kratter  and 
K.  B.  Lehmann,  it  seems  as  if  it  were  possible  by  experimental  means 
to  convert  animal  tissue  rich  in  proteins  (muscles)  into  adipocere  by  the 
continuous  action  of  water.  Irrespective  of  this,  Salkowski  has  shown 
that  in  the  formation  of  adipocere,  the  fat  itself  takes  part,  in  that  the 
olein  decomposes  with  the  formation  of  solid  fatty  acids,  still  it  must 
be  considered  that  lower  organisms  undoubtedly  take  part  in  its  forma- 
tion. The  production  of  adipocere  as  a  proof  of  the  formation  of  fat 
from  proteins  is  disputed  by  many  investigators  for  this  and  other  reasons. 

Fatty  degeneration  has  been  considered  as  another  proof  of  the 
formation  of  fat  from  proteins.  From  the  investigations  of  Bauer 
on  dogs,  and  Leo  on  frogs,  it  was  assumed  that,  at  least  in  acute  poisoning 
by  phosphorus,  a  fatty  degeneration,  with  the  formation  of  fat  from 
proteins,  takes  place.  Pfluger  has  raised  such  strong  arguments  against 
the  older  researches  as  well  as  the  more  recent  one  of  Polimanti,  who 
claims  to  have  shown  the  formation  of  fat  from  proteins  in  phosphorus 


1  Coronedi  and  Marchetti,  cited  by  Winternitz,  Zeitschr.  f.  physiol.  Chem.,  24, 
A  review  of  the  literature  on  fat  formation  may  be  found  in  Rosenfeld,  Fettbildung. 
in  Ergebnisse  der  Physiologie,  1,  Abt.  1. 


FORMATION  OF  FATS.  561 

poisoning,  that  we  cannot  consider  the  formation  of  fat  as  conclusively 
proved.  The  investigations  of  Lebedeff,  Athanasiu,  Taylor,  Schwalbe 
and  others,  have  shown  that  probably  no  new  formation  of  fat  from 
protein  took  place,  but  rather  a  fat  migration  and  that  this  is  actually 
the  case  has  been  especially  shown  by  Rosenfeld  and  recently  by  Shi- 
bata  [  in  a  conclusive  manner. 

Another  more  direct  proof  of  the  formation  of  fat  from  proteins 
has  been  given  by  Hofmann.  He  experimented  with  fly-maggots. 
A  number  of  these  were  killed  and  the  quantity  of  fat  determined.  The 
remainder  were  allowed  to  develop  in  blood  whose  proportion  of  fat 
had  been  previously  determined,  and  after  a  certain  time  they  were  killed 
and  analyzed.  He  found  in  them  from  seven  to  eleven  times  as  much 
fat  as  was  contained  in  the  maggots  first  analyzed  and  the  blood  taken 
together.  Pfluger  2  has  made  the  objection  that  a  considerable  number 
of  lower  fungi  develop  in  the  blood  under  these  conditions,  in  whose 
cell-body  fats  and  carbohydrates  are  formed  from  the  different  con- 
stituents of  the  blood  and  their  decomposition  products,  and  that  these 
serve  as  food  for  the  maggots. 

Weinland  3  has  observed  the  formation  of  higher  non-volatile  fatty 
acids  in  the  Calliphora  larvae  when  they  were  rubbed  to  a  homogeneous 
paste  after  the  addition  of  Witte's  peptone.  This  experiment  shows  a 
formation  of  fat  from  protein,  but  cannot  be  considered  as  quite  con- 
clusive. 

As  a  more  convincing  proof  of  fat  formation  from  proteins,  the 
investigations  of  Pettenkofer  and  Voit  are  often  quoted.  These 
investigators  fed  dogs  with  large  quantities  of  meat  containing  the  least 
possible  proportion  of  fat,  and  found  all  of  the  nitrogen  in  the  excreta, 
but  only  a  part  of  the  carbon.  As  an  explanation  of  these  conditions 
it  has  been  assumed  that  the  protein  of  the  organisms  splits  into  a 
nitrogenized  and  a  non-nitrogenized  part,  the  former  changing  into  the 
nitrogenized  final  product,  urea,  and  like  products,  and  the  other  part, 
on  the  contrary,  being  retained  in  the  organism  as  fat  (Pettenkofer 
and  Voit). 

Pfluger  has  arrived  at  the  following  conclusion  by  an  exhaustive 
criticism  of  Pettenkofer  and  Voit's  experiments  and  a  careful  recal- 
culation of  their  balance-sheet;  that  these  very  meritorious  invest  iga- 

1  Bauer,  Zeitschr.  f.  Biologie,  7;  Leo,  Zeitschr.  f.  physiol.  Chera.,  9;  Polimanti, 
Pfliiger's  Arch.,  70;  Pfluger,  ibid.,  51  (literature  on  the  formation  of  fat  from  protein) 
and  71;  Athanasiu,  ibid.,  74;  Taylor,  Journ.  Exp.  Medicine,  4;  see  also  footnote  2, 
p.  384;  Shibata,Bioch.  Zeitschr.,  37,  which  contains  the  literature;  Rosenfeld,  Ergebn. 
d.  Physiol.,  1. 

2  See  Rosenfeld,  Fettbildung,  Ergebnisse  der  Physiologie,  1,  Abt.  1. 

3  Zeitschr.  f.  Biol.,  51  and  52. 


562  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

tions,  which  were  continued  for  a  series  of  years,  were  subject  to  such 
great  defects  that  they  are  not  conclusive  as  to  the  formation  of  fat 
from  proteins.  He  especially  emphasizes  the  fact  that  these  investigators 
started  from  a  wrong  assumption  as  to  the  elementary  composition  of 
the  meat,  and  that  the  quantity  of  nitrogen  assumed  by  them  was  too 
low  and  the  quantity  of  carbon  too  high.  The  relation  of  nitrogen  to 
carbon  in  meat  poor  in  fat  was  assumed  by  Voit  to  be  as  1:3.68,  while 
according  to  Pfluger  it  is  1:3.22  for  fat-free  meat  after  deducting  the 
glycogen,  and  according  to  Rubner  1:3.28  without  deducting  the  gly- 
cogen. On  recalculation  of  the  figures,  using  these  coefficients,  Pfluger 
has  arrived  at  the  conclusion  that  the  assumption  as  to  the  formation 
of  fat  from  proteins  finds  no  support  in  these  experiments. 

In  opposition  to  these  objections,  E.  Voit  and  M.  Cremer  have  made 
new  feeding  experiments,  to  show  the  formation  of  fat  from  proteins, 
but  the  proof  of  these  recent  investigations  has  been  disputed  by  Pfluger. 
On  feeding  a  dog  on  meat  poor  in  fat  (containing  a  known  quantity  of 
ether  extractives,  glycogen,  nitrogen,  water,  and  ash),  Kumagawa  1 
could  not  prove  the  formation  of  fat  from  protein.  According  to  him 
the  animal  body  under  normal  conditions  has  not  the  power  of  forming 
fat  from  protein. 

Several  French  investigators,  especially  Chauveau,  Gautier,  and 
Kaufmann,2  consider  the  formation  of  fat  from  proteins  as  positively 
proved.  Kaufmann  has  recently  substantiated  this  view  by  a  method 
which  will  be  spoken  of  in  detail  in  Chapter  XVII,  in  which  he  studied 
the  nitrogen  elimination  and  the  respiratory  gas  exchange  in  conjunction 
with  the  simultaneous  formation  of  heat. 

As  we  are  agreed  that  carbohydrates  and  glycogen,  as  well  as  sugar, 
can  be  formed  from  proteins,  the  fact  cannot  be  denied  that  possibly 
an  indirect  formation  of  fat  from  proteins,  with  a  carbohydrate  as  an 
intermediate  step,  can  take  place.  The  possibility  of  a  direct  fat  for- 
mation from  proteins  without  the  carbohydrate  as  intermediary  must 
also  be  generally  admitted,  although  such  a  formation  has  not  been 
conclusively  proved. 

According  to  Chauveau  and  Kaufmann,  in  the  direct  formation  of 
fat  from  proteins,  the  fat  is  formed  besides  urea,  carbon  dioxide,  and 
water,  as  an  intermediary  product  in  the  oxidation  of  the  proteins,  while 
Gautier  considers  the  formation  of  fat  from  proteins  as  a  cleavage 
without  the  taking  up  of  oxygen.  If  fat  is  formed  from  protein  in  the 
animal  body,  then  such  formation  is  not  a  splitting  off  of  fat  from  the 


<■  Rosenfeld,  Fettbildung,  Ergebnisse  der  Physiologre,  1,  Abt.  1. 
2  Kaufniann,  Arch,  de  physiol.,  ('>)  8,  where  the  works  of  Chauveau  and  Gautier 
are  cited. 


FORMATION  OF  FATS.  503 

proteins,  but  rather  a  synthesis  from  primarily  formed  cleavage  products 

of  proteins  which  are  poor  in  carbon. 

The  formation  "/  foi  from  carbohydrates  in  the  animal  body' was 
first  suggested  by  Liebig.  This  was  opposed  for  some  time,  and  until 
lately  it  was  the  general  opinion  that  a  direct  formation  of  fat  from 
carbohydrates  not  only  had  not  been  proved,  but  also  that  it  was 
improbable.  The  undoubtedly  great  influence  of  the  carbohydrates  on 
the  formation  of  fat  as  observed  and  proved  by  Liebig  was  explained 
by  the  statement,  that  the  carbohydrates  were  consumed  instead  of 
the  absorbed  fat  or  that  derived  from  the  proteins,  hence  they  have  a 
sparing  action  on  the  fat.  By  means  of  a  series  of  nutrition  experiments  l 
with  different  animals,  with  foods  especially  rich  in  carbohydrates  it  has 
been  apparently  proved  that  a  direct  formation  of  fat  from  carbohydrates 
does  actually  occur.  The  processes  by  which  this  formation  takes  place 
are  still  unknown.  As  the  carbohydrates  do  not  contain  such  com- 
plicated carbon  chains  as  the  fats,  the  formation  of  fat  from  carbohydrates 
must  consist  of  a  synthesis,  in  which  the  group  CHOH  is  converted  into 
CH2;  hence  a  reduction  must  occur. 

After  feeding  with  very  large  quantities  of  carbohydrates  the  relation  between 
the  inspired  oxygen  and  the  expired  carbon  dioxide,  i.e.,  the  respiratory  quotient 

—p,  was  found  greater  than  1  in  certain  cases  (H.\xRiOTand  Richet,  Bleibtreu, 

Kaufmaxx.  Laclaxie  -).  This  is  explained  by  the  assumption  that  the  fat 
is  formed  from  the  carbohydrate  by  a  cleavage  setting  free  carbon  dioxide  and  water 
without  taking  up  oxygen.  This  increase  in  the  respiratory  quotient  also  depends 
in  part  on  the  increased  combustion  of  the  carbohydrate. 

When  food  contains  an  excess  of  fat,  the  superfluous  amount  is  stored 
up  in  the  fatty  tissue,  and  on  partaking  of  food  deficient  in  fat  this 
accumulation  is  quickly  exhausted;  and  it  is  very  probable  that  the 
lipase  is  of  importance  here,  as  Loevenhart3  has  found  that  all  over 
the  body  where  fat  is  deposited  in  large  amounts  lipase  also  occurs  in 
considerable  amounts.  There  is  perhaps  not  one  of  the  various  tissues 
that  decreases  so  much  in  starvation  as  the  fatty  tissue.  The  organism, 
then,  possesses  in  this  tissue  a  depot  where  there  is  stored,  during  proper 


1  Lawes  and  Gilbert,  Phil.  Transactions,  1859,  part  2;  Soxhlet,  see  Mary's  Jahresber., 
11,  51;  Tscherwinsky,  Landwirthsch.  Versuchsstaat,  29  (cited  from  Maly's  Jahresber., 
13);  Meissl  and  Stromer,  Wien.  Sitzungsber.,  88,  Abt.  3;  Schultze,  Maly's 
Jahresber.,  11,  47;  Chaniewski,  Zeitschr.  f.  Biologie,  20;  Voit  and  Lehmann,  see  C. 
v.  Voit,  Sitzungsber,  d.  k.  bayer.  Akad.  d.  Wissensch.,  1885;  I.  Munk,  Virchow's  Arch., 
101;  Rubner,  Zeitschr.  f.  Biologie,  22;  Lummert,  Pfliiger's  Arch.,  71. 

2  Hanriot  and  Richet,  Annal.  de  Chim.  et  de  Phys.  (6),  22;  Bleibtreu,  Pfliiger's 
Arch.,  56  and  So;  Kaufmann,  Arch,  de  Physiol.  (5),  8;  Laulanic,  ibid.,  791. 

3  Amer.  Journ.  of  Physiol.,  6. 


564  TISSUES  OF  THE  CONNECTIVE  SUBSTANCE. 

alimentation,  a  nutritive  substance  of  great  importance  in  the  develop- 
ment of  heat  and  vital  force,  which  substance,  on  insufficient  nutrition, 
is  given  up  as  may  be  needed.  On  account  of  their  low  conducting 
power,  the  fatty  tissues  become  of  great  importance  in  regulating  the 
loss  of  heat  from  the  body.  They  also  serve  to  fill  cavities  and  act  as 
a  protection  and  support  to  certain  internal  organs. 


CHAPTER  X. 
MUSCLES. 

STRIATED    MUSCLES. 

In  the  study  of  the  muscles  the  chief  problem  for  physiological  chem- 
istry is  to  isolate  their  different  morphological  elements  and  to  investigate 
each  element  separately.  By  reason  of  the  complicated  structure  of 
the  muscles  this  has  been  thus  far  almost  impossible,  and  we  must  be 
satisfied  at  the  present  time  with  a  few  microchemical  reactions  in  the 
investigation  of  the  chemical  composition  of  the  muscular  fibers. 

Each  muscle-tube  or  each  muscle-fiber  consists  of  a  sheath,  the 
sarcolemma,  which  seems  to  be  composed  of  a  substance  similar  to 
elastin,  and  containing  a  large  proportion  of  protein.  This  last,  which 
in  life  possesses  the  power  of  contractility,  has  in  the  inactive  muscle 
an  alkaline  reaction,  or,  more  correctly  speaking,  an  amphoteric  reac- 
tion with  a  predominating  action  on  red  litmus  paper.  Rohmann 
found  that  the  fresh,  inactive  muscle  shows  an  alkaline  reaction  with 
red  lacmoid,  and  an  acid  reaction  with  brown  turmeric.  From  the  effect 
of  various  acids  and  salts  on  these  coloring-matters,  he  concludes  that  the 
alkalinity  of  the  fresh  muscle  with  lacmoid  is  due  to  sodium  bicarbonate, 
diphosphate,  and  probably  also  to  an  alkaline  combination  of  protein 
bodies,  and  the  acid  reaction  with  turmeric,  on  the  contrary,  to  chiefly 
monophosphate.  The  dead  muscle  has  an  acid  reaction,  or,  more  cor- 
rectly, the  acidity  with  turmeric  increases  on  the  decease  of  the  muscle, 
and  the  alkalinity  with  lacmoid  decreases.  The  difference  depends  on  the 
presence  of  a  larger  quantity  of  monophosphate  in  the  dead  muscle,  and 
according  to  Rohmann  free  lactic  acid  is  found  in  neither  the  one  case 
nor  the  other.1 

If  the  somewhat  disputed  statements  relative  to  the  finer  structure 
of  the  muscles  are  disregarded,  one  can  differentiate  in  the  striated  muscles 
between  the  two  chief  components,  the  doubly  refracting — anisotropous 
— and  the  singly  refracting — isotropous — substance.  Both  contain 
abundance  of  protein,  which  form  the  chief  part  of  the  solids  of  the  muscles. 

1  The  various  reports  in  regard  to  the  reaction  of  the  muscles  and  the  cause  thereof 
are  conflicting.  See  Rohmann,  Pfluger's  Arch.,  50  and  55;  Heffter,  Arch.  f.  exp. 
Path.  u.  Pharm.,  31  and  38.     These  references  contain  the  pertinent  literature. 

065 


5€6  MUSCLES. 

If  the  muscular  fibers  are  treated  with  reagents  which  dissolve  proteins, 
such  as  dilute  hydrochloric  acid,  soda  solution,  or  gastric  juice,  they  swell  greatly 
and  break  up  into  "  Bowman's  disks."  By  the  action  of  alcohol,  chromic  acid, 
boiling  water,  or  in  general  such  reagents  as  cause  a  shrinking,  the  fibers  split 
longitudinally  into  fibrils;  and  this  behavior  shows  that  several  chemically  dif- 
ferent substances  of  various  solubilities  enter  into  the  construction  of  the  muscular 
fibers. 

The  protein  myosin  is  generally  considered  as  the  principal  constituent  of  the 
diagonal  disks,  while  the  isotropous  substance  contains  the  chief  mass  of  the 
other  proteins  of  the  muscles  as  well  as  the  chief  portion  of  the  extractives. 
According  to  the  observations  of  Danilewsky,  confirmed  by  J.  Holmgren,1 
myosin  may  be  completely  extracted  from  the  muscle  without  changing  its  struc- 
ture, by  means  of  a  5-per  cent  solution  of  ammonium  chloride,  which  fact  con- 
flicts with  the  above  view.  Danilewsky  claims  that  another  protein-like  sub- 
stance, insoluble  in  ammonium  chloride  and  only  swelling  up  therein,  enters  essen- 
tially into  the  structure  of  the  muscles.  The  proteins,  which  form^the  principal 
part  of  the  solids  of  the  muscles,  are  of  the  greatest  importance. 

Proteins  of  the  Muscles. 

Like  the  blood  which  contains  a  fluid,  the  blood-plasma,  which  sponta- 
neously coagulates,  separating  fibrin  and  yielding  blood-serum,  so  also 
the  living  muscle,  at  least  of  cold-blooded  animals,  contains,  as  first 
shown  by  Kuhne,  a  spontaneously  coagulating  liquid,  the  muscle-plasma, 
which  coagulates  quickly,  separating  a  protein  body,  myosin,  and  yield- 
ing also  a  serum.  That  liquid  which  is  obtained  by  pressing  the  living 
muicle  is  called  muscle-plasma,  while  that  obtained  from  the  dead 
muscle  is  called  muscle-serum.  These  two  fluids  contain  at  least  in  part 
different  protein  bodies. 

Muscle-plasma  was  first  prepared  by  Kuhne  from  frog-muscles,  and  later 
by  Halliburton,  according  to  the  same  method,  from  the  muscles  of  warm- 
blooded animals,  especially  rabbits.  The  principle  of  this  method  is  as  follows: 
The  blood  is  removed  from  the  muscles  immediately  after  the  death  of  the  animal 
by  passing  through  them  a  strongly  cooled  common-salt  solution  of  5-6  p.  m. 
Then  the  muscles  are  quickly  cut  and  immediately  frozen  thoroughly  so  that 
they  can  be  ground  in  this  state  to  a  fine  mass — "  muscle-snow."  This  pulp  is 
strongly  pressed  in  the  cold,  and  the  liquid  which  exudes  is  called  muscle-plasma. 
According  to  v.  Furth  2  this  cooling  or  freezing  is  not  necessary.  It  is  sufficient 
to  extract  the  muscle  free  from  blood,  as  above  directed,  with  a  6  p.  m.  common 
salt  solution. 

Muscle-plasma  forms  a  yellow  to  brownish-colored  fluid  with  an 
alkaline  reaction.  It  varies  in  different  animals.  Muscle-plasma  from 
the  frog  spontaneously  coagulates,  slowly,  at  a  little  above  0°  C,  but  more 

1  Danilewsky,  Zeitschr.  f.  physiol.  Chem.,  7;  J.  Holmgren,  Maly's  Jahresber.,  23. 

2  See  Kuhne,  Untersuchungen  iiber  das  Protoplasma,  (Leipzig,  1864),  2;  Hallibur- 
ton, Journ.  of  Physiol.,  8;  v.  Furth,  Arch.  f.  exp.  Path.  u.  Pharm.,  36  and  37;  Hof- 
meister's  Beftrage,  3,  and  Ergebnisse  der  Physiologie,  1,  Abt.  1;  Stewart  and  Soll- 
mann,  Journ.  of  Physiol.,  24. 


•  PROTEINS  OF  THE  MUSCLES.  567 

quickly  at  the  temperature  of  the  body.  Muscle-plasma  from  mammals 
coagulates  slowly,  according  to  vv.  Furth,  even  at  the  temperature  of 
the  room,  though  only  slightly,  and  it  can  hardly  be  considered  as  a 
process  comparable  with  the  coagulation  of  the  blood.  Indeed  the  ques- 
tion may  be  asked  whether  a  true  muscle-plasma  does  exist  in  warm- 
blooded animals,  or  whether  the  rluid  obtained  from  such  muscles 
exactly  represents  the  plasma  of  the  living  muscle.  According  to  Kuhne 
and  v.  Furth  the  reaction  remains  alkaline  during  coagulation,  while 
Halliburton,  Stewart  and  Sollmann  find  that  it  becomes  acid. 
Earlier  investigators  held  that  the  clot  consists  of  a  globulin  called 
myosin,  while  v.  Furth  claims  that  it  consists  of  two  coagulated  pro- 
teins, myosin-fibrin  and  myogen-fibrin. 

The  study  of  the  proteins  of  the  muscles,  as  well  as  their  nomen- 
clature, has  changed  markedly  in  the  last  few  years,  and  it  is  questionable 
whether  an  essential  difference  exists  between  the  proteins  of  the  muscle- 
plasma  and  the  muscle-serum  of  warm-blooded  animals.  Nevertheless 
it  is  necessary  to  discuss  separately  the  proteins  of  the  dead  muscle  as 
well  as  those  of  the  muscle-plasma. 

The  proteins  of  the  dead  muscle  are  in  part  soluble  in  water  or  dilute 
salt  solutions,  and  in  part  are  insoluble  therein.  Myosin  and  musculin 
and  also  myoglobulin  and  myoalbumin,  which  exist  to  a  very  slight 
extent  and  are  perhaps  only  derived  from  the  remaining  lymph,  belong 
to  the  first  group,  and  the  stroma  substances  of  the  muscle-tubes  belong 
to  the  second  group. 

Myosin  was  first  discovered  by  Kuhne,  and  constitutes  the  principal 
mass  of  the  soluble  proteins  of  the  dead  muscle.  It  is  generally  considered 
as  the  most  essential  coagulation  product  of  muscle-plasma.  The  name 
myosin,  Kuhne  also  gives  to  the  mother-substance  of  the  plasma-clot, 
and  this  mother-substance  forms,  according  to  certain  investigators, 
the  principal  mass  of  contractile  protoplasm.  The  findings  as  to  the  oc- 
currence cf  myosin  in  other  organs  besides  the  muscles  require  further 
confirmation.  The  quantity  of  myosin  in  the  muscles  of  different  animals 
varies,  according  to  Danilewsky,1  between  30  and  110  p.  m. 

Myosin,  as  obtained  from  dead  muscles,  is  a  globulin  whose  elementary 
composition,  according  to  Chittenden  and  Cummins.2  is,  on  an  average, 
the  following:  C  52.28,  H  7.11,  N  16.77,  S  1.27,  O  22.03  per  cent.  If 
the  myosin  separates  as  fibers,  or  if  a  myosin  solution  with  a  minimum 
quantity  of  alkali  is  allowed  to  evaporate  to  a  gelatinous  mass  on  a 
microscope-slide,  doubly  refracting  myosin  may  be  obtained.  Myosin 
has  the  general  properties  of  the  globulins  and  is  readily  converted  into 

1  Zeitsohr.  f.  physiol.  Chem.,  7. 

5  Studies  from  the  Physiol.  Chem.  Laboratory  of  Yale  College,  New  Haven,  3,  115. 


568  MUSCLES. 

albuminates  by  dilute  acids  or  alkalies.  It  is  completely  precipitated 
upon  saturation  with  NaCl,  also  by  MgSO-i,  in  a  solution  containing 
94  per  cent  of  the  salt  with  its  water  of  crystallization  (Halliburton). 
The  precipitated  myosin  readily  becomes  insoluble.  Like  fibrinogen  it 
coagulates  at  56°  C.  in  a  solution  containing  common  salt,  but  differs 
irom  it,  since  under  no  circumstances  can  it  be  converted  into  fibrin. 
The  coagulation  temperature,  according  to  Chittenden  and  Cummins, 
not  only  varies  for  myosins  of  different  origin,  but  also  for  the  same 
myosin  in  different  salt  solutions. 

Myosin  may  be  prepared  in  the  following  way,  as  suggested  by  Halli- 
burton: The  muscle  is  first  extracted  by  a  5-per  cent  magensium- 
sulphate  solution,  and  by  fractional  precipitation  with  magnesium  sul- 
phate the  musculin  and  then  the  myosin  are  precipitated  (see  Halli- 
burton,  1.  c). 

The  older  and  perhaps  the  usual  method  of  preparation  consists, 
according  to  Danilewsky,1  in  extracting  the  muscle  with  a  5-10  per  cent 
ammonium-chloride  solution,  precipitating  the  myosin  from  the  filtrate 
by  strongly  diluting  with  water,  and  redissolving  the  precipitate  in  ammo- 
nium-chloride solution,  and  the  myosin  obtained  from  this  solution  is 
reprecipitated  either  by  diluting  with  water  or  by  removing  the  salt 
by  dialysis. 

Musculin,2  called  paramyosinogen  by  Halliburton,  and  myosin 
by  v.  Furth,  is  a  globulin  which  is  characterized  by  its  low  coagulation 
temperature,  in  frogs  below  40°,  in  mammalia  42-48°,  and  in  birds  about 
51°  C,  and  which  may  vary  in  different  species  of  animals.  It  is  more 
easily  precipitated  than  myosin  by  NaCl  or  MgSC>4  (50  per  cent  salt, 
including  water  of  crystallization).  According  to  v.  Furth  it  is  precipi- 
tated by  ammonium  sulphate  with  a  concentration  of  12-24  per  cent 
salt.  If  the  dead  muscle  is  extracted  with  water  a  part  of  the  musculin 
goes  into  solution,  and  may  be  precipitated  therefrom  by  carefully 
acidifying.  It  separates  from  a  dilute  salt  solution  on  dialysis.  Mus- 
culin readily  passes  into  an  insoluble  modification  which  v.  Furth  calls 
myosin  fibrin.  Musculin  is  called  myosin  by  v.  Furth,  as  he  considers 
it  nothing  but  myosin.  As  musculin  has  a  lower  coagulation  temper- 
ature and  has  other  precipitating  properties  for  neutral  salts  than  the 
older  substance  called  myosin,  it  is  difficult  to  accept  this  view. 

Myoglobulin.  After  the  separation  of  the  musculin  and  the  myosin  from  the 
salt  extract  of  the  muscle  by  means  of  MgSO-i,  the  myoglobulin  may  be  precipitated 

1  Zeitschr.  f.  physiol.  Chem.,  5,  158. 

2  As  we  have  up  to  the  present  no  conclusive  basis  for  the  identity  of  the  globulins 
railed  myosin  and  paramyosinogen,  and  also  as  the  use  of  the  name  myosin  for  the 
last-mentioned  substance  may  readily  cause  confusion,  the  author  does  not  feel 
kistified  in  dropping  the  old  name  musculin  (Nasse). 


PROTEINS  OF  THE  MUSCLES.  569 

by  saturating  the  filtrate  with  the  salt.  It  is  similar  to  serglobulin,  but  coagu- 
lates at  63°  C.  (Halliburton).  MyoaUmmin,  or  muscle-albumin,  seems  to 
be  identical  with  seralbumin   (seralbumin  a,  according  to  Hallibi  bton),  and 

probably  originates  only  from  the  blood  or  the  lymph.     Proteoses  and  peptones 

do  not  seem  to  exist  in  the  fresh  muscles. 

Alter  the  complete  removal  from  the  muscle  of  all  protein  bodies  which  are 
soluble  in  water  and  ammonium  chloride,  an  insoluble  protein  remains  which 
only  swells  in  ammonium-chloride  solution,  and  which  forms  with  the  other  insoluble 
constituents  of  the  muscular  fiber  the  "  muscle-stroma."  According  to  Danilew- 
sky  the  amount  of  such  stroma  substance  is  connected  with  the  muscle  activity. 
He  maintains  that  the  muscles  contain  a  greater  amount  of  this  substance,  com- 
pared with  the  myosin  present,  when  the  muscles  are  quickly  contracted  and 
relaxed,  the  correctness  of  which  report  has  recently  been  disputed  by  Saxl.1 

According  to  J.  Holmgren,2  this  stroma  substance  does  not  belong  to  either 
the  Qucleoalbumin  or  the  nucleoprotein  group.  It  is  not  a  glucoproteid,  as  it 
does  not  yield  a  reducing  substance  when  boiled  with  dilute  mineral  acids.  It  is 
very  similar  to  the  coagulable  proteins,  and  dissolves  in  dilute  alkalies,  forming 
an  albuminate.  The  elementary  composition  of  this  substance  is  almost  the  same 
as  that  of  myosin.  There  is  no  doubt  that  the  insjluble  substances,  myofiorin 
and  myosin  fibrin,  which  are  formed,  according  to  v.  Fukth,  in  the  coagulation  of 
the  plasma,  also  occur  among  the  stroma  substances.  When  the  muscles  are 
previously  extracted  with  water,  the  stroma  substances  also  contain  a  part  of  the 
myosin  hereby  made  insoluble.  The  observations  of  Saxl  on  rabbits'  muscles 
agree  with  this  view  that  the  fresh  muscle  after  work  contains  11.5-21.6  per  cent 
of  the  total  protein  in  an  insoluble  form,  while  the  muscle  after  rigor  mortis  con- 
tains on  the  contrary  71.5-73.2  per  cent. 

To  the  proteins  insoluble  in  water,  and  neutral  salts,  belongs  the 
nucleoprotein  detected  by  Pekelharing,  which  occurs  as  traces  and  is 
soluble  in  faintly  alkaline  water,  and  which  probably  originates  from 
the  muscle  nuclei.  According  to  Bottazzi  and  Ducceschi  3  the  heart 
muscle  is  richer  in  nucleoprotein  than  the  skeletal  muscle. 

Muscle-syntonin,  which  may  be  obtained  by  extracting  the  muscles  with 
hydrochloric  acid  of  1  p.m.,  and  which,  according  to  K.  Morner,  is  less  soluble 
and  has  a  greater  aptitude  to  precipitate  than  other  acid  albumins,  seems  not 
to  occur  preformed  in  the  muscles.  Heubner's4  mytolin  is  modified  muscle- 
proteid,  chiefly  myosin,  which  has  lost  a  part  of  its  sulphur  by  the  action  of  alkali. 

Proteins  of  the  Muscle-plasma.  As  above  stated,  myosin  was  ordi- 
narily considered  as  the  coagulated  modification  of  a  soluble  protein 
existing  in  the  muscle-plasma.  As  in  blood-plasma  there  is  present 
a  mother-substance  of  fibrin,  fibrinogen,  so  also  there  exists  in  the 
muscle-plasma  a  mother-substance  of  myosin,  a  soluble  myosin  or  a 
myosinogen.     This  body  has  not  thus  far  been  isolated  with  certainty. 


1  Hofmeister's  Breitage,  9. 

2  See  footnote  1,  p.  566. 

3  Pekelharing,  Zeitschr.  f.  physiol.  Chem.,  22;  Bottazzi  and  Ducceschi,  Centraibl. 
f.  Physiol.,  12. 

4  Arch.  f.  exp.  Pathol,  u.  Pharm.,  53. 


570  MUSCLES 

Halliburton,  who  has  detected  in  the  muscles  an  enzyme-like  substance, 
11  myosin  ferment,"  which  is  related  to  fibrin  ferment  but  is  not  identical  with  it, 
has  also  found  that  a  solution  of  purified  myosin,  in  dilute  salt  solution  (5  per 
cent  MgS04),  and  sufficiently  diluted  with  water,  coagulates  after  a  certain 
time,  and  at  the  same  time  becomes  acid,  and  a  typical  myosin-clot  separates. 
This  coagulation,  which  is  accelerated  by  warming  or  by  the  addition  of  myosin ' 
ferment,  is,  according  to  Halliburton,  a  process  analogous  to  the  coagulation 
of  the  muscle-plasma.  According  to  this  same  investigator,  myosin  when  dis- 
solved in  water  by  the  aid  of  a  neutral  salt  is  reconverted  into  nvyosinogen,  while 
after  diluting  with  water  myosin  is  again  produced  from  the  nvyosinogen.  The 
musculin  (paramyosinogen)  is  carried  down,  according  to  Halliburton,  with  the 
myosin-clot,  but  has  nothing  to  do  with  the  coagulation,  as  the  myosin-clot  also 
forms  in  the  absence  of  musculin,  and  this  last  is  not  changed  into  myosin. 

Besides  the  traces  of  globulin  and  albumin,  which  perhaps  do  not 
belong  to  the  muscle-plasma,  there  occur  in  mammals,  according  to 
v.  Furth,  two  proteins,  namely,  musculin  (myosin  according  to  v.  Furth) 
and  myogen. 

Musculin  (Nasse)  =  paramyosinogen  (Halliburton)  =  myosin  (v. 
Furth)  forms  about  20  per  cent  of  the  total  proteins  of  the  muscle- 
plasma  of  rabbits.  Its  properties  have  already  been  given,  and  it  is 
sufficient  to  remark  that  its  solutions  beccme  cloudy  on  standing,  and 
a  precipitate  of  myosin  fibrin  occurs,  which  is  insoluble  in  salt  solutions. 

Myogen,  or  myosinogen  (Halliburton),  forms  the  chief  mass, 
75-80  per  cent,  of  the  proteins  of  rabbit  muscle-plasma.  It  does  not 
separate  from  its  solutions  on  dialysis  and  is  not  a  true  globulin,  but 
a  protein  sui  generis.  It  coagulates  at  55-65°  C.  and  is  precipitated 
in  the  presence  of  26-40  per  cent  ammonium  sulphate.  Myogen  solu- 
tions are  precipitated  by  acetic  acid  only  in  the  presence  of  some  salt. 
It  is  converted  into  an  albuminate  by  alkalies,  this  albuminate  being 
precipitable  by  ammonium  chloride.  Myogen  passes  spontaneously, 
especially  with  higher  temperatures  as  well  as  in  the  presence  of  salt, 
into  an  insoluble  modification,  myogen  fibrin.  A  protein,  coagulating 
at  30-40°  C,  soluble  myogen  fibrin,  is  produced  as  a  soluble  intermediate 
step.  This  substance  occurs  to  a  considerable  extent  in  native  frog- 
muscle  plasma.  It  does  not  always  occur  in  the  muscle-plasma  of 
warm-blooded  animals,  and  when  it  does  it  is  present  only  to  a  slight 
extent.  It  can  be  separated  by  precipitating  with  salt  or  by  diffusion. 
Halliburton's  assumption  as  to  the  action  of  a  special  myosin  ferment 
has  not  sufficient  basis,  according  to  v.  Furth,  nor  has  the  often-admitted 
analogy  with  the  coagulation  of  the  blood.  The  difference  between 
the  musculin  and  the  myogen  in  their  becoming  insoluble  is  that  the 
musculin   passes   into   myosin   fibrin   without   any   soluble   intermediate 

Step-. 

Myogen  may  be  prepared,  according  to  v.  Furth,  by  heating,  for  a 
short  time,  the  dialyzed  and  filtered  plasma  to  52°  C.,  separating  it  in 


PROTEINS  OF  THE  MUSCLES.  571 

this  way  from  the  rest  of  the  musculin.  The  myogen  exists  in  the  new 
nitrate  and  can  be  precipitated  by  ammonium  sulphate.  The  musculin 
may  also  be  removed  by  adding  28  per  cent  ammonium  sulphate;  and  then 
precipitating  the  myogen  from  the  filtrate  by  saturating  with  the  salt. 

Stewart  and  Sollmann  admit  of  only  two  soluble  proteins  in  the  muscles. 
One  is  the  paramyosinogen,  which  is  the  same  as  v.  Furth's  myosin+the  soluble 
myogen  fibrin.  The  other  they  call  myosinogen,  which  corresponds  to  v.  Furth's 
myogen  or  to  Halliburton's  myosinogen-)- my oglobulin.  It  is  a  typical  globulin 
which  coagulates  at  50-60°  C.  The  paramyosinogen  as  well  as  the  myosinogen 
is  readily  converted  into  an  insoluble  modification,  myosin.  The  myosin  of  the 
above  investigators  is  the  same  as  v.  Furth's  myosin  fibrin + myogen  fibrin,  and 
corresponds,  it  seems,  also  to  myosin  mixed  with  paramyosinogen  (Halliburton). 
Stewart  and  Sollmann  differ  from  Halliburton  in  considering  that  paramy- 
osinogen also  coagulates  and  is  converted  into  myosin.  According  to  them 
myosin  is  also  insoluble  in  a  NaCl  solution. 

The  views  of  the  various  investigators  differ  so  essentially  and  the 
nomenclature  is  so  complicated  (three  different  things  are  designated 
by  the  name  myosin)  that  it  is  extremely  difficult  to  give  any  correct 
review  of  the  various  opinions.1  Thorough  investigations  on  this  subject 
are  very  necessary. 

Myoproteid  is  a  protein  found  by  v.  Furth  in  the  plasma  from  fish-muscles. 
It  does  not  coagulate  on  boiling,  is  precipitated  by  acetic  acid,  and  is  considered 
as  a  compound  protein  by  v.  Furth. 

In  connection  with  v.  Furth's  work,  Przibram  has  carried  on  ivestiga- 
tions  on  the  occurrence  of  muscle-proteins  in  various  classes  of  animals.  The 
myosin  (v.  Furth)  and  myogen  occur  in  all  classes  of  vertebrates;  the  myogen 
is  always  absent  in  the  invertebrates.  Myoproteid  occurs,  at  least  in  considerable 
quantity,  only  in  fishes.  In  the  muscle  after  cutting  the  nerve,  Steyrer  2  found 
somewhat  more  musculin  and  less  myogen  in  the  muscle-juice  than  in  the  normal 
muscle. 

Muscle-pigments.  There  is  no  question  that  the  red  color  of  the 
muscles,  even  when  completely  freed  from  blood,  depends  in  part  on 
haemoglobin.  K.  Morner  has  shown  that  muscle-haemoglobin  is  not 
quite  identical  with  blood-haemoglobin.  The  statement  of  MacMunn 
that  in  the  muscles  another  pigment  occurs  which  is  allied  to  haemo- 
chromogen,  and  called  myohcematifi  by  him,  has  not  been  substantiated, 
at  least  for  muscles  of  higher  animals  (Levy  and  Morner3).  MacMunn 
claims  that  myohaematin  occurs  in  the  muscles  of  insects,  which  do  not 
contain  any  haemoglobin.  The  reddish-yellow  coloring-matter  of  the 
muscles  of  the  salmon  has  been  little  studied. 

1  For  these  reasons  the  author  is  not  sure  whether  he  has  understood  and  correctly 
given  the  work  of  the  different  investigators. 

2  Przibram,  Hofmeister's  Beitrage,  2;  Steyrer,  ibid.,  4. 

'See  MacMunn,  Phil.  Trans,  of  Roy.  Soc,  177,  part  1,  Journ.  of  Physiol.,  8  and 
Zeitsehr.  f.  Physiol.  Chem.,  13;  Levy,  ibid.,  13;  K.  Morner,  Nord.  Med.  Archiv.  Fest- 
band.,  1897,  and  Maly's  Jahresber.,  27. 


572  MUSCLES. 

Various  enzymes  have  been  found  in  the  muscles.  To  these  belong 
(besides  traces  of  fibrin  ferment  and  myosin  ferment?)  the  catalases  and 
oxidases,  which  occur  only  to  a  slight  extent  and  the  glycolytic  enzyme 
(Chapter  VII).  An  amylolytic  and  a  proteolytic  enzyme  (Hedin  and 
Rowland  x)  have  also  been  found,  and  the  hydrolytic  and  oxidizing 
enzymes  (Chapter  XIV)  active  in  the  formation  and  destruction  of  uric 
acid  are  also  present. 

Extractive  Bodies  of  the  Muscles. 

The  nitrogenous  extractives  in  the  muscles  of  higher  animals  con- 
sist chiefly  of  creatine  and  creatinine  (especially  in  fishes)  and  carnosine. 
To  these  also  belong  inosinic  acid  (and  the  closely  related  carnine),  phos- 
phocarnic  acid,  carnitine  and  purine  bases,  especially  hypoxanihine.  The 
purine  bases  occur  partly  free  (which  is  especially  the  case  with  hypoxan- 
thine)  and  partly  combined. 

Among  the  extractive  substances  is  also  found  the  acid  noticed  by  Limpricht 
in  the  flesh  of  certain  cyprinidea,  namely,  the  nitrogenized  protic  acid,  while  the 
isocreatinine  found  by  J.  Thesen  in  fish-flesh  is  nothing  but  impure  creatinine, 
according  to  Poulsson,  Schmidt  and  Korndorfer.2  The  following  have  also 
been  found  in  the  muscles,  in  certain  cases  only,  of  a  few  varieties  of  animals: 
uric  acid  (especially  in  alligators),  taurine  (in  cephalopoda  and  oysters),  glycocoll 
(in  gasteropoda),  betaine  and  methyl  guanidine,  in  fish  meat,  several  monamino 
acids  and  also  the  three  hexone  bases  histidine,  lysine  and  arginine.3  Urea  occurs 
in  large  quantities  in  the  muscle  of  the  shark  and  ray.  The  reports  are  very 
contradictory  in  regard  to  the  occurrence  of  urea  in  the  muscles  of  higher  animals. 
According  to  the  investigations  of  Kaufmann  and  Schondorff,  confirmed  by 
Brunton-Blaikie,4  urea  is  a  regular  constituent  of  the  muscles,  although  M. 
Nencki  and  Kowarski  dispute  this. 

In  regard  to  the  division  of  the  nitrogenous  extractives  of  the  muscles,  v. 
Furth  and  Schwarz  found  the  following  in  1000  grams  of  the  moist  extremity 
musculature  of  the  horse  and  dog  (after  subtracting  the  proteoses  derived  by 
secondary  cleavage  processes),  3.27-3.82  gram  extractive  nitrogen.  Of  this 
4.5-7  per  cent  was  ammonia,  6.1-11.1  per  cent  purine  bodies,  26.5-37.1  per  cent 
creatine  and  creatinine,  30.3-36.3  per  cent  carnosine  fraction,  8.2-15.3  per  cent 
base  residue  (carnitine,  methylguanidine,  etc.)  and  6.3-16  per  cent  urea,  poly- 
peptides and  amino-acids.  The  quantity  of  purine  base  nitrogen,  according 
to  Burian  and  Hall  in  fresh  meat  of  the  horse,  ox  and  calf,  was  0.55  p.  m.,  0.63 

1  Zeitschr.  f.  physiol.  Chem.,  32. 

2  See  Limpricht,  Annal.  d.  Chem.  u.  Pharm.,  127,  and  Thesen,  Zeitschr.  f.  physiol. 
Chem.,  24;  Poulsson,  Arch.  f.  exp.  Path.  u.  Pharm.,  51;  Schmidt  and  Korndorfer, 
ibid.,  51. 

3  In  regard  to  the  extractives  of  the  muscles  see  besides  the  specially  cited  works, 
Kurkenberg  and  Wagner,  Zeitschr.  f.  biol.  21;  U.  Suzuki  and  collaborators,  Zeitschr.  f. 
physiol.  Chen).  62  and  Chem.  Centralbl.  1913,  1;  Suwa,  Pfluger's  Arch.  128  and  129; 
Zunz,  Centralbl.  f.  Physiol.,  18. 

*  Kaufmann,  Arch,  de  Physiol.  (5),  6;  Schondorff,  Pfluger's  Arch.,  62;  Nencki 
and  Kowarski.,  Arch.  f.  exp.  Path.  u.  Pharm.,  36;  Brunton-Blaikie,  Journ.  of  Physiol., 
23,  Supplement. 


CREATINE.  573 

p.  m.  and  0.71  p.  m.  respectively,  which  corresponds  closely  to  the  results  found 
by  Scaffidi,  Buglia  and  Costantino  for  the  striated  muscle  of  the  calf,  namely, 
().">s  Of  is  p.  in.  According  to  Hinaldi  and  Scaffidi  '  the  lowest  values  for  the 
purine  nitrogen  occur  in  the  striated  muscles  of  the  covering  of  polype,  0.436 
p.  m.,  then  in  fishes  0.595-0.82  p.  m.  and  the  highest  L.061  p.  in.  in  birds.  Bi  QUA 
and  Costantino  have  determined  the  nitrogen  titcatable  with  formol,  and  from 
this  determined  the  amount  of  monamino-acid  nitrogen  as  well  as  diamino-acid 
nitrogen  in  various  animals.  In  oxen  they  found  in  the  moist,  striated  muscle 
0.18  p.  in.  inonamino-  and  0.40  p.  m.  diamino-nitrogen.  In  the  heart  the  cor- 
responding figures  were  0.18  and  0.18  p.  m.  In  percentage  of  the  total  nitrogen 
the  total  amino-acid  nitrogen  in  the  striated  muscle  was  1.70  per  cent  and  in 
the  heart  1.48  per  cent. 

The  most  extensively  occurring  nitrogenous  extractives  in  the  muscle 
are  creatine  and  carnosine. 

(/NH2 

Creatine,  C4H9N3O2,    C=NH  ,    or   methyl-guanidine- 

\N(CH3).CH2COOH 
acetic  acid,  occurs  in  the  striated  as  well  as  smooth  muscles.     In  the 
striated  muscle  of  vertebrates  the  amount  varies  between  2.5  and  7  p.  m. 
It  is  also  found  in  the  brain,   blood,  transudates,   amniotic  fluid,  and 
sometimes  also  in  the  urine. 

Creatine  may  be  prepared  synthetically  from  cyanamide  and  sar- 
cosine  (methylglycocoll).  On  boiling  with  baryta-water  it  decomposes, 
with  the  addition  of  water,  and  yields  urea,  sarcosine,  and  certain  other 
products.  Because  of  this  behavior  several  investigators  consider 
creatine  as  a  step  in  the  formation  of  urea  in  the  organism.  On  boiling 
with  acids,  creatine  is  easily  converted,  with  the  elimination  of  water 
into  the  corresponding  anhydride,  creatinine,  C4H7N3O,  which  is  retrans- 
formed  into  creatine  by  the  action  of  alkali. 

The  question  as  to  the  mutual  relation  of  creatine  to  creatinine  in 
metabolism  will  be  treated  in  Chapter  XIV  (urine).  In  this  chapter, 
besides  the  properties  and  reactions,  we  will  discuss  the  question  as  to 
the  origin  of  creatine  and  its  relation  to  the  metabolism  of  the  muscles. 

Of  special  interest  in  this  regard,  besides  the  relation  between  creatine 
and  muscle  work  which  will  be  discussed  below,  is  the  question  as  to 
the  occurrence  of  free  or  combined  creatine  in  the  muscle.  Urano  by 
the  aid  of  dialysis  experiments  has  shown  the  probability  that  the  crea- 
tine does  not  exist  free  in  the  muscle,  but  as  a  labile,  non-dialyzable 
combination.  Nevertheless  Gottlieb  and  Stangassinger  claim  by 
various  researches  to  have  shown  in  the  autolysis  of  muscles  and  other 
organs,. that. creatine  is  first  formed  and  then  first  changed  into  creatinine 
by  special  bodies  of  an  enzymotic  nature,  and  then  destroyed.     Seemann 

1  v.  Furth  and  Schwarz,  Bioch.  Zetschr.,  30;  Scaffidi,  ibid.,  33;  Burian  and  Hall, 
Zeitschr.  f.  physiol.  Chem.  38;  Buglia  and  Costantino,  ibid.,  81  and  82;  Rinaldi  and 
Scaffidi,  Bioch.  Zeitschr.,  41. 


574  MUSCLES. 

claims,  by  an  autolysis  of  three  months'  duration,  .to  have  obtained  two 
to  three  times  as  much  creatinine,  directly  from  the  muscle,  and  after 
the  addition  of  creatinine-free-gelatin  four  times  as  much,  which  is  an 
argument  against  the  enzymotic  destruction  of  creatinine  in  autolysis, 
and  he  admits  of  the  formation  of  creatine  (or  creatinine)  from  protein. 
The  autolytic  experiments  of  Rothmann  also  indicate  the  formation  of 
creatine  from  a  preliminary  body,  and  the  recent  experiments  of  van 
Hoogenhuyze  and  Verploegh  make  the  enzymotic  transformation 
of  creatine  and  creatinine  probable.  Mellanby  positively  denies  the 
re-formation  of  creatine  as  well  as  its  destruction  in  autolysis  entirely 
free  from  bacteria.  It  is  hard  to  draw  positive  conclusions  from  exper- 
iments with  autolysis.  The  transfusion  experiments  of  Gottlieb  and 
Stangassinger  with  the  kidneys  and  livers  of  dogs,  not  only  point  to 
the  ability  of  these  organs  to  decompose  creatine,  but  also  for  a  re-forma- 
tion of  creatine  in  the  liver.  Further  investigations  are  still  very  nec- 
essary, especially  as  the  conditions  are  probably  not  the  same  in  all  animals. 
Thus  Noel-Paton  and  Mackie  l  found  that  the  exclusion  of  the  liver 
in  birds  is  without  influence  upon  the  creatine  metabolism. 

As  will  be  discussed  in  Chapter  XIV,  no  certain  relationship 
exists  between  the  quantity  of  food  protein  and  the  extent  of  creatine 
and  creatinine  elimination.  On  the  contrary,  several  observations  speak 
for  a  relation  between  creatine  formation  and  catabolism  of  organ  pro- 
tein, especially  muscle  protein  and  according  to  Noel-Paton2  the 
elimination  of  creatine  in  bird  urine,  which  here  corresponds  to  the 
creatinine  in  the  mammalian  urine,  is  a  measure  of  the  protein  catabo- 
lism of  the  muscles. 

Under  all  circumstances  the  proteins,  and  especially  the  guanidine 
groups  contained  therein,  are  the  mother-substance  for  the  creatine  or 
creatinine.  The  guanidine  occurs  in  the  protein  molecule  as  arginine; 
but  according  to  Otori  it  is  not  improbable  that  in  the  protein  also 
other  guanidine  groups  exist.  Nevertheless  the  observations  of  Jaffe  3 
speak  against  the  assumption  as  to  a  creatine  formation  from  argin- 
ine as  he  found  that  arginine  subcutaneously  injected  did  not  cause 
any  increase  in  the  elimination  of  creatine  substances.  But  as  the 
introduced  arginine  was  probably  decomposed  by  the  enzyme  arginase, 
because  the   urea  elimination  was   greatly  increased,4  does  not  exclude 


1  Urano,  Hofmeister's  Beitrage,  9;  Gottlieb  and  Stangassinger,  Zeitschr.  f.  physiol. 
Chem.,  52  and  55;  Stangassinger,  ibid.,  55;  Seemann,  Zeitschr.  f.  Biol.,  49;  Roth- 
mann, Zeitschr.  f.  physiol.  Chem.,  57;  v.  Hoogenhuyze  and  Verploegh,  ibid.,  57; 
Mellanby,  Journ.  of  Physiol.,  36;  Noel-Paton    and  Mackie,  Journ.  of  Physiol.  45. 

2 Journ.  of  Physiol..  39. 

•Otori,   Zeitschr.   f.   physiol.   Chem.,   42,   43;   Jaffe,  ibid.,  48. 

4  See  Thompson,  Journ.  of  Physiol.,  32  and  33. 


CREATINE. 


575 


the  possibility  that  in  the  muscles,  which  according  to  Kossel  and  Dakin 
contain  only  little  arginase,  the  arginine  was  decomposed  in  other  ways. 
In  autolysis  as  well  as  perfusion  experiments  with  livers,  Inouye  '  has 
recently  shown  that  an  increase  in  the  creatine  occurs  at  the  expense  of 
the  arginine  added. 

Starting  with  the  observation  of  Jaff6  2  that  glyeocyamine  (guanidine  acetic 
acid)  in  rabbits  is  transformed  with  a  niethylation  into  creatine,  we  can  consider 
the  cleavage  of  arginine  into  creatine  in  the  following  manner,  basing  this  con- 
ception upon  the  ruling  conception  on  the  cleavage  of  amino-acids  and  fatty  acids 
in  the  animal  body. 


NH2 


HN=C 


/ 

\ 


HX=C 


XH 


/ 


CH, 


XH2 

/ 

\ 
NH 

/ 


NH2 
HX=C^ 

NH 

/ 
CH2 


NH, 


HX 


=< 


N(CH,) 
/ 


(CH2)2    — 

I 
COOH 

7-guanidine 
butyric  acid. 


->     COOH 

Ouanidine  acetic 
acid  (glyeocyamine). 


CH2 

I 
COOH 

Creatine. 


CH2 

I 

CH2        - 

CH, 

I 
CH(XH2) 

I 
COOH 

Arginine. 

The  opinions  are  not  unanimous  in  regard  to  the  organ  producing 
creatine  or  creatinine.  Based  upon  several  investigations  it  is  generally 
admitted  that  the  liver  here  plays  an  important  role.  Several  other 
organs  may  also  be  considered  and  in  the  first  place,  the  muscles.  Accord- 
ing to  Mellanby  the  creatinine  is  probably  formed  in  the  liver,  trans- 
formed into  creatine  in  the  muscles  and  there  stored  up  as  such.  Other 
observations  still  speak  for  the  fact  that  the  creatine  is  formed  in  the 
muscles  and  transformed  into  creatinine  in  the  liver,  while  according 
to  Noel-Paton  and  Mackie  the  exclusion  of  the  liver  in  birds  is  without 
effect  upon  the  creatinine  metab<  lism. 

Creatine  crystallizes  in  hard,  colorless,  monoclinic  prisms  which 
lose  their  water  of  crystallization  at  100°  C.  It  is  soluble  in  7-4  parts 
of  water  at  the  ordinary  temperature,  and  in  9419  parts  absolute  alcohol. 
It  dissolves  more  easily  with  the  aid  of  heat.  Its  watery  solution  has 
a  neutral  reaction.  Creatine  is  not  dissolved  by  ether.  If  a  creatine 
solution  is  boiled  with  precipitated  mercuric  oxide,  this  is  reduced, 
especially  in  the  presence  of  alkali,  to  mercury  and  oxalic  acid,  and  the 
foul-smelling  methyluramine    (methylguanidine)    is   developed.     A   solu- 


1  Kossel  and  Dakin,  Zeitschr.  f.  physiol.  Chem.,  41  and  42;  Inouye,  ibid.,  81. 
1  Zeitschr.  f.  physiol.  Chem.,  48;  see  also  Dorner,  ibid.,  52. 


576  MUSCLES. 

tion  of  creatine  in  water  is  not  precipitated  by  basic  lead  acetate,  but 
gives  a  white,  flaky  precipitate  with  mercurous  nitrate  if  the  acid  reac- 
tion is  neutralized.  When  boiled  for  an  hour  with  dilute  hydrochloric 
acid,  creatine  is  converted  into  creatinine,  and  may  be  identified  by  its 
reactions.  On  boiling  with  formaldehyde  it  can  be  transformed  into 
dioxymethylenecreatinine,  which  crystallizes  readily  (Jaffe  '). 

The  preparation  and  detection  of  creatine  is  best  accomplished  by  the 
following  method  of  Neubauer,2  which  was  first  used  in  the  preparation 
of  creatine  from  muscles:  Finely  cut  meat  is  extracted  with  an  equal 
weight  of  water  at  50-55°  C.  for  10-15  minutes,  pressed,  and  extracted 
again  with  water.  The  proteins  are  removed  from  the  united  extracts 
so  far  as  possible  by  coagulation  at  boiling  heat,  the  filtrate  precipitated 
by  the  careful  addition  of  basic  lead  acetate,  the  lead  removed  from  this 
filtrate  by  H2S,  and  the  solution  then  carefully  concentrated  to  a  small 
volume.  The  creatine,  which  crystallizes  in  a  few  days,  is  collected  on  a 
filter,  washed  with  alcohol  of  88  per  cent,  and  purified,  when  necessary, 
by  recrystallization.  In  the  preparation  of  large  quantities  of  creatine 
we  can  especially  start  with  meat  extracts.  The  quantitative  estimation 
of  creatine  is  performed  by  transforming  it  into  creatinine  (see  Chapter 
XIV). 

Carnosine,  C9H14N4O3,  is  a  base  first  isolated  by  Gulewitsch  and 
Amiradzibi  from  meat  extracts  and  which  subsequently  was  also  pre- 
pared directly  from  meat.  The  quantity  seems  to  be  relatively  consider- 
able, as  according  to  the  above-mentioned  determination  of  v.  Furth* 
and  Schwarz,  the  carnosine  fraction  from  the  horse  and  dog  muscles 
was  just  as  large  or  indeed  greater  than  the  creatine-creatinine  fraction 
of  the  extractive  nitrogen.  Krimberg  found  1.3  p.  m.  and  Skworzow,3 
1.76  p.  m.  (as  nitrate)  in  fresh  meat. 

Carnosine,  which  according  to  Gulewitsch  is  identical  with  the  base 
ignotine  isolated  from  meat  extracts  by  Kutscher  while  both  bases  are 
isomeric  bodies  according  to  Kutscher,4  is  a  histidine  derivative  accord- 
ing to  Gulewitsch  which  on  cleavage  yields  /3-alanine  besides  histidine. 

Carnosine  is  a  base  readily  soluble  in  water,  which  is  precipitated 
as  stellar  warts  of  short  delicate  needles  from  the  concentrated  watery 
solution  by  the  addition  of  alcohol.  The  specific  rotation  for  the  light 
X  =  546  is  according  to  Gulewitsch  in  watery  solution  where  c  =  12.925 
per  cent  and  20.1°  C.  =  -r-25.3°.     The  base  is  precipitated  by  phospho- 

1  Ber.  d.  d.  Chem.  Gesellsch.,  35. 

2  Zeitschr.  f.  analyt.  Chem.,  2  and  6. 

'Gulewitsch   and   Amiradzibi,  Zeitsdir.  f.  physiol.  Chem.,  30;  Gulewitsch,  ibid., 

50,  51,  52  and  73;  Krimberg.  ihid.,  48;  Skworzow,  ibid.,  68. 

4  Gulewitsch  and  Amiradzibi,  Zeitschr.  f.  physiol.  Chem.,  30;  Gulewitsch,  ibid.,  50, 

51,  52  and  73;  Krimberg,  ibid.,  08;  Skworzow,  ibid.,  68;   Kutscher,  ibid.,  50,  51. 


CARNITINE.     CARNINE.  577 

tungstic  acid,  by  mercuric  nitrate  and  by  silver  nitrate  with  an  excess  of 
barium  hydrate.  Carnosine-silver  is  soluble  with  difficulty  in  cold  water 
but  readily  soluble  in  hot  water.  Carnosine  nitrate  melts  at  211-212°  C. 
Carnosine  also  gives  a  crystalline  copper  salt. 

The  principle  in  preparing  this  base  consists  in  precipitat'ng  with 
phosphotungstic  acid,  separating  the  free  base  with  barium  hydrate, 
conversion  into  the  nitrate,  precipitating  with  silver  nitrate  and  barium 
hydrate,  decomposing  the  salt  with  H28  and  conversion  into  nitrate. 
From  the  latter,  which  is  readily  obtained  as  crystals,  the  base  is  precip- 
itated by  phosphotungstic  acid  and  then  set  free  by  barium  hydrate. 

Carnitine,  C7H15NO3  (or  C;HiGX03),  another  base  isolated  by  Gulewitsch 
and  Krimberg  from  meat  extracts,  has  a  strong  alkaline  reaction,  is  very 
readily  soluble  in  water,  and  was  also  found  by  Krimberg  in  fresh  meat.  Skworzow 
found  0.19  p.  m.  carnitine  in  calf's  muscles.  Carnitine  according  to  Krimberg 
is  probably  7-trimethvl-/3-oxybutyrobetaine  with  the  formula 

X) CO 

(CH3):N\  !  .     According  to  Engeland  it  is  on  the  contrarv 

\CH2— CH(OH)— CH2 

a    7-trimethyl-o;-oxybutyrobetaine    (CH3)3-N~  V         T      . 

CH2CH2CH(OH)— CO 

according  to  Krimberg  and  Engeland1  identical  with  novaine  prepared  by  Kossel 
from  meat  extracts.  It  gives  crystalline  double  compounds  with  platinum,  gold 
and  mercuric  chlorides,  among  which  the  following,  C7Hi5N032HgCl2,  with  a 
melting-point  of  196-197°  C,  is  especially  used  in  the  isolation  of  the  base. 
The  hydrochloride  and  the  nitrate  are  readily  soluble  and  the  solution  of  the  first 
isla?vo-rotatory,  about  («)D  =  —21°. 

The  inosinic  acid  has  been  discussed  in  Chapter  II.  In  close  relation  to  this 
stands  probably  the  carnine. 

Carnine,  C7H8X  iO;i+H20,  is  one  of  the  substances  found  by  Weidel  in  American 
meat  extract.  It  has  also  been  found  by  Krukenberg  and  Wagner  in  frog 
muscles  and  in  the  flesh  of  fishes,  and  by  Pouchet  in  the  urine.  Carnine  is,  accord- 
ing to  Haiser  and  Wenzel,2  probably  only  an  equimolecular  mixture  of  hypo- 
xanthine  and  the  crystalline  pentoside  (hypoxanthin-riboside)  inosine,  which  is 
readily  split  by  acid  into  hypoxanthine  and  pentose. 

Carnine  has  been  obtained  as  a  white  crystalline  mass.  It  dissolves  with 
difficulty  in  cold  water,  but  more  readily  in  warm.  It  is  insoluble  in  alcohol 
and  ether.  It  dissolves  in  warm  hydrochloric  acid  and  yields  a  salt  crystallizing 
in  shining  needles,  which  gives  a  double  compound  with  platinum  chloride.  Its 
watery  solution  is  precipitated  by  silver  nitrate,  but  this  precipitate  is  dissolved 
neither  by  ammonia  nor  by  warm  nitric  acid.  Its  watery  solution  is  precipitated 
by  basic  lead  acetate;  but  the  lead  compound  may  lie  dissolved  on  boiling. 

Phosphocarnic  acid3  is  a  complicated  substance,  first  isolated  by  Siegfried 

Gulewitsch  and  Krimberg,  Zeitschr.  f.  physiol.  Chem.  45;  Krimberg,  ibid.;  49, 
50,  53  and  56,  Ber.  d.  d.  Chem.  Gesellsch.  42;  Engeland,  ibid.,  42;  Skworzow,  1.  c. 

2  Weidel,  Annal.  d.  Chem.  u.  Pharm.,  158;  Krunkenberg  and  Wagner,  SUzungsber. 
d.  Wiirzb.  phys.-med.  Gesselsch.,  1883;  Pouchet,  cite!  from  Neubauer-Huppert, 
Analyse  des  Harnes,  10.  Aufl.,  335;  Haiser  and  Wenzel,  Monatsch.  f.  Chem.,  29. 

3  In  regard  to  carnic  acid  and  phosphocarnic  acid,  see  the  works  of  Siegfried,  Arch, 
f.  (Anat.  u.)  Physiol.,  1894,  Ber.  d.  deutsch.  chem.  Gesellsch.,  28,  and  Zeitschr.  f. 
physiol.  Chem.,  21  and  28;  M.  Miiller,  ibid.,  22;  Kriiger,  ibid.,  22  and  28;    Balke  and 


578  MUSCLES. 

from  meat  extracts,  which  yields  as  cleavage  products  succinic  acid,  paralactic 
acid,  carbon  dioxide,  phosphoric  acid,  and  a  carbohydrate  group,  besides  the 
previously  mentioned  carnic  acid,  which  is  identical  with  or  nearly  related  to 
antipeptone.  It  stands,  according  to  Siegfried,  in  close  relation  to  the  nucleins, 
and  as  it  yields  peptone  (carnic  acid),  it  is  designated  as  a  nucleon  by  Siegfried. 
Phosphocarnic  acid  may  be  precipitated  as  an  iron  compound,  carniferrine,  from 
the  extract  of  the  muscles  free  from  proteins.  The  quantity  of  phosphocarnic 
acid,  calculated  as  carnic  acid,  can  be  determined  by  multiplying  the  quan- 
tity of  nitrogen  in  the  compound  by  the  factor  6.1237  (Balke  and  Ide).  In 
this  way  Siegfried  found  0.57-2.4  p.  m.  carnic  acid  in  the  resting  muscles 
of  the  dog,  and  AI.  Muller  1-2  p.  m.  in  the  muscles  of  adults  and  a  maximum 
of  0.57  p.  m.  in  those  of  new-born  infants.  According  to  Cavazzani  nucleon 
occurs  to  a  much  greater  extent  in  oysters,  namely,  an  average  of  3.725  p.  m. 
It  also  occurs,  as  he  and  Manicardi  found,  in  the  plant  kingdom.  Phospho- 
carnic acid  has  not  been  prepared  in  the  pure  state  and  possesses  on  this  account 
a  variable  composition;  according  to  Siegfried  it  serves  as  a  source  of  energy 
in  the  muscles  and  is  consumed  during  work.  Besides,  by  means  of  its  property 
of  forming  soluble  salts  with  the  alkaline  earths,  as  also  an  iron  combination 
soluble  in  alkalies,  it  acts  as  a  means  of  transportation  for  these  bodies  in  the 
animal  body. 

Phosphocarnic  acid  is  prepared  from  the  extract  free  from  protein  by  first 
removing  the  phosphate  by  CaCl2  and  NH3.  .  The  acid  is  precipitated  as  carnifer- 
rine by  ferric  chloride  from  the  filtrate  while  boiling. 

From  Liebig's  extract  of  beef  Kutscher  has  isolated  besides  the  above- 
mentioned  ignotine  and  novaine,  several  other  bodies,  neosine,  CeH^NCK,  which 
according  to  Kutscher  and  Ackermann  is  a  homologue  of  choline,  vitiatine 
(as  gold  salt,  CoHi4X6.2HC1.2AuCls),  carnomuscarine,  methylguanidine  (also  found 
by  Gulewitsch),  oblitine,  CisHssXoOs,  which  probably  contains  two  novaine 
groups,  which  corresponds  well  with  Krimberg's  view,  and  also  choline  and 
murine.  From  dog  muscles  Ackermann1  has  isolated  a  platinum  compound, 
CnH3oX204PtCl6,  of  a  base  called  myocynine,  which  seems  to  be  a  hexamethyl- 
ornithine.  Micro2  found  in  meat  extracts  small  quantities  of  alanine,  glutamic 
acid,  taurine  and  inosite,  but  no  dipeptides.  In  crab  extract  Kutscher  and  Ack- 
ermann found  no  creatine  and  creatinine,  but  among  others  betaine  and  two  new 
bases,  crangitine,  CY(Hi0X2O4,  and  crangonine,  C13H26X2O3.  In  crab  muscles  Suzuki  3 
and  collaborators  found  a  base,  canirine  which  although  it  has  the  same  composi- 
tion. C6H14X2O2,  as  lysine,  is  not  identical  therewith. 

The  base  musculamine,  isolated  by  Etard  and  Vila  on  the  hydrolysis  of  veal, 
is  nothing  but  cadaverine,  according  to  Posternak.1 

We  must  also  include  among  the  nitrogenous  extractives  those  bodies  which 
were  first  discovered  by  Gautier,6  and  which  occur  only  in  very  small  quantities, 
namely,  the  leucomaines,  xanthocreatinine,  C5H10H4O,  crusocreatinine,  C5H8N4O, 
amphicreatine,  C9H19X7O4,  and  pseudoxanthine,  C4H5X5O. 


Ide,  ibid.,  21,  and  Balke,  ibid.,  22;  Macleod,  ibid.,  28;  E.  Cavazzani,  Centralbl.  f. 
Physiol.,  18,  666;  Panella,  Maly's  Jahresber.,  34. 

1  Kutscher,  Zeitschr.  f.  Unters.  d.  Nahrungs-  u.  Genussmittel,  10,  11,  Centralbl. 
f.  Physiol.,  19  and  21,  Zeitschr.  f.  physiol.  Chem.,  48,  49,  50,  51,  with  Ackermann, 
ibid.,  56;  Gulewitsch.  ibid.,  47;  Krimberg,  ibid.,  56;  Ackermann  (on  myocynine). 
Zeitschr.  f.  biol.  59. 

2  Zeitsfhr.  f.  physiol.  Chem.,  56. 

■■  Kutscher  and  Ackermann,  Zeitschr.  f.  Unters.  d.  Nahrungs-  u.  Genuarnittel,  13 
and  14;  Suzuki,  Chem.  Centralbl.  1913,  1. 

4  Etard  and  Vila,  Cornpt.  Rend.,  135;  Posternak,  ibid.,  135. 
8  See  M:.ly's  Jahresber.,  16,  523. 


INOSITE.  579 

In  the  analysis  of  moat,  and  for  the  detection  and  separation  of  the  various 
extractive  bodies  of  meat,  we  make  use  of  the  systematic  method  as  suggested 
by  Gautier,1  for  details  of  which  the  reader  is  referred  to  the  original  article 
Bfl  well  as  for  the  Kutscht  r  method  for  working  the  meat  extracts. 

The  non-nitrogenous  extractive  bodies  of  the  muscles  are  inosite,  gly- 
cogen, sugar,  and  lactic  aval. 

Inosite,  CgHi20c+H20  =  C6Hg(OH)6+H20.  This  body,  discovered 
by  Scherer,  is  not  a  carbohydrate,  but  belongs  to  the  hydroaromatic 
compounds,  and  is  a  hexahydroxybenzene  (Maquenne2).  That  it 
stands  in  certain  relation  to  the  carbohydrates  follows  from  the  fact  that 
Neuberg  obtained  some  furfurol  from  inosite  by  distillation  with  phos- 
phoric anhydride,  and  also  that  P.  Meyer3  found  fermentation  lactic 
acid  in  the  urine  of  rabbits  after  the  introduction  of  inosite  per  os.  It 
has  been  known  for  some  time  that  inosite  undergoes  lactic  acid  fermenta- 
tion. The  acid  formed  thereby  is  sarcolactic  acid  according  to  Hilger 
and  fermentation  lactic  acid  according  to  Vohl.4 

Inosite  is  found  in  the  muscles,  liver,  spleen,  leucocytes,  kidneys, 
suprarenal  capsule,  lungs,  brain,  testicles,  and  in  the  urine  in  pathological 
cases,  and  as  traces  in  normal  urine.  It  is  found  very  widely  dis- 
tributed in  the  vegetable  kingdom,  especially  in  the  unripe  fruit  of  green 
beans  (Phaseolus  vulgaris),  and  therefore  it  is  also  called  phaseomannite. 
In  the  plant  kingdom  another  substance  occurs  which  is  called  phytin 
and  which  is  the  Mg  and  Ca  compound  of  inosite  and  phosphoric  acid 
and  which  was  first  isolated  by  Posternak.  Winterstein  identified  this 
as  an  inosite-phosphoric  acid.  This  inosite-phosphoric  acid  can  be  split 
into  phosphoric  acid  and  inosite  by  the  plant  enzyme  phytase  (Suzuki, 
Yoshimura  and  Takaishi)  as  well  as  by  enzymes  of  the  animal  tissues 
(Starkenstein).  Inosite  is  found  in  plants,  especially  in  the  develop- 
ing organs  (Meillere),  and  according  to  Starkenstein5  it  occurs  to  a 
greater  extent  in  the  organs  of  young  animals  as  compared  with  those  of 
older  animals.  From  this  it  follows  that  inosite  is  probably  not  a  decom- 
position product  of  metabolism,  but  rather  a  body  necessary  for  the  devel- 
opment of  the  cells  (Meillere);  but  according  to  Starkenstein  the 
facts  are  different. 


1  Maly's  Jahresb.,  22. 

2  Bull.  soc.  chem.  (2),  47  and  48;  Compt.  Rend.,  104. 
1  Neuberg,  Bioch.  Zeitschr.,  9;  P.  Meyer,  ibid.,  9. 

*  Hilger,  Annal.  d.  Chem.  u.  Pharm.,  160;  Vohl,  Ber.  d.  d.  Chem.  Gesellsch.,  9. 

5  Winterstein,  Ber.  d.  d.  chem.  Gesellsch.,  30;  :>.nd  Zeitschr.  f.  physiol.  chem.,  58; 
Posternak,  Contribution  a  l'dtude  chim.  de  I'assimilation  chlorophyllienne.  Revue 
cenerale  botanique,  Tome  12  (1900),  and  Compt.  Rend.,  137;  Suzuki,  Yoshimura  and 
Takaishi,  Bull,  agric.  Univers.  Tokio,  7;  Starkenstein,  Bioch.  Zeitschr.,  30. 


580  MUSCLES. 

According  to  Starkenstein  the  free  inosite  is  without  importance 
and  is  only  a  decomposition  product  of  metabolism;  of  importance, 
especially  for  young,  growing  individuals  is  according  to  this  worker 
only  the  phytin,  which  is  decomposed  in  the  intestine  by  bacteria,  and  in 
the  tissues  by  enzymes,  and  correspondingly  supplies  phosphoric  acid  and 
lime  to  the  organism  while  the  inosite  is  excreted  as  a  valueless  cleavage 
product.  The  free  inosite  in  the  animal  body  originates  according  to 
Starkenstein  from  the  inositephosphoric  acid  and  in  this  sense  the 
assumption  of  Rosenberger1  as  to  the  occurrence  of  an  inositogen  in  the 
animal  body,  is  substantiated. 

Inosite,  which  almost  without  exception  is  inactive  mesoinosite, 
crystallizes  in  large,  colorless,  rhombic  crystals  of  the  monoclinic  sys- 
tem, or,  if  not  pure  and  if  only  a  small  quantity  crystallizes,  it  forms 
groups  of  fine  crystals  similar  to  cauliflower.  It  loses  its  water  of  crys- 
tallization at  110°  C,  also  if  exposed  to  the  air  for  a  long  time.  Such 
exposed  crystals  are  non-transparent  and  milk-white.  The  crystals 
melt  at  225°  C.  when  dry.  Inosite  dissolves  in  7.5  parts  of  water  at 
ordinary  temperature,  and  the  solution  has  a  sweetish  taste.  It  is  insoluble 
in  strong  alcohol  and  in  ether.  It  dissolves  cupric  hydrate  in  alkaline 
solutions,  but  does  net  reduce  on  boiling.  It  gives  negative  results  with 
Moore's  test  and  with  Bottger-Almen's  bismuth  test.  It  does  not 
ferment  with  beer-yeast,  but  may  undergo  lactic-  and  butyric-acid  fer- 
mentation. With  an  excess  of  nitric  acid  inosite  is  oxidized  to  rhodizonic 
acid,  and  the  following  reaction  depends  upon  this. 

If  inosite  is  evaporated  to  dryness  on  paltinum-foil  with  nitric  acid 
and  the  residue  treated  with  ammonia  and  a  drop  of  calcium  chloride 
solution  and  carefully  re-evaporated  to  dryness,  a  beautiful  rose-red 
residue  is  obtained  (Sherer's  inosite  test).  If  we  evaporate  an  inosite 
solution  to  incipient  dryness  and  moisten  the  residue  with  a  little  mer- 
curic nitrate  solution,  there  is  obtained  a  yellowish  residue  on  drying 
which  becomes  a  beautiful  red  on  strongly  heating.  The  coloration 
disappears  on  cooling,  but  it  reappears  on  gently  warming  (Gallois' 
inosite  test) .  Other  inosite  reactions  have  been  suggested  by  Deniges  2 
and  others.3 

To  prepare  inosite  from  a  liquid  or  from  a  watery  extract  of  a  tissue, 
the  proteins  are  first  removed  by  coagulation  at  boiling  heat.     The  filtrate 


1  Meillere,  Journ.  d.  Chim.  et  Pharm.  (6)  28;  Starkenstein,  Zeitschr.  f.  exp.  Path, 
u.  Therap.  5,  Bioch.  Zeitschr.  30  and  Zeitschr.  f.  physiol.  Chem.  58;  Rosenberger, 
ibid.,  56,  57  and  58. 

2  Compt.  rend.  soc.  biol.,  62. 

3  In  regard  to  the  salts  of  phytin  and  compounds  of  inosite  see  Anderson,  Journ. 
of  biol.  Chem.  11  and  12. 


GLYCOGEN.  581 

is  precipitated  by  sugar  of  Lead,  this  filtrate  boiled  with  basic  lead  acetate 
and  allowed  to  stand  24-48  hours.  The  precipitate  thus  obtained, 
which  contains  all  the  inosite,  is  decomposed  in  water  by  HfeS.  The 
nitrate  is  strongly  concentrated,  treated  with  2-4  vols,  hot  alcohol,  and 
the  liquid  removed  as  soon  as  possible  from  the  tough  or  flaky  masses 
which  ordinarily  separate.  If  no  crystals  separate  from  the  liquid  within 
twenty-four  hours,  then  treat  with  ether  until  the  liquid  has  a  milky 
appearance  and  allow  it  to  stand.  In  the  presence  of  a  sufficient  quantity 
of  ether,  crystals  of  inosite  separate  within  twenty-four  hours.  The 
crystals  thus  obtained,  as  also  those  which  are  directly  obtained  from  the 
alcoholic  solution,  are  recrystallized  by  redissolving  in  very  little  boiling 
water  and  adding  2-A  vols,  of  alcohol.  Meillere  l  and  others  have 
suggested  mollifications  in  the  methods  for  detecting  and  quantitatively 
estimating  inosite. 

Scyllite  is  a  body  which  is  isomeric  with  inosite,  according  to  Joh.  Muller,2 
and  which  was  found  long  ago  in  the  kidneys,  liver  and  spleen  of  Plagiostomata 
and  also  in  the  plant  kingdom  as  cocosite  and  quercinite.  Scyllite  crystallizes 
in  shining  prisms,  is  soluble  in  water  1:100  at  18°  C,  is  similar  to  inosite  in  its 
reactions,  but  has  a  much  higher  melting-point,  namely  about  360°  C.  From 
the  adductor  muscles  of  the  Mytilus  Janssen  3  has  isolated  a  substance,  called 
mytilite  which  is  crystalline,  soluble  with ,  difficulty  in  cold  water  and  readily  sol- 
uble in  hot  water,  and  having  the  formula  C6Hi2Oo.2H20.  He  claims  that  it  is 
stereisometric  with  the  alcohol  quercite. 

Glycogen  is  a  constant  constituent  of  the  living  muscle,  while  it  may 
be  absent  in  the  dead  muscle.  The  quantity  of  glycogen  varies  in  the 
different  muscles  of  the  same  animal  and  according  to  Maignon  this 
is  not  only  true  for  the  same  muscles  in  both  halves  of  the  body  but  also 
for  different  parts  of  the  same  muscle.  Bohm  found  10  p.  m.  glycogen 
in  the  muscles  of  cats,  and  moreover  he  found  a  smaller  amount  in  the 
muscles  of  the  extremities  than  in  those  of  the  rump.  Moscati  found  an 
average  of  4  p.  m.  in  human  muscles,  and  Schondorff4  has  found  a 
maximum  of  37.2  p.  m.  in  the  dog-muscle.  Reports  as  to  the  quantity 
of  glycogen  in  the  heart  are  conflicting;  although  the  heart  is  considered 
as  somewhat  poorer  in  glycogen  than  the  other  muscles,  still  this  difference 
is  not  very  great,  and  can  be  explained  by  the  ready  disappearance  of 
glycogen  from  the  heart  after  death,  as  well  as  after  starvation  and 
after  strong  work  (Boruttatj,  Jensen5).  Work  and  food  have  a  great 
influence  upon  the  quantity  of  glycogen.  Bohm  found  1-4  p.  m. 
glycogen  in  the  muscles  of  fasting  animals,  and  7-10  p.  m.  after  partak- 

^ompt.  rend.  soc.  biol.,  60,  and  Journ.  d.  Chim.  et  Pharni.  (6),  24;  see  also 
Starkenstein,  Zeitschr.  f.  exp.  Path.  u.  Ther.,  5. 

2  Ber.  d.  d.  chem.  Gesellsch.,  40. 

3  Zeitschr.  f.  physiol.  Chem.,  85. 

4  Maignon,  Journ.  de  physiol.  et  d.  path.  10  Bohm,  Pfluger's  Arch.,  23,  44;  Schon- 
dorff, ibid.,  99;  Moscati,  Hofmeister's  Beitrage,  10. 

6  Boruttau,  Zeitschr.  f.  physiol.  Chem.,  18;  Jensen,  ibid.,  35. 


582  MUSCLES. 

ing  of  food.  As  stated'  in  Chapter  VII,  work,  starvation,  and  lack  of 
carbohydrates  in  the  food  cause  the  glycogen  to  disappear  earlier  from 
the  liver  than  from  the  muscles. 

The  sugar  of  the  muscles,  of  which  only  traces  occur  in  the  living  mus- 
cle, and  which  is  probably  formed  after  the  death  of  the  muscle  from 
the  muscle-glycogen,  is,  according  to  the  investigations  of  Panormoff,  in 
part  glucose,  but  consists  principally  of  maltose  (Osborne  and  Zobel  l) 
with  some  dextrin. 

Lactic  Acids.  Of  the  oxypropionic  acids  with  the  formula  C3H6O3 
there  is  one,  ethylene  lactic  acid,  CH2(OH).CH2.COOH,  which  is  not 
found  in  the  animal  body,  and  therefore  has  no  physiological  chemical 
interest. 

CH3 
Indeed  only  a-oxypropionic  acid  or  ethylidene   lactic  acid,  CH(OH),  of 

COOH 
which  there  are  two  physical  isomers,  namely,  the  dextrorotatory  par- 
alactic  or  sarcolactic  acid,  and  the  levolactic  acid  obtained  by 
Schardinger  by  the  fermentation  of  cane-sugar  by  means  of  a  special 
bacillus.  This  levolactic  acid,  which  is  formed  by  the  typhoid  bacillus 
and  various  vibriones  2  need  not  be  discussed  here,  and  we  will  only  treat 
here  the  d-Z-lactic  acid  (the  inactive  fermentation  lactic  acid)  and  the 
dextrolactic  acid. 

The  fermentation  lactic  acid,  which  is  formed  from  lactose  by  allow- 
ing milk  to  sour,  and  by  the  acid  fermentation  of  other  carbohydrates, 
is  considered  to  exist  in  small  quantities  in  the  muscles  (Heintz),  in  the 
gray  matter  of  the  brain  (Gscheidlen),  and  in  diabetic  urine.  The 
occurrence  of  fermentation  lactic  acid  in  the  brain  and  other  organs 
is  still  very  improbable  and  has  been  disputed  by  Moriya.3  During 
digestion  this  acid  is  also  found  in  the  contents  of  the  stomach  and  intestine, 
and  as  alkali  lactate  in  the  chyle.  The  parallactic  acid,  is  at  all  events, 
the  true  acid  of  meat  extracts,  and  this  alone  has  been  found  with  certainty 
in  dead  muscle.  The  lactic  acid  which  is  found  in  the  brain,  spleen,, 
lymphatic  glands,  thymus,  thyroid  gland,  blood,  bile,  pathological 
transudates,  osteomalacial  bones,  in  perspiration  in  puerperal  fever, 
in  the  urine  after  fatiguing  marches,  in  acute  yellow  atrophy  of  the  liver, 


1  Panormoff,  Zeitschr.  f.  physiol.  Chem.,  17;  Osborne  and  Zobel,  Journ.  of  Physiol., 
29. 

1  See  Schardinger,  Monatshefte  f.  Chem.,  11;  Blachstein,  Arch,  des  sciences  biol. 
de  St.  Petersbourg,  1,  199 ;  Kuprianow,  Arch.  f.  Hygiene,  19,  and  Gosio,  ibid.,  21; 
Herzog  and  Horth,  Zeitschr.  f.  physiol.  Chem.,  60.  I 

1  Heintz,  Annal.  d.  Chem.  u.  Pharm.,  157,  and  Gscheidlen,  Pfliiger's  Arch.,  8,. 
171;  Moriya,  Zeitschrift  f.  physiol.  Chem.,  43. 


LACTIC  ACIDS.  583 

in  poisoning  by  phosphorus,  and  especially  after  extirpation  of  the  liver 
seems  to  be  paralactic  acid. 

The  origin  of  paralactic  acid  in  the  animal  organism  has  been  sought 
by  several  investigators,  who  took  for  basis  the  researches  of  Gaglio, 
Minkowski,  and  Araki,  in  a  decomposition  of  protein  in  the  tissues 
Gaglio  claims  a  lactic-acid  formation  by  passing  blood  through  the  sur- 
viving kidneys  and  lungs.  He  also  found  0.3-0.5  p.  m.  lactic  acid  in  the 
blood  of  a  dog  after  protein  food,  and  only  0.17-0.21  p.  m.  after  fast- 
ing for  forty-eight  hours.  According  to  Minkowski  the  quantity  of  lactic 
acid  eliminated  by  the  urine  in  animals  with  extirpated  livers  is  increased 
with  protein  food,  while  the  administration  of  carbohydrates  has  no 
effect.  Araki  has  also  shown  that  if  we  produce  a  scarcity  of  oxygen 
in  animals  (dogs,  rabbits,  and  hens)  by  poisoning  with  carbon  monoxide, 
by  the  inhalation  of  air  deficient  in  oxygen,  or  by  any  other  means,  a 
considerable  elimination  of  lactic  acid  (besides  sugar  and  also  often 
albumin)  takes  place  through  the  urine,  an  observation  which  has  been 
confirmed  by  Saito  and  Katsuyama.1  As  a  scarcity  of  oxygen,  accord- 
ing to  the  ordinary  statements,  produces  an  increase  of  the  protein 
catabolism  in  the  body,  the  increased  elimination  of  lactic  acid  in  these 
cases  must  be  due  in  part  to  an  increased  protein  destruction  and  in  part 
to  a  diminished  oxidation. 

Araki  has  not  drawn  such  a  conclusion  from  his  experiments,  but 
he  considers  the  abundant  formation  of  lactic  acid  to  be  due  to  a  cleavage 
of  the  sugar  formed  from  the  glycogen.  He  found  that  in  all  cases  where 
lactic  acid  and  sugar  appeared  in  the  urine  the  quantity  of  glycogen 
in  the  liver  and  muscles  was  always  diminished.  Without  denying 
the  possibility  of  a  formation  of  lactic  acid  from  protein,  he  states  that 
with  lack  of  oxygen  we  have  to  deal  with  an  incomplete  combustion 
of  the  lactic  acid  derived  by  a  cleavage  of  the  sugar.  Although  the 
abundant  formation  of  lactic  acid  under  these  circumstances  can  be 
explained  in  different  ways,  still  there  are  other  conditions  which  make 
the  formation  of  lactic  acid  from  proteins  very  probable.  To  this 
belongs  the  lactic  acid  formation  from  alanine,  in  the  liver,  as  mentioned 
in  a  previous  chapter,  and  recently  further  substantiated  by  Embden 
and  F.  Kraus.2 

The  carbohydrates  are  also  considered  as  the  mother-substance 
of  the  lactic  acid,  as  it  is  now  generally  admitted  that  the  cleavage  of  the 


1  Gaglio,  Arch  f  (Anat.  u.)  Physiol.,  1886;  Minkowski ,  Arch  exp.  Path,  u.  Pharm., 
21  and  31;  Araki,  Zeitschr.  f.  physiol.  Chem.,  15,  16,  17,  and  19;  Saito  and  Katsuyama. 
ibid.,  32. 

2  Neuberg  and  Langstein,  Arch.  f.  (Anat.  u.)  Physiol.  1903;  Embden  and  F.  Kraus, 
Bioch  Zeitschr.  45. 


584  MUSCLES. 

sugar  in  the  animal  body  occurs,  or  at  least  can  occur,  with  lactic  acid  as 
an  intermediary  step  The  views  are  indeed  different1  as  to  the  closer 
mechanism  of  this  cleavage,  but  there  does  not  exist  any  doubt  that  a 
formation  of  lactic  acid,  and  in  fact  paralactic  acid,  can  take  place  from 
carbohydrates  in  the  animal  body.  Hoppe-Seyler  2  held  the  view  that 
the  formation  of  lactic  acid,  in  the  absence  of  free  oxygen,  from  gly- 
cogen or  glucose  was  probably  a  function  of  all  living  protoplasm  and  in 
the  anaerobic  metabolism  of  the  animal  cells,  according  to  the  investiga- 
tions of  Stoklasa3  and  his  collaborators  on  alcoholic  fermentation  in 
the  tissues,  a  formation  of  alcohol  and  carbon  dioxide  takes  place  from 
the  sugar  with  lactic  acid  as  intermediary  step.  The  correctness  of  these 
statements  is  now  disputed  from  many  sides,  but  we  have  direct  observa- 
tions which  speak  positively  for  a  lactic  acid  formation  from  glycogen 
or  sugar.  Thus  Embden4  and  co-workers  have  found  that  on  transfus- 
ing blood  through  the  liver  rich  in  glycogen,  a  formation  of  lactic  acid 
takes  place,  and  an  abundance  of  lactic  acid  is  formed  when  blood  rich  in 
sugar  is  transfused  through  a  glycogen  free  liver,  while  a  blood  poor  in 
sugar  led  only  to  a  very  inconsiderable  formation  of  lactic  acid. 

Certain  investigators  (see  page  333)  admit  of  the  occurrence  of  glyceric 
aldehyde  (and  also  dioxyacetone)  as  intermediary  products  in  the  forma- 
tion of  lactic  acid  from  sugar.  Another  intermediary  product  in  the 
lactic  acid  formation  has  been  shown  by  recent  thorough  investigations 
to  be  methylglyoxal,  CH3.CO.CHO.  An  abundant  formation  of  lactic 
acid  from  methylglyoxal  has  been  obtained  by  certain  investigators, 
such  as  Dakin  and  Dudley,  and  by  Neuberg,  in  experiments  with 
tissues,  organ  extracts  and  organ  pulp,  and  by  Levene  and  Meyer5  in 
experiments  with  leucocytes  or  kidney  tissue.  The  process  is  of  an 
enzymotic  nature  and  the  active  enzyme,  which  also  converts  phenyl- 
glyoxal  into  mandelic  acid  has  been  called  ghjoxylase  by  Dakin  and 
Dudley.  The  process  is  reversible  according  to  these  experimenters, 
in  that  they  have  been  able  to  show  a  retransformation  of  lactic  acid 
into  methylglyoxal.  They  also  found  that  lactic  acid  as  well  as  methyl- 
glyoxal could  form  glucose  in  diabetic  animals.  The  detailed  procedure 
in  the  cleavage  of  sugar  to  lactic  acid  is  still  undecided. 

The  carbohydrates,  as  well  as  the  proteins,  it  seems,  must  be  con- 
sidered as  the  material  from  which  the  lactic  acid  is  formed  in  the  body. 


»See  Embden  und  Oppenheimer,  Bioch.  Zeitschr.,  45;  Parnas  and  Baer,  ibid.,  41. 

2  Yirchow's  Festschrift,  also  Her.  d.  deutsch.  chem.  Gesellsch.,  25,  Referatb.,  685. 

3  Simdeek,  Centralbl.  f.  Physiol.,  1";  Stoklasa,  Jelinek,  and  Cerny,  ibid.,  16.      In 
regard  to  opposed  statements  see  Harden  and  Mac  Lean,  Journ.  of  Physiol.,  42. 

4  Embden  and  Almagia  with  F.  Kraus,  Bioch.  Zeitschr.  45;  S.  Oppenheimer,  ibid.,  45. 

5  Dakin  and  Dudley,  Journ.  of    biol.  Chem.,  14;  Neuberg,  Bioch.   Zeitschr.,  49; 
Levene  and  Meyer,  Journ.  of  biol.  Chem.,  14. 


LACTIC  ACIDS.  585 

The  phosphocarnic  acid  (Siegfried)  and  the  inosite  are  also  considered 
as  possible  mother-substances  for  sarcolactic  acid.  Further  research 
will  show  whether  also  other  mother-substances  for  this  acid  occur.  The 
autolytic  experiments  of  Tuhkel  x  with  livers  and  the  formation  of  lactic 
acid  in  the  muscles,  not  from  carbohydrates,  inosite  or  alanine,  as  observed 
by  Embden  2  and  his  collaborators  seem  to  indicate  this. 

The  lactic  acids  are  amorphous.  They  have  the  appearance  of 
colorless  or  faintly  yellowish,  acid-reacting  syrups,  which  mix  in  all  pro- 
portions with  water,  alcohol,  or  ether.  The  salts  are  soluble  in  water, 
and  most  of  them  also  in  alcohol.  The  two  acids  are  differentiated  from 
each  other  by  theit  different  optical  properties — paralactic  acid  being 
dextrogyrate,  while  fermentation  lactic  acid  is  optically  inactive — also 
by  their  different  solubilities  and  the  different  amounts  of  water  of  crys- 
tallization cf  the  calcium  and  zinc  salts.  The  zinc  salt  of  fermentation 
lactic  acid  dissolves  in  58-63  parts  of  water  at  14-15°  C,  and  contains 
18.18  per  cent  water  of  crystallization,  corresponding  to  the  formula, 
Zn(C3Hs03)2+3H20.  The  zinc  salt  of  paralactic  acid  dissolves  in  17.5 
parts  of  water  at  the  above  temperature  and  contains  ordinarily  12.9 
per  cent  water,  corresponding  to  the  formula,  Zn(C3H503)o+2H20. 
The  calcium  salt  of  fermentation  lactic  acid  dissolves  in  9.5  parts  water 
and  contains  29.22  per  cent  (=5  molecules)  water  of  crystallization, 
while  calcium  paralactate  dissolves  in  12.4  parts  water  and  contains  24.83 
or  26.21  per  cent  (  =  4  or  4^  molecules)  water  of  crystallization.  Both 
calcium  salts  crystallize,  not  unlike  tyrosine,  in  spears  or  tufts  of  very 
fine  microscopic  needles.  Hoppe-Seyler  and  Araki,  who  have  closely 
studied  the  optical  properties  of  the  lactic  acids  and  lactates,  consider 
the  lithium  salt  as  best  suited  for  the  preparation  and  quantitative  estima- 
tion of  the  lactic  acids.  The  lithium  salt  contains  7.29  per  cent  Li.  For 
further  information  as  to  the  salts  and  specific  rotation  of  the  lactic  acids 
see  Hoppe-Seyler-Thierfelder's  Handbuch.  8.  Aufl.,  1909.3 

Lactic  acids  may  be  detected  in  organs  and  tissues  in  the  following 
manner:  After  complete  extraction  with  water,  the  protein  is  removed 
by  coagulation  at  boiling  temperature  and  the  addition  of  a  small  quan- 
tity of  sulphuric  acid.  The  liquid  is  then  exactly  neutralized,  while 
boiling,  with  caustic  baryta,  and  then  evaporated  to  a  syrup  after  filtra- 
tion. The  residue  is  precipitated  with  absolute  alcohol,  and  the  pre- 
cipitate completely  extracted  with  alcohol.  The  alcohol  is  entirely 
distilled  from  the  united  alcoholic  extracts,  and  the  neutral  residue  is 

1  Tiirkel,  Bioch.  Zeitsehr.,  20.  The  statements  on  the  formation  of  lactic  acid 
in  the  muscle  autolysis  are  rather  conflicting;  see  Fletcher,  Journ.  of  Physiol.,  43. 

2  Embden,  Kalberlah  and  Engel,  Bioch.  Zeitsehr.  45;  Kondo,  Und.,  45. 

3  See  also  E.  Jungfleisch,  Compt.  Rend.,  139,  140,  and  142;  Herzog  and  Slansky, 
Zeitsehr.  f.  physiol.  Chem.,  73. 


586  MUSCLES. 

shaken  with  ether  to  remove  the  fat.  The  residue  is  dissolved  in  water 
and  phosphoric  acid  is  added,  and  the  solution  repeatedly  shaken  with  fresh 
quantities  of  ether,  which  dissolves  the  lactic  acid.  The  ether  is  new 
distilled  from  the  united  ethereal  extracts,  the  residue  dissolved  in  water, 
and  this  solution  carefully  warmed  on  the  water-bath  to  remove  the  last 
traces  of  ether  and  volatile  acids.  A  solution  of  zinc  lactate  is  prepared 
from  this  filtered  solution  by  boiling  with  zinc  carbonate,  and  this  is 
evaporated  until  crystallization  commences,  and  is  then  allowed  to  stand 
over  sulphuric  acid.  An  analysis  of  the  salts  is  necessary  in  careful 
work.  In  regard  to  methods  for  the  detection  and  quantitative  estima- 
tion of  lactic  acid  we  must  refer  to  larger  hand-books. 

Fat  is  never  absent  in  the  muscles.  Some  fat  is  always  found  in  the 
intermuscular  connective  tissue;  but  the  muscle-fibers  themselves  also 
contain  fat.  The  quantity  of  fat  in  the  real  muscle  substance  is  always 
small,  usually  amounting  to  about  10  p.  m.  or  somewhat  more.  A  con- 
siderable quantity  of  fat  in  the  muscle-fibers  is  found  only  in  fatty  degenera- 
tion. A  part  of  the  muscle-fat  can  be  readily  extracted,  while  another 
part  can  be  extracted  only  with  the  greatest  difficulty.  This  latter 
part,  it  is  claimed,  exists  finely  divided  in  the  contractile  substance 
itself  and  is  richer  in  free  fatty  acids,  standing,  according  to  Zuntz  and 
Bogdanow,1  in  close  relation  to  the  activity  of  the  muscles  because 
it  is  consumed  during  work.  Lecithin  is  a  regular  constituent  of  the 
muscles,  and  it  is  quite  possible  that  the  fat  which  is  difficult  of  extrac- 
tion and  which  is  rich  in  fatty  acids  depends  in  part  on  a  decomposition 
of  the  lecithin  and  the  phosphatides.  Erlandsen  has  shown  that 
phosphatides  of  various  kinds  occur  in  the  muscles,  the  quantities 
varying  in  different  muscles.  According  to  him  the  ox-heart  muscle 
is  richer  in  phosphatides  than  the  muscle  of  the  thigh,  and  Rubow2 
claims  that  the  heart  of  the  dog  is  richer  in  phosphatides  than  the  striated 
muscle.  Erlandsen  found  lecithin  and  diamino-phosphatide  in  the 
heart  as  well  as  the  thigh-muscle,  while  the  monoamido-phosphatide 
cuorin,  which  occurs  abundantly  in  the  heart,  is  found  as  traces  in  the 
thigh-muscle.  Costantino  3  has  carried  on  investigations  on  the  divi- 
sion of  the  inorganic  and  organic  phosphorus  in  striated  and  smooth 
muscles. 

The  Mineral  Bodies  of  the  Muscles.  The  ash  remaining  after  burning 
the  muscle,  which  amounts  to  about  10-15  p.  m.,  calculated  on  the  moist 
muscle,  is  acid  in  reaction.  The  largest  constituent  of  the  ash  is  potas- 
sium, whose  occurrence,  according  to  Macallum,4  is  restricted  to  the  dark 

1  Arch.  f.  ( Anat.  u.)  Physiol.,  1897. 

2  Erlandsen,  Zeitschr.  f.  physiol.  Chem.,  51;  Rubow,  Arch.  f.  exp.  Path.  u.  Pharm., 
52. 

1  Bioch.  Zeitschr.,  43. 
♦Journ.  of  Physiol.,  32. 


MINERAL  BODIES.  587 

diagonal  bundles,  and  phosphoric  acid.  Next  in  amount  we  have  sodium 
and  magnesium,  and  lastly  calcium,  chlorine,  and  iron  oxide.  Sulphates 
exist  only  as  traces  in  the  muscles,  but  are  formed  by  the  burning  of  the 
proteins  of  the  muscles,  and  therefore  occur  in  abundant  quantities  in  the 
ash.  The  muscles  contain  such  a  large  quantity  of  potassium  and  phos- 
phoric acid,  that  potassium  phosphate  seems  to  be,  unquestionably,  the 
predominating  salt.  Chlorine  is  found  in  such  insignificant  quantities 
that  it  is  perhaps  derived  from  a  contamination  with  blood  or  lymph. 
The  quantity  of  magnesium  is,  as  a  rule,  considerably  greater  than  that 
of  calcium.  Iron  occurs  only  in  very  small  amounts.  The  water  of  the 
muscle  occurs  in  part  free  and  partly  as  imbibition  water  of  the  colloids. 
According  to  the  investigations  of  Jensen  and  Fischer1  only  a  small  part, 
a  few  per  cent,  of  the  total  water  exists  in  this  condition. 

Urano  2  has  removed  the  salts  of  the  intermediary  fluid  (blood, 
lymph)  from  frogs'  muscles  by  treating  them  with  an  isotonic  cane-sugar 
solution  (of  6  per  cent)  and  in  this  manner  found  that  the  sodium  did 
not  belong  to  the  muscle  substance  itself,  but  to  the  intermediary  fluid, 
while  at  least  a  small  part  of  the  chlorine  is  a  true  muscle  constituent. 
He  also  calculated,  from  the  quantity  of  sodium,  that  the  intermediary 
fluid,  if  it  has  about  the  same  composition  as  the  muscle  plasma,  makes 
up  about  one-sixth  of  the  volume  of  the  muscle.  According  to  further 
investigations  of  Urano  the  possibility  of  a  disturbance  in  the  osmotic 
propertiesof  the  muscle-fibers  by  the  sugar  solution  is  not  entirely  excluded, 
and  the  question  whether  the  muscle-fibers  are  free  from  sodium  or  not 
has  therefore  not  been  positively  decided.  Fahr's3  researches  make 
the  absence  of  sodium  in  frog's  muscle  very  probable. 

The  importance  of  the  various  mineral  bodies  for  the  function  of  the 
muscles  has  been  the  subject  of  numerous  investigations  and  by  many 
of  these  we  have  obtained  further  proof,  as  mentioned  in  a  previous 
chapter,  of  the  ion  action  of  the  electrolytes  and  the  antagonism  of 
different  ions.  These  researches  also  indicate  that  each  of  the  ions 
Na,  Ca,  and  K  plays  a  certain  part  in  the  maintenance  of  the  excitability, 
in  the  contraction  and  in  the  fatigue  of  the  muscle  (heart);  still  these 
investigations  have  not  led  to  concordant  results,  so  that  we  are  not  yet 
clear  as  to  the  action  of  these  ions.  Nevertheless  it  seems  to  be  estab- 
lished that  the  combined  action  of  various  ions  is  a  necessity  for  the  nor- 
mal function  of  the  muscles.  It  has  also  been  shown  that  it  is  possible 
to  maintain  the  muscle  (the  heart)  in  regular  activity  for  a  long  time  by 
means  of  a  transfusion  of  liquid  saturated  with  oxygen,  and  which  con- 


Jensen  and  Fischer,  Bioch.  Zeitschr.,  20. 

*  Zeitschr.  f.  Biol.,  50. 

3  Urano,  ibid.,  51;  P'ahr.,  ibid.,  52. 


588  MUSCLES. 

tained  about  7  p.  m.  NaCl,  besides  small  amounts  of  Cad2  (0.2  p.  m.), 
KC1  (0.1  p.  m.),  and  NaHC03  (0.1  p.  m.). 

The  gases  of  the  muscles  consist  of  large  quantities  of  carbon  dioxide 
besides  traces  of  nitrogen. 

In  regard  to  the  permeability  of  the  muscles  for  various  bodies  there 
are  the  complete  investigations  of  Overton.1  The  different  sheaths  of 
the  muscles,  the  sarcolemma  and  perimysium  internum,  offer  no  very 
great  resistance  to  the  diffusion  of  the  most  soluble  crystalloid  com- 
pounds, while  the  muscle-fibers,  on  the  contrary  (exclusive  of  the  sar- 
colemma), are  almost  if  not  entirely  impervious  to  most  inorganic  com- 
pounds and  to  many  organic  compounds.  The  muscle-fibers  themselves 
are  actually  semipermeable  structures  which  are  permeable  to  water 
but  not  to  the  molecules  or  ions  of  sodium  chloride  and  of  potassium  phos- 
phate. The  muscle-fibers,  as  well  as  the  various  sheaths,  are  impermeable 
to  colloids. 

The  behavior  of  the  numerous  bodies  investigated  cannot  be  discussed 
in  this  work.  The  general  rule  is  as  follows:  All  compounds  which, 
besides  having  a  marked  solubility  in  water,  are  readily  soluble  in  ethyl 
ether,  in  the  higher  alcohols,  in  olive-oil  and  in  similar  organic  solvents, 
or  are  not  much  less  soluble  in  the  last-mentioned  solvents  than  in  water, 
pass  through  the  living  muscle-fibers  with  great  ease.  The  greater  the 
difference  between  the  solubility  of  a  compound  in  water  and  in  the  other 
solvents  mentioned,  the  slower  does  the  passage  into  the  muscle-fibers 
take  place.  The  permeability  changes  essentially  on  the  death  of  the 
muscle. 

The  living  muscle-fibers  are  readily  permeable  to  oxygen,  carbon 
dioxide,  and  ammonia,  while  the  hexoses  and  disaccharides  do  not  readily 
pass  into  them.  It  is  very  remarkable  that  a  great  portion  of  those 
compounds  which  take  part  in  the  normal  metabolism  of  plants  and 
animals  belongs  to  those  bodies  to  which  the  muscle-fibers  (and  also  other 
cells)  are  entirely  or  at  least  nearly  impermeable.  On  the  contrary, 
derivatives  can  be  prepared  from  these  bodies  which  pass  into  the  cells 
very  readily,  and  Overton  finds  that  it  is  not  impossible  that  the  organ- 
ism in  part  makes  use  of  a  similar  artifice  in  order  to  regulate  the  concen- 
tration of  the  nutritive  bodies  within  the  protoplasm.     (See  Chapter  I.) 

Rigor  Mortis  of  the  Muscles.  If  the  influence  of  the  circulating 
oxygenated  blood  is  removed  from  the  muscles,  as  after  the  death  of 
the  animal  or  by  ligature  of  the  aorta  or  the  muscle-arteries  (Stenson's 
test),  rigor  mortis  sooner  or  later  takes  place.  The  ordinary  rigor 
appearing  under  these  circumstances  is  called  the  spontaneous  or  the 


1  Pfliiger's  Arch.,  92.     See  also  Hober,  ibid.,   100,  and  Hamburger,  Osmotischer 
Druck  und  Ionenlehre.  Bd.  3. 


RIGOR  MORTIS.  589 

fermentative  rigor,  because  it  seems  to  depend  in  part  on  the  action  of 
an  enzyme.  A  muscle  may  also  become  stiff  or  other  reasons.  The 
muscles  may  become  momentarily  stiff  by  warming,  in  the  case  of  frogs 
to  40°,  in  mammalia  to  48-50°,  and  in  birds  to  53°  C.  Distilled  water 
may  also  produce  a  rigor  in  the  muscles  (water-rigor).  Acids,  even  very 
weak  ones,  such  as  carbon  dioxide,  may  quickly  produce  a  rigor  (acid- 
rigor),  or  hasten  its  appearance.  A  number  of  chemically  different 
substances,  such  as  chloroform,  ether,  alcohol,  ethereal  oils,  caffeine, 
and  many  alkaloids,  produce  a  similar  effect. 

When  the  muscle  passes  into  rigor  mortis  it  becomes  shorter  and 
thicker,  harder  and  non-transparent,  and  less  ductile.  The  acid  part 
of  the  amphoteric  reaction  becomes  stronger,  which  is  explained  by  most 
investigators  by  the  assumption  of  a  formation  of  lactic  acid.  There  is 
hardly  any  doubt  that  this  increase  in  acidity  may  at  least  in  part  be 
due  to  a  transformation  of  a  part  of  the  diphosphate  into  monophosphate 
by  the  lactic  acid.  The  statements  as  to  whether  in  the  rigor  mortis 
muscles,  besides  acid  phosphate  also  free  lactic  acid  exists  or  not  are 
rather  contradictory;1  that  an  acid  formation  precedes  the  rigor  is  gen- 
erally admitted  and  this  acid  formation  is  now  accepted  as  being  in  close 
relation  to  the  rigor.  While  we  used  to  consider  the  appearance  of  a  clot 
consisting  of  myosin  (Kuhne)  or  cf  myogen-  and  myosin  fibrin  (v.  Furth) 
as  the  essential  moment  for  the  rigor,  we  now  admit,  based  upon  the 
investigations  of  Meigs,  v.  Furth  and  Lenk,2  that  the  most  essential 
factor  is  the  imbibition  of  the  disdiaclasts,  which  become  broader  or 
shorter,  by  their  taking  up  of  water  from  the  sarcolemma  fluid  and  this 
action  produced  by  the  acid  formation.  This  view  stands  in  accord  with 
the  experience  on. the  imbibition  of  colloids  and  muscles  in  water  or  calt 
solutions,  in  the  presence  and  absence  of  acid,  as  well  as  the  fact  that  the 
rigor  can  be  retarded  by  the  artificial  circulation  of  blood  or  by  the  action 
of  salt  solutions,  namely  by  those  which  contain  small  amounts  of 
NaHCOs.  This  also  agrees  well  with  the  old  experience,  that  the  muscle 
work,  which  is  also  connected  with  a  formation  of  acid,  accelerates  the 
appearance  of  rigor. 

On  further  post-mortal  changes,  namely  by  a  further  accumulation 
of  acid,  a  progressive  coagulation  of  the  proteins  gradually  occurs.  In 
this    coagulation    the    ability   of  the   colloid  systems  to   imbibe  water 


1  It  is  impossible  to  enter  into  the  details  of  the  disputed  theories  as  to  the  reac- 
tion of  the  muscles,  etc.  We  shall  only  refer  to  the  works  of  Rohmann,  Pfluger's 
Arch.,  50  and  55,  and  Heffter,  Arch.  f.  exp.  Path.  u.  Pharm.,  31  and  38.  These 
works  contain  also  the  researches  of  the  earlier  investigators  more  or  less  completely. 

2  Meigs,  Journ.  of  Physiol.  39  and  especially,  Amer.  Journ.  of  Physiol.,  24  and 
26;  v.  Furth  and  Lenk,  Bioch.  Zeitschr.,  33,  and  Wien.  klin.  Wochenschr.,  24  (1911). 


590  MUSCLES. 

diminishes,  water  is  given  off,  and  a  re-imbibition  takes  place  and  the 
so-called  "  solution  of  the  rigor  "  appears  (v.  Furth  and  Lenk). 

The  ordinary  rigor  is  an  acid  rigor  and  the  same  applies,  according 
to  Meigs,1  to  the  water  rigor  as  a  shortening  of  the  muscles  takes  place 
when  placed  in  distilled  water,  by  a  formation  of  lactic  acid,  and  because 
when  such  a  muscle  is  placed  in  Ringer's  solution  the  acid  is  removed 
and  the  muscle  again  expands. 

The  views  are  rather  contradictory  in  regard  to  the  production  of  heat 
rigor.  According  to  v.  Furth  this  rigor  depends  upon  the  coagulation 
of  certain  proteins,  and  its  occurrence  at  lower  temperatures  in  cold- 
blooded as  compared  with  warm-blooded  animals  is  due,  according  to 
v.  Furth,  to  the  fact  that  in  the  first  a  soluble  myogen  fibrin  occurs 
preformed  in  the  muscle  which  coagulates  at  30-40°  C,  while  in  the 
warm-blooded  animals  the  coagulating  substance  is  musculin  (myosin 
of  v.  Furth)  which  coagulates  at  a  higher  temperature.  According 
to  Inagaki  2  the  various  stages  in  contractions  occurring  on  heating  a 
muscle  (frog)  do  not  correspond  to  those  of  the  coagulation  of  the  pro- 
tein which  would  occur  on  heating  the  muscle  plasma,  and  Meigs  has 
arrived  at  a  similar  view.  It  must  be  remarked  that  also  a  lactic  acid 
formation  takes  place  on  heating  a  muscle,  and  this  prevents  an  exact 
comparison  of  the  coagulation  of  the  proteins  within  and  outside 
of  the  muscle.  The  observations  of  Vernon  that  the  striated  and 
the  smooth  muscles  on  heating  to  between  40  and  50°  behave  differently, 
in  that  the  striated  become  shorter  and  the  smooth  become  longer, 
while  both  kinds  become  shorter  at  higher  temperatures,  indicates  against 
a  coagulation  at  these  low  temperatures.  According  to  Meigs  3  we  must 
here  also  admit  of  an  imbibition  rigor,  due  to  the  formation  of  lactic  acid, 
and  the  different  behavior  of  the  two  kinds  of  muscle  depends  upon  a 
different  arrangement  of  their  anatomical  elements. 

The  chemical  rigor  produced  by  different  chemically  active  substances 
is  also  produced,  according  to  Meigs  as  well  as  to  v.  Furth  and  Lenk, 
upon  a  formation  of  acid,  causing  a  chemical  damage  of  the  muscles,  and 
is  to  be  considered  as  an  iml  ibition  rigor. 

As  it  is  now  generally  admitted  that  the  formation  of  lactic  acid  dur- 
ing the  death  of  the  muscle  is  the  cause  of  the  muscle  rigor,  the  question 
arises,  from  what  constituents  of  the  muscle  is  this  acid  derived?  The 
most  probable  explanation  is  that  the  lactic  acid  is  produced  from  the 
glycogen,  as  certain  investigators,  such  as  Nasse  and  Werther,  have 
observed  a  decrease  in  the  quantity  of  glycogen  in  rigor  of  the  muscle. 


1  Journ.  of  Physiol.,  39. 

2  Inagaki,  Zeitschr.  f.  Biol.,  48;  Meigs,  Journ.  of  Physiol.,  24. 

3  Vernon,  Journ.  of  Physiol.,  24;  Meigs,  Amer.  Journ.  of  Physiol.,  24. 


METABOLISM  IN  THE  MUSCLES.  591 

On  the  other  side,  Eohm  has  observed  cases  in  which  no  consumption 
of  glycogen  took  place  in  rigor  of  the  muscle,  and  he  also  found  that  the 
quantity  of  lactic  acid  produced  is  not  proportional  to  the  quantity  of 
glycogen.  According  to  Moscati  l  the  diminution  in  the  glycogen  is- 
independent  of  the  appearance  of  rigor.  It  is  therefore  possible  that  the 
consumption  of  glycogen  and  the  formation  of  lactic  acid  in  the  muscles 
are  two  processes  independent  of  each  other,  and,  as  above  stated  in  regard 
to  the  formation  of  paralactic  acid,  the  origin  of  the  lactic  acid  in  the 
muscle  is  still  not  positively  known.  The  phosphocarnic  acid  must 
also  be  considered  as  a  mother-substance  of  the  lactic  acid,  and  of  the 
carbon  dioxide,  also  formed  in  the  rigor,  as  it  yields  lactic  acid  as  well  as 
carbon  dioxide  on  its  cleavage. 

Metabolism  in  the  Inactive  and  Active  Muscles.  It  is  admitted 
by  a  number  of  prominent  investigators,  Pfluger  and  Colasanti, 
Zuntz  and  Rohrig  2,  and  others,  that  the  metabolism  in  the  muscles 
is  regulated  by  the  nervous  system.  When  at  rest,  when  there  is  no 
mechanical  exertion,  there  exists  a  condition  which  Zuntz  and  Rohrig 
have  designated  "  chemical  tonus."  This  tonus  seems  to  be  a  reflex 
tonus,  for  it  may  be  reduced  by  discontinuing  the  connection  between  the 
muscles  and  the  central  organ  of  the  nervous  system  by  cutting  through 
the  spinal  cord  or  the  muscle-nerves.  The  possibility  of  reducing  the 
chemical  tonus  of  the  muscles  in  various  ways  offers  an  important  means 
of  deciding  the  extent  and  kind  of  chemical  processes  going  on  in  the 
muscles  when  at  rest.  In  comparative  chemical  investigation  of  the 
processes  in  the  active  and  the  inactive  muscles  several  methods  of  pro- 
cedure have  been  adopted.  The  same  active  and  inactive  muscles  have 
been  compared  after  removal,  also  the  arterial  and  venous  muscle-blood 
in  rest  and  activity,  and  lastly  the  total  exchange  of  material,  the  receipts 
and  expenditures  of  the  organism,  have  been  investigated  under  these 
two  conditions. 

By  investigations  according  to  these  several  methods  it  was  found 
that  the  resting  muscle  takes  up  oxygen  from  the  blood  and  returns  to 
it  carbon  dioxide,  and  also  that  the  quantity  of  oxygen  taken  up  is  greater 
than  the  oxygen  contained  in  the  carbon  dioxide  eliminated  at  the  same 
time.  The  muscle,  therefore,  holds  in  some  form  of  combination  a  part 
of  the  oxygen  taken  up  while  at  rest.  During  activity  the  exchange  of 
material  in  the  muscle,  and  therewith  the  exchange  of  gas,  is  increased. 

1  Nasse,  Beitr.  z.  Physiol,  der  kontrakt.  Substanz,  Pfluger's  Arch.,  2;  Werther, 
ibid.,  46;  Bohm,  ibid.,  23  and  46;  Moscati,  Hofmeister's  Beitrage,  10. 

2  See  the  works  of  Pfliiger  and  his  pupils  in  Pfluger's  Arch.,  4,  12,  14,  16,  and  18; 
Rohrig,  ibid.,  4.  See  also  Zuntz,  ibid.,  12.  In  regard  to  the  metabolism  after  curare 
poisoning,  see  also  Frank  and  Voit,  Zeitschr.  f.  Biologie,  42,  and  Frank  and  Geb- 
hard,  ibid.,  43. 


592  MUSCLES. 

The  animal  organism  takes  up  much  more  oxygen  in  activity  than 
when  at  rest,  and  eliminates  also  considerably  more  carbon  dioxide.  The 
quantity  of  oxygen  which  leaves  the  body  as  carbon  dioxide  during 
activity  is  much  larger  than  the  quantity  of  oxygen  taken  up  at  the  same 
time;  and  the  venous  muscle-blood  is  poorer  in  oxygen  and  richer  in 
carbon  dioxide  during  activity  than  during  rest.  The  exchange  of  gases 
in  the  muscles  during  activity  is  the  reverse  of  that  at  rest,  for  the  active 
muscle  gives  up  a  quantity  of  carbon  dioxide  which  does  not  correspond 
to  the  quantity  of  oxygen  taken  up,  but  is  considerably  greater.  It 
follows  from  this  that  in  muscular  activity  not  only  does  oxidation  take 
place,  but  also  splitting  processes  occur.  This  also  results  from  the  fact 
that  removed  blood-free  muscles  when  placed  in  an  atmosphere  devoid  of 
oxygen  can  labor  for  some  time  and  still  yield  carbon  dioxide  (Hermann1). 

During  muscular  inactivity,  in  the  ordinary  sense,  a  consumption 
of  glycogen  takes  place.  This  is  inferred  from  the  observations  of  sev- 
eral investigators,  that  the  quantity  of  glycogen  is.  increased  and  its  cor- 
responding consumption  reduced  in  those  muscles  whose  chemical  tonus 
is  reduced  either  by  cutting  through  the  nerve  or  for  other  reasons 
(Bernard,  Chandelon,  Vay,2  and  others).  In  activity  this  consump- 
tion of  glycogen  is  increased,  and  it  has  been  positively  proved  by  the 
researches  of  numerous  investigators3  that  the  quantity  of  glycogen 
in  the  muscles  in  activity  decreases  quickly  and  freely.  The  sugar  is 
removed  from  the  blood  and  consumed  during  activity.4  The  recent 
investigations  of  Joh.  Muller,  Locke  and  Rosenheim  and  Camis  5 
have  given  direct  proof  of  the  consumption  of  sugar  during  muscular 
activity.  In  experiments  on  surviving  hearts  of  different  animals  through 
which  was  perfused  a  salt  solution  containing  sugar,  they  could  detect 
an  undoubted  consumption  of  sugar  which  was  quite  considerable  and 
which  to  all  appearances  was  used  as  material  for  muscle  work. 

The  amphoteric  reaction  of  the  inactive  muscles  is  changed  during 

1 L.  Hermann,  Unters.  iiber  d.  Stoffwechsel  der  Muskeln,  etc.,  Berlin,  1867. 
In  regard  to  gas  exchange  in  removed  muscles,  see  also  J.  Tissot,  Arch,  de  Physiol. 
(5),  6  and  7,  and  Compt.  Rend..  120. 

1  Chandelon,  Pfluger's  Arch..  13;  Vay,  Arch.  f.  exp.  Path.  u.  Pharm.,  34,  which 
also  contains  the  pertinent  literature. 

*  Nasse,  Pfluger's  Arch.,  2;  Weiss,  Wien.  Sitzungsber.,  64;  Kiilz,  in  Ludwig's 
Festschrift,  Marburg,  1890;  Marcuse,  Pfluger's  Arch.,  39;  Manchd,  Zeitschr.  f.  Bio- 
logie,  25;  Moratand  Dufour,  Arch  dc  Physiol.  (5),  4. 

4  Chauveau  and  Kaufmann,  Compt.  Rend.,  103,  104,  and  105;  Quinquaud,  Maly's 
Jahresber.,  16;  Morat  and  Dufour,  1.  c;  Cavazzani,  Centralbl.  f.  Physiol.,  8;  Seegen, 
''  Die  Zuckerbildung  im  Thicrkorpcr,"  Berlin,  1890,  Centralbl.  f.  Physiol.,  8,  9,  and 
10;  Arch.  f.  <  Anat.  u.)  Physiol .  1895  and  1896;  Pfluger's  Arch.,  50. 

6  Joh.  Muller,  Zeitschr.  f  allgem.  Physiol.,  3;  Camis,  ibid.,  8;  Locke  and  Rosen- 
heim, Journ.  of  Physiol ,  36. 


LACTIC  ACID  FORMATION   IN  ACTIVE  MUSCLES.  593 

activity  to  an  acid  reaction  (Du  Bois-Reymond  and  others),  and  the 
acid  reaction  increases,  to  a  certain  point,  with  the  work.  The  quickly 
contracting  pale  muscles  produce,  according  to  Gleiss,1  more  acid  dur- 
ing activity  than  the  more  slowly  contracting  red  muscles.  Numerous 
investigations  have  been  carried  out  on  the  cause  of  this  increased  acid 
reaction,  using  the  muscles  in  situ  and  also  upon  removed  muscles  and 
rather  contradictory  results  have  been  obtained.  Some  have  found  a 
diminution  in  the  amount  of  lactic  acid  in  the  active  muscle  while  others 
have  found  an  increase.2  The  work  of  Fletcher  and  Hopkins3  is  of 
great  importance  in  this  disputed  question,  in  which  they  show  that  in 
the  removal  of  the  muscle,  and  in  its  preparation  for  the  testing  for  lactic 
acid  several  sources  of  error  are  possible.  The  mechanical  irritation 
as  well  as  warming  or  treating  the  muscle  with  alcohol  (not  ice-cold) 
can  lead  to  a  formation  of  lactic  acid.  It  was  also  shown  that  the 
absence  of  oxygen  accelerated  the  formation  or  accumulation  of  lactic 
acid,  while  an  abundance  of  oxygen  had  the  opposite  effect. 

It  is  evident  that  the  experiments  with  the  muscles  in  situ — in  other 
words,  with  muscles  through  which  blood  is  passing — cannot  yield  any 
conclusion  to  the  above  question,  as  the  lactic  acid  formed  during  work 
may  perhaps  be  removed  by  the  blood.  The  following  objections  can 
be  made  against  those  experiments  in  which  lactic  acid  has  been  found, 
after  moderate  work,  in  the  blood  or  the  urine,  as  also  especially  against 
the  experiments  with  removed  active  muscles,  namely,  that  in  these  cases 
the  supply  of  oxygen  to  the  muscles  was  not  sufficient,  and  that  the 
lactic  acid  formed  thereby  is  not,  in  accordance  with  the  views  of  Hoppe- 
Seyler,  a  perfectly  normal  process.  The  same  is  probably  true  also  for 
the  formation  of  lactic  acid  with  excessive  work  during  life,  and  Zillessen  4 
has  found  that  the  artificial  cutting  off  of  the  oxygen  supply  in  the 
muscles  during  life,  that  more  lactic  acid  was  formed  than  under  normal 
conditions.  Other  observations  indicate  a  formation  of  lactic  acid  dur- 
ing activity.  Thus  Spiro  and  recently  also  H.  Fries5  found  an  increase 
in  the  quantity  of  lactic  acid  in  the  blood  during  work.  Colasanti 
and  Moscatelli  found  small  quantities  of  lactic  acid  in  human  urine 
after  strenuous  marches,  and  Werther6  observed  an  abundance  of  lactic 
acid  in  the  urine  of  frogs  after  tetanization. 

1  Pfliiger's  Arch.,  41. 

2  Astaschewsky,  Zeitschr.  f.  physiol.  Chem.,  4;  Warren,  Pfliiger's  Arch.,  24, 
Monari,  Maly's  Jahresber.,  19;  Heffter,  Arch.  f.  exp.  Path.  u.  Pharm.,  31;  Marcuse, 
1.  c;  Werther,  Pfliiger's  Arch.,  46;  Spiro,  Zeitschr.  f.  physiol.  Chem.,  1;   Colasanti. 

3  Journ.  of  Physiol.,  35. 

4  Hoppe-Seyler,  1.  c.  and  Zeitschr.  f.  physiol.  Chem.,  19,  476;  Zillessen,  ibid.,  15. 
6  Spiro,  Zeitschr.  f.  physiol.  Chem.  1;  Fries,  Bioch.  Zeitschr.,  35. 

•Colasanti  and  Moscatelli;  Maly's  Jahresb..  17,  212;  Werther,  Pfliiger's  Arch.,  46. 


594  MUSCLES. 

According  to  Siegfried  the  amount  of  phosphocarnic  acid  is  dimin- 
ished during  activity.  Macleod  *  claims  that  this  is  true  only  for 
intense  muscular  activity,  and  the  mother-substance  of  lactic  acid  can 
at  least  in  part  be  phosphocarnic  acid.  The  question  as  to  the  forma- 
tion of  lactic  acid  during  activity,  and  the  origin  of  the  phosphocarnic 
acid  is  certainly  in  many  points  somewhat  undecided;  the  general 
view  seems  to  be,  that  during  work  lactic  acid  is  formed,  which  transforms 
a  part  of  the  diphosphates  into  monophosphates. 

The  amount  of  proteins  in  the  removed  muscles  is,  according  to  the 
earlier  investigators,  decreased  by  work.  The  correctness  of  this  state- 
ment is,  however,  disputed  by  other  investigators.  Earlier  reports  in 
regard  to  the  nitrogenous  extractive  bodies  of  the  muscle  in  rest  and 
in  activity  are  likewise  uncertain.  According  to  the  recent  researches 
of  Monari  2  the  total  quantity  of  creatine  and  creatinine  is  increased  by 
work,  and  indeed  the  amount  of  creatinine  is  especially  augmented 
by  an  excess  of  muscular  activity.  The  creatinine  is  formed  essentially 
from  the  creatine.  The  investigations  of  Graham  Brown  and  Cathcart 
on  removed  nerve-muscle  preparations  of  frogs,  and  those  of  S.  Weber3 
on  hearts,  indicate  an  increase  in  the  formation  of  creatine  and  creatinine 
during  work.  Weber  found  that  the  working  heart  gave  up  creatine 
(and  creatinine)  to  Ringer's  solution,  and  indeed  much  more  when 
strongly  active  than  during  a  lesser  activity.  An  increased  creatinine 
elimination  after  work  does  not  occur  according  to  several  investigators 
(see  Chapter  XIV)  and  according  to  Pekelharing  and  v.  Hoogenhuyze 
with  ordinary  muscle  activity  neither  an  increased  creatine  formation 
nor  an  increased  creatinine  elimination  takes  place.  In  the  tonic 
contraction  the  creatine  is  formed  from  the  proteins,  and  correspond- 
ingly according  to  Pekelharing  and  Harkink4  the  creatinine  elimina- 
tion is  increased  under  the  influence  of  the  muscle  tonus.  The  purine 
bases  are  produced,  according  to  Burian,  in  the  muscles  themselves, 
also  in  activity,  and  an  increased  formation  takes  place  during  work 
due  to  a  re-formation.  Scaffidi5  found  on  the  contrary,  with  frogs  and 
tortoise,  during  work  that  a  diminution  of  the  total  quantity  of  purine 
bases  occurred  and  indeed  not  the  free  but  the  combined  purines. 

Attempts  have  been  made  to  solve  the  question  relative  to  the 
behavior  of  the  nitrogenized  constituents  of  the  muscle  at  rest  and  during 

1  Siegfried,  Zeitschr.  f.  physiol.  Chem.,  21;  Macleod,  ibid.,  28. 

5  Maly's  Jahresber.,  19,  296. 

» Cathcart  and  Graham  Brown,  Journ.  of  Physiol.,  37;  Weber,  Arch.  f.  exp.  Path, 
u.  Pharm.,  58. 

*  Pekelharing  and  v.  Hoogenhuyze,  Zeitschr.  f.  physiol.  Chem..  64,  with  Harkink, 
ibid.,  75. 

6  Burian,  Zeitschr.  f.  physiol.  Chem.,  43;  Scaffidi;  Bioch.  Zeitschr.,  30. 


NITROGEN   CATABOLI8M  IN  ACTIVE  MUSCLES.  595 

activity  by  determining  the  total  quantity  of  nitrogen  eliminated  under 
these  different  conditions  of  the  body.  While  formerly  it  was  held  with 
Liebig  that  the  elimination  of  nitrogen  by  the  urine  was  increased  by 
muscular  work,  the  researches  of  several  experimenters,  especially  those 
of  Voit  on  dogs,  and  Pettenkofer  and  Voit  on  men,  have  led  to  quite 
different  results.  They  have  shown,  as  has  also  lately  been  confirmed 
by  other  investigators,  especially  I.  Munk  and  Hirschfeld,1  that  during 
work  no  increase,  or  only  a  very  insignificant  increase,  in  the  elimination 
of  nitrogen  takes  place. 

We  should  not  omit  to  mention  the  fact  that  a  series  of  experiments 
has  been  made  showing  a  significant  increase  in  the  metabolism  of  pro- 
teins during  or  after  work.  There  are  for  example  the  observations 
of  Flint  and  of  Pavy  on  a  pedestrian,  v.  Wolff,  v.  Funke,  Kreuzhage, 
and  Kellner  on  a  horse,  and  Dunlop  and  his  collaborators  on  working 
human  beings,  and  of  Krummacher,  Pfluger,  Zuntz  and  his  pupils,2 
and  others.  The  researches  on  the  elimination  of  sulphur  during  rest 
and  activity  also  belong  to  this  category.  The  elimination  of  nitrogen 
and  sulphur  runs  parallel  with  the  metabolism  of  proteins  in  resting  and 
active  persons,  and  the  quantity  of  sulphur  excreted  by  the  urine  is  there- 
fore also  a  measure  of  the  protein  catabolism.  The  earlier  researches 
of  Engelmann,  Flint,  and  Pavy,  as  well  as  the  more  recent  ones  of  Beck 
and  Benedict,3  and  Dunlop  and  his  collaborators,  show  an  increased 
elimination  of  sulphur  during  or  after  work,  and  this  indicates  an  increased 
protein  metabolism  because  of  muscular  activity. 

That  an  increased  destruction  of  protein  is  not  necessarily  produced 
by  work  follows  from  the  observations  of  Caspari,  Bornstein,  Kaup, 
Wait,  A.  Loewy,  Atwater  and  Benedict,4  that  a  retention  of  nitrogen 
and  a  deposition  of  protein  occur  during  work.  The  discordant  observa- 
tions on  the  protein  destruction  during,  and  caused  by,  work  are  not 
directly  in  opposition  to  each  other,  because  the  extent  of  protein 
metabolism  is  dependent  upon  many  conditions,  such  as  the  quantity 

1  Voit,  Untersuchungen  iiber  den  Einfluss  des  Kochsalzes,  des  Kaffees  und  der 
Muskelbewegungen  auf  den  Stoffwechsel  (Miinchen,  1860),  and  Zeitschr.  f.  Biologie, 
2;  J.  Munk,  Arch.  f.  (Anat.  u.)  Physiol.,  1890  and  1896;  Hirschfeld,  Virchow's  Arch., 
121. 

2  Flint,  Journ.  of  Anat.  and  Physiol.,  11  and  12;  Pavy,  The  Lancet,  1876  and  1877; 
v,  Wolff,  v.  Funke,  Kellner,  cited  from  Voit,  Hermann's  Handb.,  86,  197;  Dunlop 
Noel-Paton,  Stockman,  and  Maccadam,  Journ.  of  Physiol.,  22;  Krummacher,  Zeitschr. 
f.  Biologie,  33;  Pfluger,  Pfliiger's  Arch.,  50;  Zuntz,  Arch.  f.  (Anat.  u.)  Physiol.,  1894. 

3  Engelmann.  Arch.  f.  (Anat.  u.)  Physiol.,  1871;  Beck  and  Benedict,  Pfliiger's 
Arch.,  54,  and  also  footnote  2. 

"Caspari,  Pfluecr's  Arch.,  83;  Bornstein,  ibid.;  Kaup,  Zeitschr.  f.  Biologie,  43; 
Wait,  U.  S.  Depart  Agricult.  Bulletin,  89;  (1901)  Atwater  and  Benedict,  ibid.,  Bull., 
69  (1S99);  Loewy,  Arch.  f.  (Anat.  u.)  Physiol.,  1901. 


596  MUSCLES. 

and  composition  of  the  food,  the  condition  of  the  adipose  tissue  of  the 
body,  the  action  of  the  work  upon  the  respiratory  mechanism,  etc.,  all 
of  which  have  an  influence  on  the  results  of  the  experiments. 

What  has  been  said  above  in  regard  to  the  protein  catabolism  dur- 
ing muscular  activity  only  applies  for  the  metabolism  experiments 
carried  on  in  the  generally  accepted  manner.  Thomas  1  has  made  an 
experiment,  under  Rubner's  direction,  on  the  action  of  work  upon  the 
nitrogen  elimination  upon  a  person  when  the  nitrogen  minimum  was 
reduced  to  the  wrear  and  tear  quota  (see  Chapter  XVII),  and  this  experi- 
ment seems  to  indicate  a  small  increase  in  the  nitrogen  elimination  due 
to  work. 

The  older  investigations  on  the  amount  of  fat  in  muscles  removed 
after  activity  and  after  rest  have  not  led  to  any  definite  results.  Accord- 
ing to  the  investigations  of  Zuntz  and  Bogdanow,2  the  fat  belonging 
to  the  muscle-fibers,  which  is  extracted  with  difficulty,  takes  part  in  work. 
Besides  these  there  are  several  researches  by  Voit,  Pettenkofer  and 
Voit,  J.  Frentzel,3  and  others  which  make  an  increased  destruction 
of  fat  during  work  probable  or  proved. 

If  the  results  of  the  investigations  thus  far  made  of  the  chemical 

processes  going  on  in  the  active  and  inactive   muscles  were  collected, 

we  would  find  the  following  characteristics  for  the  active  muscle:     The 

active  muscle  takes  up  more  oxygen  and  gives  off  more  carbon  dioxide 

than  the  inactive  muscle;  still  the  elimination  of  carbon  dioxide  is  increased 

considerably    more    than   the    absorption    of    oxygen.     The    respiratory 

COo 
quotient,  ~pr~,  is  found  to  be  regularly  raised  during  work;   yet  this  rise, 

which  will  be  explained  in  detail  in  a  following  chapter  on  metabolism, 
can  hardly  be  conditioned  on  the  kind  of  processes  going  on  in  the  muscle 
during  activity  with  a  sufficient  supply  of  oxygen.  In  work  a  consump- 
tion of  carbohydrates,  glycogen,  and  sugar  takes  place.  The  acid  reac- 
tion of  the  muscle  becomes  greater  with  work.  In  regard  to  the  extent 
of  a  re-formation  of  lactic  acid  opinion  is  divided.  An  increased  con- 
sumption of  fat  has  occasionally  been  observed.  On  the  behavior  of 
creatine  Cor  creatinine)  and  purine  bodies  the  statements  are  some- 
what divergent.  Protein  metabolism  has  been  found  increased  in  cer- 
tain series  of  experiments  and  not  in  others;  but  an  increased  elimina- 
tion of  nitrogen  as  a  direct  consequence  of  muscular  exertion  has  thus 
far  not  been  positively  proved. 

In   close   connection   with    the    above-mentioned   facts   there   is   the 

1  Arch.  f.  (Anat.  u.)  Physiol.  1910,  Supplelbd- 

*Ibid.,  1897. 

•  Pfliiger's  Arch.,  68. 


MUSCLE  WORK.  597 

question  as  to  the  material  basis  of  muscular  activity  so  far  as  it  has 
its  origin  in  chemical  processes.  In  the  past  the  generally  accepted 
opinion  was  that  of  Liebig,  that  the  source  of  muscular  action  con- 
sisted of  a  catabolism  of  the  protein  bodies;  to-day  another  generally 
accepted  view  prevails.  FlCK  and  Wislicenus  1  climbed  the  Faulhorn 
and  calculated  the  amount  of  mechanical  force  expended  in  the  attempt. 
With  this  they  compared  the  mechanical  equivalent  transformed  in  the 
same  time  from  the  proteins,  calculated  from  the  nitrogen  eliminated  in 
the  urine,  and  found  that  the  work  really  performed  was  not  by  any 
means  compensated  by  the  consumption  of  protein.  It  was,  therefore, 
proved  by  this  that  proteins  alone  cannot  be  the  source  of  muscular 
activity  and  that  this  depends  in  great  measure  on  the  metabolism  of 
non-nitrogenous  substances.  Many  other  observations  have  led  to  the 
same  result,  especially  the  experiments  of  Voit,  of  Pettenkofer  and  Voit, 
and  of  other  investigators,  whose  observations  show  that  while  the 
elimination  of  nitrogen  remains  unchanged,  the  elimination  of  carbon 
dioxide  during  work  is  very  considerably  increased.  It  is  also  gen- 
erally considered  as  positively  proved  that  muscular  work  is  produced, 
at  least  in  greatest  part,  by  the  catabolism  of  non-nitrogenous  substances. 
Nevertheless  there  is  no  warrant  for  the  statement  that  muscular  activity 
is  produced  entirely  at  the  cost  of  the  non-nitrogenous  substances, 
and  that  the  protein  bodies  are  without  importance  as  a  source  of 
energy. 

The  investigations  of  Pfluger  2  are  of  great  interest  in  this  connec- 
tion. He  fed  a  bulldog  for  more  than  seven  months  with  meat  which 
alone  did  not  contain  sufficient  fat  and  carbohydrates  even  for  the  pro- 
duction of  heart  activity,  and  then  let  him  work  very  hard  for  periods 
of  14,  35,  and  41  days.  The  positive  result  obtained  by  these  series 
of  experiments  was  that  "  complete  muscular  activity  may  be  effected 
to  the  greatest  extent  in  the  absence  of  fat  and  carbohydrates,"  and  the 
ability  of  proteins  to  serve  as  a  source  of  muscular  energy  cannot  be 
denied. 

The  nitrogenous  as  well  as  the  non-nitrogenous  nutriments  may  serve 
as  a  source  of  energy;  but  the  views  are  divided  in  regard  to  the  relative 
value  of  these.  Pfluger  claims  that  no  muscular  work  takes  place 
without  a  decomposition  of  protein,  and  the  living  cell-substance  prefers 
always  the  protein  and  rejects  the  fat  and  sugar,  contenting  itself  with 
these  only  when  proteins  are  absent.  Other  investigators,  on  the  con- 
trary, believe  that  the  muscles  first  draw  on  the  supply  of  non-nitrogenous 


1  Vierteljahrsschr.  d.  Zurich,  naturf.  Gesellsch.,  10,  cited  from  Centralbl.  f.  d.  med. 
Wiss.,  18G6,  309. 

2  Pfliiger's  Arch.,  50. 


598  MUSCLES. 

nutriments,  and  according  to  Seegen,  Chauveau,  and  Laulani6  1 
the  sugar  is  the  only  direct  source  of  muscular  force.  The  last-men- 
tioned investigator  holds  that  the  fat  is  net  directly  utilized  for  work, 
but  only  after  a  previous  conversion  into  sugar.  Zuntz  and  his  collabora- 
tors have  made  strong  objections  to  the  correctness  of  such  a  view.  If 
according  to  Zuntz,  the  fat  must  be  first  transformed  into  sugar  before 
it  can  serve  as  the  source  of  muscular  work,  a  definite  expenditure  of 
force  must  require  about  30  per  cent  more  energy  with  fatty  food  than 
it  does  with  carbohydrates;  but  this  is  not  the  case.  The  investiga- 
tions of  Zuntz  (together  with),  Loeb,  Heinemann,  Frentzel  and  Reach 
show  that  all  foodstuffs  have  nearly  the  same  power  of  serving  as  the 
material  for  the  work  of  the  muscles.  The  extensive  metabolism  investi- 
gations of  Atwater  and  Benedict2  have  also  led  to  similar  results 
as  to  the  fats  being  a  source  of  muscular  energy.  The  law  of  the  sub- 
stitution of  the  foodstuffs,  according  to  their  combustion  equivalents, 
is  also  true  for  muscular  work,  and  fat  correspondingly  acts  with  its  full 
amount  of  energy  without  previously  being  transformed  into  sugar. 
The  question  which  of  the  foodstuffs  the  muscle  prefers  is  dependent  upon 
the  relative  quantities  of  the  same  at  the  disposal  of  the  muscle.  A 
direct  substitution  of  the  body  material  by  the  bodies  supplied  as  food 
does  not  take  place  in  the  muscular  activity  in  the  ordinary  nutritive 
condition.  According  to  Johansson  and  Koraen3  the  CO2  excretion 
produced  by  certain  work  is  not  influenced  by  the  supply  of  foodstuffs 
(protein  or  sugar). 

Siegfried  considers,  as  above  stated,  the  phosphocarnic  acid  as  a  source  of 
energy.  According  to  his  and  Kruger's4  researches,  phosphocarnic  acid,  which 
yields  on  cleavage,  among  other  bodies,  carbon  dioxide,  occurs  in  part  preformed 
in  the  muscle,  and  in  part  as  a  hypothetical  aldehyde  compound  of  the  same — 
a  compound  which  forms  phosphocarnic  acid  on  oxidation.  Siegfried  therefore 
makes  the  suggestion  that  in  the  resting  muscle,  which  requires  more  oxygen 
than  exists  in  the  carbon  dioxide  eliminated,  this  reducing  aldehyde  substance  is 
gradually  oxidized  to  phosphocarnic  acid,  which  is  used  in  the  activity  of  the 
muscle  with  the  splitting  off  of  carbon  dioxide. 

Quantitative  Composition  of  the  Muscle.  A  large  number  of  analyses 
have  been  made  of  the  flesh  of  various  animals  for  purely  practical 
purposes,  in  order  to  determine  the  nutritive  value  of  different  varieties 


1  See  Seegen,  footnote  4,  page  592.  The  works  of  Chauveau  and  his  collaborators 
are  found  in  Compt.  Rend.,  121,  122,  and  123;  Laulanie,  Arch,  de  Physiol.  (5),  8. 

-  Loeb,  Arch.  f.  (Anat.  u.)  Physiol.,  1894;  Heinemann,  Pfliiger's  Arch.,  83;  Frentzel 
and  Reach,  ibid.;  Atwater  and  Benedict,  U.  S.  Dept.  of  Agric,  Bull.  136,  and  Ergeb- 
nisse  der  Physiologic,  3. 

•  Skand.  Arch.  f.  Physiol.,  13. 

*  Zeitschr.  f.  physiol.  Chera.,  22. 


QUANTITATIVE  COMPOSITION   OF  THE  MUSCLES. 


599 


of  meat;  but  their  are  no  exacl  scientific  analyses  with  sufficient  regard 
to  the  quantity  of  different  protein  bodies  and  the  remaining  muscle 
constituents,  that  is,  these  analyses  are  incomplete  or  of  little  value. 
We  will  only  give  a  few  of  the  results  of  the  work  of  various  investigators. 
The  figures  are  parts  per  1000. 

Muscles  of  Muscles  of  Muscles  of 

Mammals.  Birds.  Cold-boolded 

Animals. 

Solids 217-278  225-282                     200 

Water 722-783  717-773                     800 

Organic  bodies 207-263  217-263  180-190 

Inorganic  bodies 10-15  '0-19                       10-20 

Myosin 30-106  29 .8-1 10  29 . 7-87 

Struma  substance  (D\nilewski) 78-161  88.0-184  70.0-121 

Creatine 2-4.5  3-4.9  2.3-7 

Carnosine 1 . 3-4  —  — 

Carnitine 0.19  —  — 

Purinebases 1.3-1.7  0.7-1.3  0.53-0.88 

Inosinic  acid  (barium  salt) 0.1  0.1-0.3  — 

Phosphocarnic  acid 0 .  57-2 .4  —  — 

Inosite 0.03  —  — 

Glycogen 1-37  —  — 

lactic  acid 0 . 4-0 .7  —  — 


Of  the  mineral  substances  the  largest  part  consists  of  phosphoric 
acid  3.4-4.8  p.  m.  and  potassium  3-4  p.  m.  The  amount  of  sodium 
is  ordinarily  only  |-J  of  that  of  the  potassium.  Pork,  according  to 
Katz,1  who  has  carried  out  complete  investigations  as  to  the  quantity 
of  mineral  constituents  of  the  human  muscle  and  of  other  animals,  is 
considerably  richer  than  other  varieties  of  meat,  in  sodium  than  potas- 
sium. The  quantity  of  chlorine,  which  is  also  variable,  was  found  by 
Magnus-Levy  to  be  2.4  p.  m.  (calculated  as  NaCl)  for  the  human  heart 
muscle  and  1.004  p.  m.  in  other  muscles.  The  amount  of  Ca  and  Mg 
was  found  by  him  to  be  equal  to  0.019  and  0.174  p.  m.  respectively  in  the 
heart  muscle  and  0.065  and  0.215  p.  m.  respectively  in  other  muscles, 
v.  Moraczewski  obtained  higher  results  for  the  Ca  content  of  the  human 
heart  muscle,  namely  0.07  p.m.  Gley  and  Richaud  2  found  0.25-0.26 
p.  m.  Ca  in  the  heart  muscle  of  the  dog,  and  0.089-0.248  p.  m.  Ca  in  that 
from  the  rabbit.  The  magnesium  content  of  the  muscles  seems,  with 
the  exception  of  the  haddock,  eel  and  pike  (Katz),  to  be  greater  than  the 
calcium  content.  The  statements  differ  very  considerably  in  regard 
to  the  iron  content.  Thus  Schmey3  found  0.0793  p.  m.  iron  in  the  human 
muscle,  while  Magnus-Levy  found  0.253  p.  m.,  and  in  the  human  heart 


1  Katz,  Pfluger's  Arch.,  63;  see  also  Schmey,  Zeitschr.  f.  physiol.  Chem.,  39. 

2  Magnus-Levy,  Bioch.  Zeitschr.  24;   v.  Moraczewski,  Zeitschr.   f.  physiol.  Chem. 
23;  Gley  and  Richaud,  Journ.  de  Physiol,  et  de  Path.,  12. 

3  Zeitschr.  f.  physiol.  Chem.  39;  Magnus-Levy,  1.  c. 


600  MUSCLES. 

muscle  only  0.067  p.  m.  iron.  Other  investigators  have  only  found  0.014- 
0.035  p.  m.  iron  in  the  muscle. 

In  the  table  which  is  given  above,  no  results  are  given  as  to  the  estimates 
of  fat.  Owing  to  the  variable  quantity  of  fat  in  meat  it  is  hardly  possible 
to  quote  a  positive  average  for  this  substance.  After  most  careful 
efforts  to  remove  the  fat  from  the  muscles  without  chemical  means,  it 
has  been  found  that  a  variable  quantity  of  intermuscular  fat,  which  does 
not  really  belong  to  the  muscular  tissue,  always  remains.  The  smallest 
quantity  of  fat  in  the  muscles  from  lean  oxen  is  6.1  p.  m.  according  to 
Grouven,  and  7.6  p.  m.  according  to  Petersen.  This  last  observer 
also  regularly  found  a  smaller  quantity  of  fat,  7.6-8.6  p.  m.,  in  the 
fore  quarters  of  oxen,  and  a  greater  amount,  30.1-34.6  p.  m.,  in  the  hind 
quarters  of  the  animal,  but  this  could  not  be  substantiated  by  Steil.1 
A  small  quantity  of  fat  has  also  been  found  in  the  muscle  of  wild  animals. 
B.  Konig  and  Farwick  found  10.7  p.  m.  fat  in  the  muscles  of  the  extrem- 
ities of  the  hare,  and  14.3  p.  m.  in  the  muscles  of  the  partridge.  The 
muscles  of  pigs  and  fattened  animals  are,  when  all  the  adherent  fat  is 
removed,  very  rich  in  fat,  amounting  to  40-90  p.  m.  The  muscles  of 
certain  fishes  also  contain  a  large  quantity  of  fat.  According  to  Almen, 
in  the  flesh  of  the  salmon,  the  mackerel,  and  the  eel  there  are  contained 
respectively  100,  164,  and  329  p.  m.  fat.2 

The  quantity  of  water  in  the  muscle  is  liable  to  considerable  variation. 
The  quantity  of  fat  has  a  special  influence  on  the  quantity  of  water,  and 
one  finds,  as  a  rule,  that  the  flesh  which  is  deficient  in  water  is  correspond- 
ingly rich  in  fat.  The  quantity  of  water  does  not  depend  upon  the  amount 
of  fat  alone,  but  upon  many  other  circumstances,  among  which  must 
be  mentioned  the  age  of  the  animal.  In  young  animals,  the  organs 
in  general,  and  therefore  also  the  muscles,  are  poorer  in  solids  and  richer 
in  water.  In  man  the  quantity  of  water  decreases  until  mature  age, 
but  increases  again  toward  old  age.  Different  muscles  have  also  a 
different  water  content  and  the  uninterruptedly  active  heart  is  the 
richest  muscle  in  water.  In  man,  Magnus-Levy  found  748  p.  m.  water 
in  the  heart,  and  722  p.  m.  in  the  other  muscles.  That  the  quantity 
of  water  may  vary  independently  of  the  amount  of  fat  is  strikingly  shown 
by  comparing  the  muscles  of  different  species  of  animals.  In  cold-blooded 
animals  the  muscles  generally  have  a  greater  quantity  of  water,  in  birds 
a  lower.  The  comparison  of  the  flesh  of  cattle  and  fish  shows  very  strik- 
ingly the  different  amounts  of  water  (independent  of  the  quantity  of  fat) 


1  See  Steil,  Pfluger's  Arch.,  61. 

2  In  regard  to  the  literature  and  complete  reports  on  the  composition  of  flesh  of 
various  animals,  see  Konig,  Chemie  der  menschlichen  Nahrungs-  und  Genussmittel, 
5.  Aufl. 


SMOOTH  MUSCLES.  601 

in  the  flesh  of  different  animals.  According  to  the  analysis  of  Almen  l 
the  muscles  of  lean  oxen  contain  15  p.  m.  fat  and  707  p.  m.  water:  the 
flesh  of  the  pike  contains  only  1.")  p.  m.  fat  and  839  p.  m.  water. 

For  certain  purposes,  as,  for  example,  in  experiments  mi  metabolism,  it  is 
important  to  know  the  elementary  composition  of  flesh.  In  regard  t<>  the  quan- 
tity of  nitrogen  we  generally  accept  Vorr's  figure,  namely,  3.4  per  cent,  as  an 
average  for  fresh  lean  meat.  According  to  Nowak  and  BuPPERT  -  this  quantity 
may  vary  about  0.6  per  cent,  and  in  nunc  exact  investigations  it  is  therefore 
necessary  to  specially  determine  the  nitrogen.  Complete  elementary  analyses 
of  flesh  have  been  made  with  greal  care  by  AaGUTiNSKy.  The  average  for  ox- 
flesh  dried  in  vacuo  and  free  from  fat  and  with  the  glvcogen  deducted  was  as  fol- 
lows: C  49.G;  H  6.9;  X  15.3;  O+S  23.0;  and  ash  5.2  per  cent.  Kohler 
found  as  an  average  for  water  and  fat-free  beef  C  49.86;  H  6.78;  N  15.68;  O+S 
2l'.;i  per  cent,  which  are  very  similar  results.  This  investigator  also  made  similar 
analyses  of  the  flesh  of  various  animals  and  determined  the  calorific  value  of  the 
ash-  and  fat-free  dried  meat  substance.  This  value  was,  per  gram  of  substance, 
5509-5677  cal.  The  relation  of  the  carbon  to  nitrogen,  which  ARGUTixsKy  calls 
the  "flesh  quotient,"  is  on  an  average  3.24  :  1.  From  Kohler's  analyses  the 
average  for  beef  is  3.15  :  1  and  for  horse-flesh  3.38: 1.  Max  Muller  has  shown 
with  experiments  on  dogs,  that  the  flesh  of  the  same  individual  shows  some  varia- 
tion in  this  quotient  after  different  foods.  According  to  Salkowski,  of  the  total 
nitrogen  of  beef  77.4  per  cent  was  insoluble  proteins,  10.08  per  cent  soluble  pro- 
teins, and  12.52  per  cent  other  soluble  bodies.  Frextzel  and  Schreuer  3  find 
that  about  7.74  per  cent  of  the  total  nitrogen  belongs  to  the  nitrogenous 
extractives. 

Smooth  Muscles. 

The  smooth  muscles  have  a  neutral  or  alkaline  reaction  (Du  Bois- 
Reymond)  when  at  rest.  During  activity  they  are  acid,  which  is  inferred 
from  the  observations  of  Bernstein,  who  found  that  the  almost  con- 
tinually contracting  sphincter  muscle  of  the  Anodonta  is  acid  during 
life.  The  smooth  muscles  may  also,  according  to  Heidenhain  and 
Kuhne,4  pass  into  rigor  mortis  and  thereby  become  acid.  A  spontaneous 
but  slowly  coagulating  plasma  has  also  been  observed  in  several  cases. 

In  regard  to  the  proteins  of  the  smooth  muscles  we  have  the  earlier 
accounts  of  Heidenhain  and  Hell  wig;5  but  they  were,  first  carefully 
studied  according  to  newer  methods  by  Munk  and  Velichi.6     These 

1  Nova  Act.  Reg.  Soc.  Scient.  Upsal.,  Vol.  extr.  ord.,  1877;  also  Maly's  Jahresber.,  7. 
2Voit,  Zeitschr.  f.  Biologie,  1;  Huppert,  ibid.,  7;  Nowak,  Wien.  Sitzungsber.,  64, 
Abt.  2. 

3  Argutinsky,  Pfliiger's  Arch.,  55;  Kohler,  Zeitschr.  f.  physiol.  Chem.,  31;  Sal- 
kowski, Centralbl.  f.  d.  mcd.  Wissensch.,  1S94;  Frentzel  and  Schreuer,  Arch.  f.  (Anat. 
u.)  Physiol.,  1902;  Muller,  Pfliiger's  Arch.,  116. 

4  Du  Bois-Reymond  in  Xasse,  Hermann's  Handb.,  1,  339;  Bernstein,  ibid.,  Heiden- 
hain, ibid.,  340,  with  Helhvig,  ibid.,  339;  Kuhne,  Lehrbuch,  331. 

5  Heidenhain  in  Xasse,  Hermann's  Handb.,  1,  340,  with  Helhvig,  ibid.,  339;  Kuhne, 
Lehrbuch,  331. 

8  Munk  and  Velichi,  Centralbl.  f.  Physiol.,  12. 


602  MUSCLES. 

experimenters  prepared  a  neutral  plasma  from  the  gizzard  of  geese, 
according  to  v.  Furth's  method.  This  plasma  coagulated  spon- 
taneously at  the  temperature  of  the  room,  although  slowly.  It  con- 
tained a  globulin,  precipitated  by  dialysis,  which  coagulated  at  55-60° 
C.  and  also  showed  certain  similarities  with  Kuhne's  myosin.  A  spon- 
taneously coagulating  albumin,  which  differed  from  myogen  (v.  Furth) 
by  coagulating  at  45-50°  C,  and  which  passes  by  spontaneous  coagula- 
tion into  the  coagulated  modification  without  a  soluble  intermediate 
product,  exists  in  still  greater  quantities  in  this  plasma.  Alkali  albu- 
minates do  not  occur,  but  a  nucleoprotein  is  found,  which  exists  in  about 
five  times  the  quantity  as  compared  with  striated  muscles.  Nucleon 
is,  according  to  Panella,1  a  normal  constituent  of  smooth  muscles  and 
occurs  in  larger  amounts  than  in  striated  muscles. 

Recent  investigations  of  Bottazzi  and  Cappelli,  VincENT  and 
Lewis,  Vincent  and  v.  Furth,2  some  on  the  muscles  of  warm-blooded 
and  some  on  those  of  lower  animals,  have  led  to  dissimilar  results,  but 
they  substantiate,  as  a  whole,  the  observations  of  Munk  and  Velichi. 
Besides  the  nucleoproteins  the  smooth  muscles  contain  two  bodies 
corresponding  in  coagulation  temperature  to  musculin  and  myosinogen 
(myogen,  v.  Furth),  but  they  are  not  identical  therewith.  Haemo- 
globin occurs  in  the  smooth  muscles  of  certain  animals,  but  is  absent  in 
others.  In  the  smooth  muscles  (in  certain  varieties  of  animals)  creatine, 
creatinine,  hypoxanthine,  taurine,  inosite,  glycogen,  and  lactic  acid  have 
been  found.  Purine  bases,  especially  xanthine  also  occur  accord- 
ing to  Buglia  and  Costantino  but  the  quantity  is  smaller  than  in 
striated  muscles.  This  applies  at  least  to  the  total  quantity  while  the 
amount  of  free  purine  bases,  according  to  Scafpidi,3  in  the  smooth 
muscles  is  greater  than  in  the  striated  muscles.  Creatine  and  carnosine 
are  less  abundant  in  the  smooth  muscles  than  in  the  striated  muscles. 
The  first  are  richer  in  diamino-acid  than  in  monamino-acid-nitrogen 
than  the  striated  muscles  (Buglia  and  Costantino). 

In  regard  to  the  mineral  constituents,  Costantino  has  found  that 
the  smooth  muscles  are  richer  in  chlorine,  namely  0.84-1.3  p.  m.,  than 
the  striated  muscles  with  0.25-0.46  p.  m.  According  to  older  state- 
ments the  sodium  compounds  exceed  the  potassium  compounds  but 
Costantino  4  could  not  substantiate  this.    He  found,  namely,  no  general 

1  Maly's  Jahresber.,  34. 

1  Bottazzi,  Centralbl.  f.  Physiol.,  15;  Vincent  and  Lewis,  Journ.  of  Physiol.,  26; 
Vincent,  Zeitschr.  f.  physiol.  Chern.,  34;  v.  Furth,  ibid.,  31. 

'  Scaffidi,  Bioch.  Zeitschr.  33;  Buglia  and  Costantino,  Zeitschr.  f.  physiol.  Chem. 
83,  81  and  82. 

4  Costantino,  Bioch.  Zeitschr.  37;  See  also  Meigs  and  Ryan,  Journ.  of  biol. 
Chem.  11. 


SMOOTH  MUSCLES.  603 

difference  in  the  proportion  K  :  Na  in  the  smooth  and  striated  muscles. 
According  to  Saiki  1  magnesium  does  not  occur  to  a  greater  extent 
than  calcium  in  the  smooth  muscles  of  the  stomach  or  the  bladder  of 
pigs.  The  same  investigator  found  801-811  p.  m.  water  and  199-189 
p.  m.  solids  in  these  muscles. 

Henze  found  abundance  of  taurine  in  the  muscles  of  Octopods,  5  p.  m.,  but 
no  creatine,  which,  according  to  Fre.my  and  Valenciennes,2  occurs  in  the  muscles 
of  Cephalopods.  He  also  found  no  glycogen  and  no  paralactic  acid,  but,  on  the 
contrary,  small  amounts  of  fermentation  lactic  acid.  The  muscles  of  Octopods 
are  richer  in  mineral  bodies  than  the  muscles  of  vertebrates,  and  are  nearly  twice 
as  rich  in  sulphur  as  these. 

1  Journ.  of  Biol.  Chem.,  4. 

2  Henze,  ibid.,  43;  Fremy  and  Valenciennes,  cited  from  Kiihne's  Lehrbuch,  p.  333. 


CHAPTER  XI. 
BRAIN  AND  NERVES. 

On  account  of  the  difficulty  in  making  a  mechanical  separation 
and  isolation  of  the  different  tissue-elements  of  the  central  nervous 
organ  and  the  nerves,  we  must  resort  to  a  few  microchemical  reactions, 
pr  ncipally  to  qualitative  and  quantitative  investigations  of  the  different 
parts  of  the  brain,  in  order  to  study  the  varied  chemical  composition 
of  the  cells  and  the  nerve-axes.  This  study  is  accompanied  with  the 
greatest  difficulty,  and  although  our  knowledge  of  the  chemical  com- 
position of  the  brain  and  nerves  has  been  somewhat  extended  by  the 
investigations  of  modern  times,  still  it  must  be  admitted  that  this  sub- 
ject is  as  yet  one  of  the  most  obscure  and  complicated  in  physiological 
chemistry. 

Proteins  of  different  kinds  have  been  shown  to  be  chemical  constit- 
uents of  the  brain  and  nerves,  and  these  are  representatives  of  the  same 
chief  groups  as  occur  in  the  protoplasm.  In  the  brain  there  occur  some 
proteins  which  are  insoluble  in  water  and  neutral  salt  solutions,  and 
which  resemble  the  stroma  substances  of  the  muscles  and  cells,  while 
other  proteins  are  soluble  in  water  and  neutral  salt  solutions.  Among 
the  latter  we  find  mainly  nucleoproteins  and  globulins.  The  nucleo- 
protein  found  by  Halliburton  and  also  by  Levene1  in  the  gray  substance 
contains  0.5  per  cent  phosphorus  and  coagulates  as  55-60°.  Levene 
obtained  adenine  and  guanine  but  no  hypoxanthine  as  cleavage  prod- 
ucts. According  to  Halliburton  there  are  two  globulins,  namely, 
the  neuroglobulin  a,  which  coagulates  at  47°,  or  as  in  the  case  of 
birds,  50-53°,  and  the  neuroglobulin  /3,  whose  coagulation  temperature 
is  70-75°,  but  which  varies  somewhat  in  different  animals.  In  the  frog 
still  another  protein  body  occurs,  which  coagulates  at  a  still  lower  tem- 
perature, about  40°.  It  must  be  remarked  that  the  coagulation  tempera- 
ture of  a-globulin  corresponds  with  the  temperature  of  the  first  heat 
contraction  of  the  nerves  of  different  classes  of  animals  (Halliburton). 


1  Halliburton,  On  the  Chemical  Physiology  of  the  Animal's  Cell,  King's  College, 
London,  Physiological  Laboratory,  Collected  Papers  No.  1,  1S93,  and  Ergebnisse  der 
Phyeiologie,  4;  Levene,  Arch,  of  Neurology  and  Psychopathology,  2  (1899). 

604 


CONSTITUENTS  OF  THE   NERVOUS   SYSTEM.  605 

The  gray  substance  is  only  slightly  richer  in  proteins  than  the  white 
substance;  but  as  the  neurokeratin,  which  forms  the  neurolgia,  and  as 
a  double  sheath  envelops  the  outside  of  the  nerves,  belongs  in  great 
part,  or  according  to  Koch,  entirely,  to  the  white  substance  (Kuhne, 
and  Chittenden,  Baumstark  l),  the  gray  substance  is  actually  richer 
in  protein.  The  same  is  true  also  for  the  nucleoprotein  or  at  least  for 
the  nucleins  which  v.  Jaksch  found  in  large  amounts  in  the  gray  sub- 
stance. The  mixture  of  amino-acids  obtained  from  the  proteins  of 
the  gray  and  white  substances  has  about  the  same  composition  (Abder- 
halden  and   Weil2).     Glycocoll  could  not  be  detected  in  this  mixture. 

The  so-called  protagon  has  been  considered  as  one  of  the  chief  con- 
stituents, perhaps  the  only  constituent  (Baumstark),  of  the  white 
substance.  This  protagon,  according  to  most  investigators,  is  only  a 
mixture  of  phosphatides  with  cerebron  or  with  a  mixture  of  cerebrosides 
(see  below).  Protagon  belongs  to  the  so-called  brain  lipoids,  which 
include  three  chief  groups,  phosphatides,  cerebrosides  and  cholesterin  and 
which  are  contained  to  a  greater  extent  in  the  white  than  in  the  gray 
substance.  Among  the  closely  studied  phosphatides  the  cephalin 
seems  to  occur  to  the  greatest  extent  in  the  brain.  The  lecithin,  accord- 
ing to  Frankel,3  does  not  occur  in  the  human  brain  and  only  in  very 
small  quantities  in  other  brains  (of  sheep  and  beef).  Other  brain 
phosphatides  especially  described  by  Thudichum  and  by  Frankel,4 
have  not  been  positively  proved  as  chemical  individuals.  The  same 
is  true  for  the  jecorin  and  the  sulphurized  lipoids  isolated  from  the 
human  brain  and  from  ox  brains.  Cholesterin  occurs  chiefly  in  the 
white  substance.  Fatty  acids  and  neutral  fats  may  be  prepared  from 
the  brain  and  nerves;  but  as  these  may  be  readily  derived  from  a  decom- 
position of  phosphatides,  which  exist  in  the  fatty  tissue  between  the 
nerve- axes,  it  is  difficult  to  decide  what  part  the  fatty  acids  and  neutral 
fats  play  as  constituents  of  the  real  nerve-substance. 

By  allowing  water  to  act  on  the  contents  of  the  medulla,  round  or  oblong 
double-contoured  drops  or  fibers,  not  unlike  double-contoured  nerves,  are  formea. 
These  remarkable  formations,  which  can  also  be  seen  in  the  medulla  of  the  dead 
nerve,  have  been  called  "  myelins  forms,"  and  they  were  formerly  considered  a;s 
produced  from  a  special  body,  "  myeline."  Myeline  forms  may,  however,  be 
obtained  from  other  bodies,  such  as  impure  protagon,  lecithin,  and  impure  choles- 
terin, and  they  depend  upon  a  decomposition  of  the  constituents  of  the  medulla. 

1  Koch,  Amer.  Journ.  of  Physiol.,  11;  Kuhne  and  Chittenden,  Zeitschr.  f  Biologie, 
26;  Baumstark,  Zeitschr.  f.  physiol.  Chem.,  9. 

2  v.  Jaksch,  Pfliiger's  Arch.  13;  Abderhalden  and  Weil,  Zeitschr.  f.  physiol.  Chem. 
81  and  83. 

3  Bioch.  Zeitschr.,  24. 

4  Thudichum,  Die  chemische  Konstitution  des  Gehirns  des  Menschen  und  der 
Tiere,  Tubingen,   1901;  S.  Frankel,  and  collaborators,   Bioch.  Zeitschr.,   24  and  28. 


606  BRAIN  AND  NERVES. 

The  extractive  bodies  seem  to  be  almost  the  same  as  in  the  muscles. 
One  finds  creatine,  which  may,  however,  be  absent  (Baumstark),  purine 
bases,  inosite,  choline,  paralactic  acid  (Moriya),  phosphocarnic  acid,  uric 
acid,  and  the  diamine  neuridine,  C5H14N2,  discovered  by  Brieger  l 
and  which  is  most  interesting  because  of  its  appearance  in  the  putrefac- 
tion of  animal  tissues  or  in  cultures  of  the  typhoid  bacillus.  Among 
the  enzymes  we  must  mention  catalases,  peroxidases,  lipases  and  amylases 
(Wr6blewski).  According  to  the  autolytic  experiments  of  Simon2 
a  proteolytic  enzyme,  and  an  enzyme  acting  upon  the  organic  phos- 
phorized  substance  with  the  splitting  off  of  phosphoric  acid  also  occur. 
Under  pathological  conditions  leucine  and  urea  have  been  found  in  the 
brain.  Urea  is  also  a  physiological  constituent  of  the  brain  of  cartilagi- 
nous fishes. 

Several  of  the  lipoids  occurring  in  the  brain  have  been  discussed  in 
previous  chapters,  and  we  will  here  only  speak  of  the  protagon  and  the 
cerebrosides. 

Protagon.  Under  this  name  Liebreich  described  a  crystalline, 
nitrogenous  and  phosphorized  substance,  which  has  been  found  in  the 
brain  of  man,  mammalia  and  also  birds  (Argiris)  but  not  in  the  brain 
of  fishes  (Argiris).  Its  elementary  composition,  according  to  Gamgee 
and  Blankenkorn,  is  C  66.39,  H  10.69,  N  2.39  and  P  1.07  per  cent. 
The  results  obtained  by  Cramer  correspond  well  with  these  figures  and 
he  found  that  protagon  also  contained  sulphur  which  had  previously  been 
found  by  Ruppel  and  by  Kossel.  Recently  Wilson  and  Cramer  3  have 
reported  more  recent  analyses  and  they  find  for  protagon,  recrystallized 
4-5  times,  almost  the  same  figures  as  Gamgee  and  Blankenhorn,  namely, 
C  66.53,  H  10.97,  N  2.37,  P  0.95  and  S  0.73  per  cent.  They  consider 
protagon  as  a  unit  substance. 

Gies,  Posner  and  Rosenheim  and  Tebb  4  dispute  the  unit  nature 
of  protagon.  They  have  found,  on  fractional  precipitation  or  on  recrys- 
tallization,  that  protagons  can  be  obtained  from  the  various  solvents, 
having  variable  composition,  especially  different  P  and  N  contents.     They 


1  Brieger,  Ueber  Ptomaine,  Berlin,  1885  and  1886. 

2  \Yr6blewski,  Compt.  Rend.,  152;  Fr.  Simon,  Zeitschr.  f.  physiol.  Chem.,  72. 

3  Liebreich,  Annal.  d.  Chem.  u.  Pharm.,  134;  Argiris,  Zeitschr.  f.  physiol.  Chem., 
57;  Gamgee  and  Blankenhorn,  ibid.,  3;  Kossel  and  Freytag,  ibid.,  17;  Ruppel, 
Zeitschr.  f.  Biol.,  31;  Cramer,  Journ.  of  Physiol.,  31,  with  R.  A.  Wilson,  Journ.  of 
exp.  Physiol.,  1,  with  Lockhead,  Bioch.  Journ.,  2;  also  Cramer,  Quarterly  Journ.  of 
exp.  Physiol.  3,  and  Bioch.  Handlexikon  (Abderhalden)  Bd.  3,  which  contains  the 
literature. 

*  Gies  and  Lesem,  Amer.  Journ.  of  Physiol.,  8;  Posner  and  Gies,  Journ.  of  biol. 
Chem.,  1;  Gies,  ilid..  3;  Rosenheim  and  Tebb,  Journ.  of  Physiol.,  36  and  37,  Quarterly 
Journ.  of  exp.  Physiol.,  2,  and  Bioch.  Zeitschr.,  25. 


PROTAGON.  607 

are,  therefore,  as  are  Lesem,  Thudichum,  WdBNEB  and  Thierfelder,1 
and  others,  of  the  opinion  that  protagon  does  not  exist  as  a  chemical 
individual,  but  as  a  mixture  of  cerebrosides  and  phosphatides.  It  is 
not  easy  to  come  to  any  decision  on  this  disputed  question.  On  the 
one  hand  it  must  be  recalled  that  several  investigators  call  the  impure 
mixture  of  brain  lipoids,  protagon,  which  they  obtain  from  the  solution 
in  warm  alcohol  on  cooking,  and  which  is  not  purified,  and  this  mixture 
is  claimed  to  be  identical,  without  sufficient  basis,  with  the  substance 
isolated  and  analyzed  by  Gamgee  and  Cramer.  On  the  other  hand 
it  cannot  be  denied  that  certain  investigations,  especially  those  of  ROSEN- 
HEIM and  Tebb  speak  against  the  chemical  individuality  of  protagon. 
These  investigations  do  not  exclude  the  possibility  that  protagon  is  a 
loose  chemical  combination  between  cerebroside  and  phosphatide,  which 
like  other  readily  dissociable  combinations,  exist  only  under  certain 
conditions  or  in  certain  solvents.  It  is  difficult  to  understand  how  a 
mixture  of  amorphous  or  only  difficultly  crystallizable  bodies  can  be 
so  easily  crystallized  and  yield  a  product,  which  with  proper  care,  can  be 
recrystallized  repeatedly  without  changing  its  composition,  and  physical 
properties.  According  to  Rosenheim  and  Tebb  if  the  proper  quantity 
is  used  in  solution,  a  crystalline  product  can  be  obtained  from  the  decom- 
position products  cf  protagon,  which  has  the  same  specific  rotation  as 
protagon  and  can  be  repeatedly  recrystallized  without  changing  its 
composition  or  its  optical  activity.2  A  further  study  of  these  con- 
ditions would  naturally  be  of  great  interest. 

As  we  are  not  decided  whether  protagon  is  only  a  mixture  or  is  a  body 
contaminated  with  other  substances,  it  is  difficult  to  decide  as  to  how 
far  the  so-called  decomposition  products  exist  as  preformed  constituents 
of  the  mixture  or  whether  they  are  true  decomposition  products.  On 
boiling  with  baryta-water  protagon  yields  cerebrosides  (see  below)  and 
the  decomposition  products  of  lecithin,  namely,  fatty  acids,  glycerophos- 
phoric  acid,  and  choline.  Kossel  and  Freytag  found  three  cerebro- 
sides, namely,  cerebrin,  kerasix  (homocerebrin),  and  encephalix. 
According  to  Koch  3  the  protagon  molecule  contains  cerebroside,  lecithin 
and  sulphuric  acid  (in  ester-like  combination  with  the  cerebroside) 
besides  excess  of  cerebroside.  Of  interest  is  the  finding  of  Kitagawa 
and  Thierfelder4  that  protagon  dissolved  in  methyl  alcohol  contain- 
ing chloroform,  deposits  crusts  of  cerebron  (not  pure)   after  a  time  at 


1  Lesem,   1.   c;  Thudichum,   1.   c;  Worner  and  Thierfelder,   Zeitschr.  f.   physiol. 
Chem.,  30. 

1  Journ.  of  Physiol.,  3";  Proc.  physiol.  Soc,  January,  1908,  p.  3. 

*  Zeitschr.  f.  physiol.  Chem.,  53. 

*  Kitagawa  and  Thierfelder,  ibid.,  49;  Rosenheim  and  Tebb,  Journ.  of  Physiol.    37 
341  and  348. 


608  BRAIN  AND  NERVES. 

ordinary  temperature,  and  that  as  shown  by  Rosenheim  and  Tebb, 
on  dissolving  in  pyridine  at  30°  C.  and  heating  or  cooling  the  solution 
deposits  a  precipitate  of  a  substance  rich  in  phosphorus.  Although  we 
generally  consider  the  phosphorized  component  of  protagon  as  lecithin, 
still,  according  to  Rosenheim  and  Tebb,  it  is  probably  a  diamido- 
phosphatide,  called  sphingomyelin  by  Thudichum.  On  boiling  protagon 
with  dilute  mineral  acids  it  yields  galactose,  due  to  the  decomposition 
of  the  cerebrosides. 

Protagon  appears,  when  dry,  as  a  loose  white  powder.  It  dissolves 
in  alcohol  of  85  vols,  per  cent  at  45°  C,  but  separates  on  cooling  as  a 
snow-white,  flaky  precipitate,  consisting  of  globules  or  groups  of  fine 
crystalline  needles.  On  heating  to  150°  it  becomes  yellowish,  softens 
at  180°  and  melts  sharply  at  200°  forming  a  brown,  oily  liquid  (Cramer). 
It  is  difficultly  soluble  in  cold  alcohol  or  ether,  but  dissolves,  at  least 
when  freshly  precipitated,  in  ether  on  warming.  It  dissolves  in  methyl 
alcohol  containing  chloroform  and,  as  above  stated,  separates  cerebron. 
Protagon  is  soluble  in  pyridine  at  30°  C,  yielding  a  clear  solution,  and 
this  solution  has  a  specific  rotation  (a)D  =  +6.9  to  7.7°  according  to 
the  concentration  of  the  solution  (Wilson  and  Cramer).  On  warm- 
ing or  cooling  according  to  Rosenheim  and  Tebb,  the  rotation  changes 
with  the  separation  of  sphingomyelin  so  that  it  first  diminishes  in  rota- 
tion, then  is  zero,  and  then  becomes  strongly  levorotatory  until  it  reaches 
—  242°,  and  finally,  when  nearly  all  the  sphingomyelin  has  separated 
out  it  becomes  constant  at  about  -13.3°.  The  strong  levorotation 
depends  upon  the  accumulations  of  doubly  refracting  spheroid  crystals 
of  sphingomyelin.  With  little  water  protagon  swells  up  and  is  partly 
decomposed.  With  more  water  it  forms  a  jelly  or  pasty-like  mass  which, 
with  the   addition   of   considerable  water,   forms   an  opalescent  liquid. 

Protagon  can  be  prepared  in  the  following  way:  The  finely  ground 
brain-mass,  as  free  as  possible  from  blood  and  membrane,  is  dehydrated, 
which  is  best  done  by  cold  acetone  or  by  grinding  with  burned  plaster-of- 
paris  or  anhydrous  sodium  sulphate,  and  then  extracted  with  ether. 
The  mass  is  then  extracted  at  45°  C.  with  85  vol.  per  cent  alcohol  until 
the  filtrate  when  cooled  to  0°  C.  gives  no  more  precipitate.  All  the 
precipitates  obtained  on  cooling  to  0°  C.  are  extracted  with  ether  and 
recrystallized  from  alcohol.  Further  details  can  be  found  in  the  cited 
works  of  Cramer,  Wilson,  Gies,  Rosenheim  and  Tebb. 

Among  the  phosphatides  occurring  in  the  brain  we  must  mention  besides  the 
lecithin  and  ccphalin,  the  following  substances. 

Myelin,  C.,oII7:,XPOio,  according  to  Thudichum,  is  not  well  known  but  is  char- 
acterized by  the  fact  that  itfl  alcoholic  solution  is  not  precipitated  by  CdCl2  or 
PtCl4.  On" the  contrary  an  alcoholic  solution  of  lead  acetate  gives  a  precipitate. 
The  existence  of  a  second  monaminomonophosphatide,  paramyelin,  C38H26NPO9, 
according  to  Thudichum,  is  very  improbable. 


•  CEREBROSIDES.  609 

Sphingomyelin,  is  a  diaminomonophosphatide  which  Thudichum  prepared 
from  the  brain  and  is  the  chief  phosphatide  obtainable  from  the  impure  protagon 
mixtures.  Rosenheim  and  Tebb  obtained  it,  as  above  mentioned,  from  the 
protagon.  It  has  been  given  the  formula  (  ,JHl„1Xd)09+H20.  As  cleavage 
products  an  alcohol,  Bphingol,  iieurin,  cholin,  according  to  ROSENHEIM  and  Tebb, 
the  base  sphingosin  (see  cerebron)  and  sphingostearic  acid  have  been  obtained. 
Sphingomyelin  is  soluble  with  difficulty  in  cold  alcohol  but  readily  soluble  in  hot 
alcohol  and  crystallizes  therefrom  in  needles.  It  is  insoluble  in  ether.  In  regard 
to  the  specific  rotation  see  above  in  reference  to  protagon.  Amidomyelin  (Thud- 
ichum) is  another  diaininomonophosphatide  of  an  unknown  constitution  and  of 
an  uncertain  composition.     Its  existence  is  uncertain. 

Sahidin  was  found  by  Fu.vxkel1  in  the  brain,  and  is  a  triaminodiphosphatide, 
whose  cadmium  compound  has  the  formula  CsoHic7X3P20i2.3CdCl2.  It  is  a  crys- 
talline powder  which  is  insoluble  in  water,  cold  ethyl  or  methyl  alcohol  and  in 
ether.  It  is  soluble  with  difficulty  in  warm  alcohol  but  readily  soluble  in  chloro- 
form and  hot  benzene.  It  yields  saturated  and  unsaturated  fatty  acids,  choline 
and  glycerophosphoric  acid. 

Leucopoliin  is  an  unsaturated  phosphatide  found  b}r  Frankel  and  Elias  2  in 
the  brain  and  which  is  a  decaminodiphosphatide  or  a  pentaminomonophosphatide. 
It  crystallizes  from  boiling  alcohol  on  cooling.  It  does  not  contain  any  methylated 
base  but  does  contain  a  carbohydrate  group. 

Sulphatide  is  the  name  given  by  Koch  3  to  a  sulphurized  and  phosphorized 
product  obtained  from  the  human  brain  which  separates  from  warm  pyridine 
on  cooling  as  a  crystalline,  granular  mass.  It  contains  phosphatide,  sulphuric 
acid  and  cerebroside  and  is  claimed  to  be  phosphatidesulphuric  acid  cerebroside. 

Cerebrosides. 

On  decomposing  protagon  (or  the  protagons),  or  the  brain  substance 
by  the  gentle  action  of  alkalies  we  obtain,  as  cleavage  products,  as  above 
stated,  one  or  more  bodies  which  Thudichum  has  embraced  under  the 
name  cerebrosides.  The  cerebrosides  are  nitrogenous  substances  free 
from  phosphorus,  which  yield  galactose  on  boiling  with  dilute  mineral 
acids.  With  concentrated  sulphuric  acid  they  first  give  a  yellow  and 
then  a  purple-red  coloration.  With  sulphuric  acid  and  cane-sugar 
they  give  a  purple  coloration  directly.  The  cerebrosides  isolated  from 
the  brain  are  cerebrin,  homocerebrin,  phrenosin,  kerasin,  encephalin, 
and  cerebron,  but  it  must  be  remarked  that  there  is  no  doubt  that 
sometimes  the  same  body  of  varying  purity  has  received  different  names. 
According  to  Levene  and  Jacobs4  it  must  be  admitted  that  the  cere- 
brosides are  mixtures  of  stereoisomeric  substances. 

Cerebrin.  Under  this  name  W.  Muller5  first  described  a  nitrog- 
enous substance,  free  from  phosphorus,  which  he  obtained  by  extracting, 
with  boiling  alcohol,  a  brain-mass  which  had  been  previously  boiled  with 

1  Bioch.  Zeitschr.  24. 

2  Frankel  and  Elias,  Bioch.  Zeitschr.  28. 

3  Zeitschr.  f.  physiol.  Chem.  70. 

4  Journ.  of  biol.  Chem.  12. 
BAnnal.  d.  Chem.  u.  Pharm.,  105. 


610  BRAIN  AND  NERVES. 

baryta-water.  Following  a  method  essentially  the  same,  but  differing 
slightly,  Geoghegan  prepared,  from  the  brain,  a  cerebrin  with  the 
same  properties  as  Muller's,  but  containing  less  nitrogen.  Accord- 
ing to  Parous  1  the  cerebrin  isolated  by  Geoghegan,  as  well  as  by 
Muller,  consists  of  a  mixture  of  three  bodies,  "  cerebrin,"  "  homo- 
cerebrin," and  "  encephalin."  Kossel  and  Freytag  isolated  two 
cerebrosides  from  protagon  which  were  identical  with  the  cerebrin  and 
homocerebrin  of  Parous.  According  to  these  investigators,  the  two 
bodies  phrenosin  and  kerasin,  as  described  by  Thudichum,  seem  to  be 
identical  with  cerebrin  and  homocerebrin. 

Cerebrin,  according  to  Parous,  has  the  following  composition:  C 
69.08,  H  11.47,  N  2.13,  0  17.32  per  cent,  which  corresponds  with  the 
analyses  made  by  Kossel  and  Freytag.  No  formula  has  been  given  to 
this  body.  In  the  dry  state  it  forms  a  pure  white,  odorless,  and  tasteless 
powder.  On  heating  it  melts,  decomposes  gradually,  smells  like  burned 
fat,  and  burns  with  a  luminous  flame.  Melting-point  is  170-176°  C.  It 
is  insoluble  in  water,  dilute  alkalies,  or  baryta-water;  also  in  cold  alcohol 
and  in  cold  or  hot  ether.  On  the  contrary,  it  is  soluble  in  boiling  alcohol 
and  separates  as  a  flaky  precipitate  on  cooling,  and  this  is  found  to  con- 
sist of  a  mass  of  globules  or  grains  on  microscopical  examination.  Cere- 
brin forms  a  compound  with  baryta,  which  is  insoluble  in  water  and  is 
decomposed  by  the  action  of  carbon  dioxide.  The  variety  of  sugar 
split  off  on  boiling  with  mineral  acids — the  so-called  brain-sugar — is, 
as  Thierfelder2  first  showed,  galactose.  On  cleavage  with  nitric  acid 
fatty  acids  (stearic  acid)  were  obtained. 

Kerasin  (Thudichum),  or  homocerebrin  (Parous),  has  the  following 
composition:  C  70.06,  H  11.60,  N  2.23,  and  O  16.11  per  cent.  Enceph- 
alin has  the  composition  C  68.40,  H  11.60,  N  3.09,  and  O  16.91  per  cent. 
Both  bodies  remain  in  the  mother-liquor  after  the  impure  cerebrin  has 
precipitated  from  the  warm  alcohol.  These  bodies  have  the  tendency 
of  separating  as  gelatinous  masses.  Kerasin  is  similar  to  cerebrin,  but 
dissolves  more  easily  in  warm  alcohol  and  also  in  warm  ether.  It  may 
be  obtained  as  extremely  fine  needles.  Encephalin  is,  Parous  thinks, 
a  transformation  product  of  cerebrin.  In  the  perfectly  pure  state  it 
crystallizes  in  small  lamellae.     It  swells  in  warm  water  into  a  pasty  mass. 

As  the  purity  and  the  chemical  individuality  of  the  above-mentioned  bodies 
is  questionable,  it  is  perhaps  sufficient  in  regard  to  their  preparation  to  simply 
call  attention  to  the  cited  works  of  Muller,  Geoghegan,  Kossel  and  Freytag. 
All  these  methods  split  with  barium  hydroxide  and  purify  the  cerebroside  by 
solution  in  hot  alcohol  and  a  precipitation  by  cooling. 


1  Geoghegan,  Zeitschr.  f.  physiol.  Chem.,  3;  Parcus,  Ueber  einige  neue  Gehrinstoffe, 
Inaug.-Diss.  Leipzig,  1881. 

1  Zeitschr.  f.  physiol.  Chem.,  14. 


CEREBRON.  611 

Whether  the  above-described  cerebrosides  are  chemical  individuals  or 
mixtures,  i.  e.,  impure  substances,  is  still  undecided.  The  purest  cere- 
broside  thus  far  investigated  is  undoubtedly  Thierfelder's  cerebron, 
and  there  is  hardly  any  doubt  that  the  above-mentioned  cerebrosides 
consist  essentially  of  this  body. 

Cerebron.  This  cerebrin,  isolated  by  Thierfelder  and  Worner 
and  then  especially  studied  by  Thierfelder,  was  first  isolated  by  Gam- 
gee  and  called  pseudocerebrin  by  him.  Thudichum's  phrenosin  is, 
according  to  Gies,1  identical  with  cerebron.  Cerebron  can  be  prepared 
directly  from  the  brain  without  saponification  with  baryta,  by  treat- 
ment with  alcohol  containing  benzene  or  chloroform  at  a  temperature 
of  50°,  and  hence  it  is  considered  as  existing  preformed  in  the  brain. 
According  to  Thierfelder,  cerebron  has  the  formula  C48H93XO9;  it 
melts  at  212°,  dissolves  in  warm  alcohol,  and  separates  out  on  cooling. 
From  proper  solvents  (acetone  or  methyl  alcohol  containing  chloroform) 
it  may  be  separated  as  small  needles  or  plates.  If  cerebron  is  suspended 
in  85-per  cent  alcohol  at  a  temperature  of  50°  C.  it  balls  together  in 
amorphous  masses,  and  from  these  needle-  and  leaf-shaped  crystals 
gradually  form.  It  is  dextrorotatory,  and  in  about  a  5-per  cent  solu- 
tion in  methyl  alcohol  (containing  75  per  cent  chloroform)  is  (o0d  =  +7.6o 
(Kitagawa  and  Thierfelder).  According  to  Thierfelder  it  yields 
as  cleavage  products,  galactose,  cerebronic  acid  (Thudichum  "  neuro- 
stearic  acid  ")  and  sphingosin  which  is  in  part  obtained  as  such  and  part 
as  dimethylsphingosin.  The  base  sphingosin,  C17H35XO2,  discovered 
by  Thudichum,  is,  according  to  Thierfelder  and  to  Levene  and  Jacobs,2 
an  unsaturated,  diatomic,  monoamino-alcohol  which  is  readily  soluble 
in  alcohol,  ether,  acetone  and  petroleum  ether  but  insoluble  in  water, 
nas  an  alkaline  reaction  and  has  not  been  obtained  in  a  crystalline 
state.  The  sulphate  of  dimethylsphingosin  crystallizes,  on  the  contrary, 
from  alcohol.  Cerebronic  acid  is  an  oxyacid  with  the  formula  C25H50O3, 
which  is  crystalline  and  which  gives  a  crystalline  methyl  ester  which 
melts  at  65°  C.  It  has  been  obtained  by  Levene  and  Jacobs  3  in  part 
in  a  dextrorotatory  and  in  part  as  an  inactive  form.  The  first  melts 
at  106-108°  and  the  other  at  82-85°  C. 

Cerebron  can  best  be  prepared,  according  to  Thierfelder  and 
Kitagawa,  by  decomposing  the  protagon  in  methyl  alcohol  containing 

1  Thierfelder  and  Worner,  Zeitschr.  f.  physiol.  Chem.,  30;  Thierfelder,  ibid.,  43, 
44,  4G,  with  Kitagawa,  ibid.,  49;  with  H.  Loening,  ibid.,  68,  74,  77;  Gamgee,  Text- 
book of  Physiol.  Chem.,  London,  1880;  Thudichum,  1.  c;  Gies,  Journ.  of  Biol. 
Chem.,  1  and  2. 

2  Thierfelder  and  O.  Riesser  and  K.  Thomas,  Zeitschr.  f.  physiol.  Chem.,  77; 
Levene  and  Jacobs,  Journ.  of  biol.  Chem.  11. 

5  Journ.  of  biol.  Chem.,  12. 


612  BRAIN  AND  NERVES. 

chloroform  (see  page  607),  and  purifying  the  separated  cerebron  from 
contaminating  phosphatides  by  precipitating  these  with  an  ammoniacal 
solution  of  zinc  hydroxide  in  methyl  alcohol,  and  recrystallizing  the  cere- 
bron from  methyl  alcohol  containing  chloroform.  Thierfelder  and 
Loening  have  devised  another  method  of  purification  and  at  the  same 
time  they  have  suggested  another  method  for  preparing  cerebron.  This 
method  is  based  upon  the  resistance  of  the  cerebrosides  to  baryta  and 
their  solubility  in  hot  acetone.  It  consists  in  boiling  the  impure  protagon 
mixture  with  baryta-water  and  boiling  the  insoluble  residue  with  acetone. 

Neuridine,  C6Hi4N2,  is  a  non-poisonous  diamine  discovered  by  Brieger,  and 
obtained  by  him  in  the  putrefaction  of  meat  and  gelatin,  and  from  cultures  of 
the  typhoid  bacillus.  It  also  occurs  under  physiological  conditions  in  the  brain, 
and  as  traces  in  the  yolk  of  the  egg. 

Neuridine  dissolves  in  water  and  yields  on  boiling  with  alkalies  a  mixture 
of  dimethylamine  and  trimethylamine.  It  dissolves  with  difficulty  in  amyl 
alcohol.  It  is  insoluble  in  ether  or  absolute  alcohol.  In  the  free  state,  neuridine 
has  a  peculiar  odor,  suggesting  semen.  With  hydrochloric  acid  it  gives  a  compound 
crystallizing  in  long  needles.  With  platinic  chloride  or  gold  chloride  it  gives 
crystallizable  double  compounds  which  are  valuable  in  its  preparation  and  detec- 
tion. 

The  so-called  corpuscula  amylacea,  which  occur  on  the  upper  surface  of  the 
brain  and  in  the  pituitary  gland,  are  colored  more  or  less  pure  violet  by  iodine 
and  more  blue  by  sulphuric  acid  and  iodine.  They  perhaps  consist  of  the  same 
substance  as  certain  prostatic  calculi,  but  they  have  not  been  closely  investigated. 

Quantitative  Composition  of  the  Brain.  The  quantity  of  water  is 
greater  in  the  gray  than  in  the  white  substance,  and  greater  in  new-born 
or  young  individuals  than  in  adults.  The  brain  of  the  foetus  contains 
879-926  p.  m.  water.  The  observations  of  Weisbach1  show  that  the 
quantity  of  water  in  the  several  parts  of  the  brain  (and  in  the  medulla) 
varies  at  different  ages.  The  following  figures  are  in  1000  parts — A  for 
men  and  B  for  women: 

20-30  years.  30-50  years.  50-70  years.  70-94  years. 

A.  B.  A.  B.  A.  B.  A.  B. 

White  brain-substance.  .  695.6  682.9  683.1  703.1  701.9  689.6    726.1  722.0 

Gray                  "                 833.6  826.2  836.1  830.6  838.0  838.4  .  847. S  839.5 

Gyri 784.7  792.0  795.9  772.9  796.1  796.9    802.3  801.7 

Cerebellum 788.3  794.9  778.7  789.0  787.9  784.5     803.4  797.9 

Pons  Varolii 734.6  740.3  725.5  722.0  720.1  714.0    727.4  724.4 

Medulla  oblongata 744.3  740.7  732.5  729.8  722.4  730.6    736.2  733.7 

The  recent  investigations  of  K.  Linnert2  correspond  to  the  above 
in  that  the  pons  and  the  medulla  were  found  to  be  next  to  the  white  sub- 
stance, the  poorest  in  water,  of  the  human  brain. 

Quantitative  analyses  of  human  brains  at  different  ages,  namely 
6  weeks,  2  and  19  years,  have  been  made  by  Koch  and  Mann.3  These 
analyses  show  that  with  increasing  age  the  water,  proteins,  extractives 

1  Cited  from  K.  B.  Hoffmann's  Lehrbuch  d.  Zioch.,  Wien,  1877,  p.  121. 

2  Wien.  klin.  Wochenschr.,  23. 

•  Journ.  of  Physiol.,  36,  Proc.  physiol.  Soc,  1907. 


COMPOSITION  OF  THE   BRAIN.  613 

and  salts  diminish  relatively,  while  the  phosphatides,  cerebrosidea  and 
especially  cholesterin  strikingly  increase.  The  sulphur  of  the  lipoids 
increased  to  the  second  year,  but  then  existed  in  the  same  amounts  as  at 
nineteen  years. 

Baumstark  claims  to  have  found  that  a  part  of  the  cholesterin  in  the  brain 
occurs  in  a  combined  state,  perhaps  as  ester;  this  view  has  been  found  to  be 
incorrect  by  the  recent  investigations  of  Bunz.  He  obtained  from  the  brain 
neither  esters  of  cholesterin  with  higher  fatty  acids  nor  other  compounds  of 
cholesterin  which  split  on  saponification.  Tebb  "i  has  also  found  only  free 
cholesterin. 

According  to  Frankel,2  who  has  fractionally  extracted  the  human 
brain  with  various  solvents,  found  230  p.  m.  solids  in  the  brain  and  this 
consisted  of  §  lipoids  and  §  proteins.  Of  the  lipoids  about  17  per  cent 
was  cholesterin,  34.482  per  cent  saturated  and  48.293  per  cent  unsat- 
urated compounds.  The  amount  of  cholesterin  in  the  different  parts 
of  the  brain  was  as  follows,  according  to  Frankel,  Kirschbaum  and 
Linnert.  In  the  cortex  11.5  p.  m.,  in  the  white  substance  24.7  p.  m., 
in  the  cerebellum  13.1  p.  m.,  and  in  the  bridge  and  medulla  40.3  p.  m., 
all  calculated  in  the  moist  substance. 

The  analysis  of  the  brain  of  an  epileptic  made  by  Koch  3  is  of  very  great 
interest.  As  the  protagon  is  considered  by  Koch  as  a  mixture,  no  results  for 
the  quantity  of  protagon  are  given.  As  no  accurate  methods  for  the  estima- 
tion of  the  little  known  bodies  cephalin,  myelin,  phrenosin  and  kerasin  are 
available,  the  figures  given  for  these  are  of  little  value.  The  following  results 
are  calculated  to  1000  parts: 

Corpua  Cortex 

Calloaum  (prefrontal). 

Water 679.7  841.3 

Protein 32.0  50.0 

Nucleoproteins 37.0  30.0 

Neurokeratin 27.0  (Chittenden)        4.0  (Chittenden) 

Extractives  (water-soluble) 15. 1  15.8 

Lecithins 51.9  31.4 

Cephalin  and  myelin 34.9  7.4 

Phrenosin  and  kerasin 45.7  15.5 

Cholesterin 48.6  7.0 

Sulphurized  substance 14.0  14.5 

Mineral  bodies 8.2  8.7 

Pighini  and  Carbone  found  that  the  brains  of  paralytics  were  richer  in  water, 
considerably  richer  in  cholesterin,  but  poorer  in  cephalin  than  healthy  brains. 
This  last  corresponds,  to  the  observations  of  Koch  and  Mann  4  that  the  quan- 
tity of  lipoid  phosphorus  was  diminished  in  paralytics. 


1  Baumstark,  Zeitschr.  f.  physiol.   Chem.  9;  R.  Bunz    ibid.,  46;  Tebb,  Joum.  of 
Physiol.,  34. 

2  Bioch.  Zeitschr.,  19,  with  Kirschbaum  and  Linnert,  ibid.,  46. 

3  Amer.  Joum.  of  Physiol.,  11. 

4  Pighini  and  Carbone,  Bioch.  Zeitschr.,  46;  Koch  and  Mann,  Arch,  of  Neurol, 
and  Psychol.,  1910. 


614  BRAIN  AND  NERVES. 

According  to  Fr.  Falk  j  the  cerebrosides  occur  in  the  medullary 
nerve  fibers  as  well  as  in  the  nerves  without  medullas.  These  latter 
yielded  much  less  substance  on  extraction  than  the  medullary,  namely, 
11.51  per  cent  extract  as  compared  to  46.59  per  cent.  The  extract  of 
the  first  was  poorer  in  cerebrosides,  but  richer  in  cholesterin,  cephalin 
and  lecithin,  as  shown  by  the  following  figures. 

Non-medullary  fibers     Medullary  fibers  in 

in  p.  m.  of  the  total      p.  m.  of  the  total 

extract  extract 

Cholesterin 470  250 

Cephalin 237  124 

Cerebrosides 60  182 

Lecithins 98  29 

S.  Frankel  and  L.  Dimitz  2  find  that  the  spinal  marrow  contains  on 
an  average  740  p.  m.  water,  180  p.  m.  lipoids  and  80  p.  m.  protein.  The 
quantity  of  cholesterin  (in  the  fresh,  spinal  marrow  containing  water) 
is  40  p.  m.,  the  unsaturated  phosphatide  120  p.  m.,  and  the  saturated 
15  p.  m.  The  spinal  marrow  is  the  richest  part  of  the  nervous  system 
in   unsaturated   phosphatides   and   it   contains   abundance   of   cephalin. 

According  to  Noll  the  white  substance  of  the  spinal  marrow  is  some- 
what richer  in  protagon  than  the  brain,  and  in  nerve  degeneration  the 
quantity  of  protagon  diminishes.  The  method  used  by  him  would  not 
allow  of  an  exact  determination  of  the  disputed  substance  protagon. 
Mott  and  Halliburton  3  have  also  shown  that  in  degenerative  diseases 
of  the  nervous  system,  the  quantity  of  substances  containing  phosphorus 
diminishes,  and  that  in  these  cases,  especially  in  general  paralysis,  choline 
passes  into  the  cerebrospinal  fluid  and  the  blood.  In  degenerated  nerves, 
the  quantity  of  water  increases,  and  the  phosphorus  decreases.  On 
comparative  investigations  of  the  central  nervous  system  of  normal 
persons,  and  those  afflicted  with  dementia  prsecox  (5  cases),  Koch4  found 
that  the  variation  from  the  normal  composition  was  not  great  enough 
nor  so  constant  that  positive  conclusions  could  be  drawn  therefrom. 

The  quantity  of  neurokeratin  in  the  nerves  and  the  different  parts 
of  the  brain  has  been  carefully  determined  by  Kuhne  and  Chittenden.5 
They  found  3.16  p.  m.  in  the  plexus  brachialis,  3.12  p.  m.  in  the  cortex 
of  the  cerebellum,  22.434  p.  m.  in  the  white  substance  of  the  cerebrum, 
25.72-29.02  p.  m.  in  the  white  substance  of  the  corpus  callosum,  and 
3.27  p.  m.  in  the  gray  substance  of  the  cortex  of  the  cerebrum  (when 

1  Bioch.  Zeitschr.,  13. 

*  Ibid.,  28. 

1  Noll,  Zeitschr.  f.  physiol.  Chem.,  27;  Mott  and  Halliburton,  Philos.  Transactions. 
Ser.  B.,  191  (1899),  and  194  (1901). 
4  Arch,  of  Neurology,  3. 

*  Zeitschr.  f .  Biologje,  26. 


VISUAL  PURPLE.  C15 

free  as  possible  from  white  substance).  The  white  is  decidedly  richer 
in  neurokeratin  than  the  peripheral  nerves  or  the  gray  substance.  '  Accord- 
ing to  Griffiths,1  neurochitin  replaces  neurokeratin  in  insects  and  Crus- 
tacea, the  quantity  of  the  first  being  10.6-12  p.  m. 

The  quantity  of  mineral  constituents  in  the  brain  amounts  to  2.95- 
7.08  p.  m.  according  to  Geoghegan.  He  found  in  1000  parts  of  the 
fresh,  moist  brain  0.43-1.32  CI,  0.956-2.01G  P04,  0.244-0.796  C03, 
0.102-0.220  S04,  0.01-0.098  Fe2(P04)2,  0.005-0.022  Ca,  0.016-0.072 
Mg,  0.58-1.778  K,  and  0.450-1.114  Na.  The  gray  substance  yields  an 
alkaline  ash,  the  white  an  acid  ash.  Magnus-Levy  2  found  in  fresh  brain 
substance  1.305  p.  m.  CI,  0.166  p.  m.  Ca,  0.139  p.  m.  Mg,  and  0.083  p.m. 
Fe. 

Appendix. 

THE   TISSUES  AND   FLUIDS    OF  THE  EYE. 

The  retina  contains  in  all  865-899.9  p.  m.  water,  57.1-84.5  p.  m. 
protein  bodies — myosin,  albumin,  and  mucin  (?),  9.5-28.9  p.  m.  lecithin, 
and  8.2-11.2  p.  m.  salts  (Hoppe-Seyler  and  Cahn  ;;).  The  mineral  bodies 
consist  of  422  p.  m.  Na2HPC>4  and  352  p.  m.  NaCl.  The  retina  con- 
tains, according  to  Barbieri,4  also  cholesterin  but  no  cerebrosides  and  in 
fact  none  of  the  specific  constituents  of  the  brain  substance. 

Those  bodies  which  form  the  different  segments  of  the  rods  and  cones 
have  not  been  closely  studied,  and  the  greatest  interest  is  therefore  con- 
nected with  the  coloring-matters  of  the  retina. 

Visual  purple,  also  called  rhodopsin,  erythropsin,  or  visual  red,  is 
the  pigment  of  the  rods.  Boll,5  in  1876,  observed  that  the  layer  of  rods 
in  the  retina  during  life  had  a  purplish-red  color  which  was  bleached 
by  the  action  of  light.  Kuhne  6  later  showed  that  this  red  color  might 
remain  for  a  long  time  after  the  death  of  the  animal  if  the  eye  was  pro- 
tected from  daylight  or  investigated  by  a  sodium  light.  Under  these 
conditions  it  was  also  possible  to  isolate  and  closely  study  this  substance. 

Visual  red  (Boll)  or  visual  purple  (Kuhne)  has  become  known  mainly  by 
the  investigations  of  Kuhne.  The  pigment  occurs  mainly  in  the  rods  and  only 
in  their  outer  parts.     In  animals  whose  retina  has  no  rods  the  visual  purple  is 

^ompt.  Rend.,  115. 

2  Geoghegan,   Zeitschr.   f.   physiol  Chem..    1;  Magnus- Levy,   Bioch.   Zeitschr.   24. 

*  Zeitschr.  f.  physiol.  Chem.,  5. 

4Compt.  Rend.,  154. 

5  Monatsber.  d.  Kgl.  Preuss.  Akad.,  12.  Nov.,  1876. 

6  The  investigations  of  Kuhne  and  his  pupils,  Evvald  and  Ayres,  on  the  visual  purple 
will  be  found  in  ..Untersuchungen  aus  dem  physiol.  Institut  der  Universitat  Heidel- 
berg, 1  and  2,  and  in  Zeitschr.  f.  Biologie,  32. 


616  BRAIN  AND  NERVES. 

absent,  and  is  also  necessarily  absent  in  the  macula  lutea.  In  a  variety  of  bat 
(Rhinolophus  hipposideros),  in  hens,  pigeons  and  new-born  rabbits,  no  visual 
purple  has  been  found  in  the  rods. 

A  solution  of  visual  purple  in  water  which  contains  2-5  per  cent  crys- 
tallized bile,  which  is  the  best  solvent  for  it,  is  purple-red  in  color,  quite 
clear,  and  not  fluorescent.  On  evaporating  this  solution  in  vacuo  we 
obtain  a  residue  similar  to  ammonium  carminate  which  contains  violet 
or  black  grains.  If  the  above  solution  is  dialyzed  with  water,  the  bile 
diffuses  and  the  visual  purple  separates  as  a  violet  mass.  Under  all 
circumstances,  even  when  still  in  the  retina,  the  visual  purple  is  quickly 
bleached  by  direct  sunlight,  and  with  diffused  light  with  a  rapidity  cor- 
responding to  the  intensity  of  the  light.  It  passes  from  red  and  orange 
to  yellow.  Red  light  bleaches  the  visual  purple  slowly;  the  ultra-red 
light  does  not  bleach  it  at  all.  A  solution  of  visual  purple  shows  no  special 
absorption  bands,  but  only  a  general  absorption  which  extends  from  the 
red  side,  beginning  at  D  and  extending  to  the  G  line.  The  strongest 
absorption  is  found  at  E. 

Koettgen  and  Abelsdorf"  r  have  shown  that  there  are,  in  accordance  with 
Kuhne's  views,  two  varieties  of  visual  purple,  the  one  occurring  in  mammals, 
birds,  and  amphibians,  and  the  other,  which  is  more  violet-red,  in  fishes.  The 
first  has  its  maximum  absorption  in  the  green  and  the  other  in  the  yellowish- 
green. 

Visual  purple  when  heated  to  52-53°  C.  is  destroyed  after  several 
hours,  and  almost  instantly  when  heated  to  76°  C.  It  is  also  destroyed 
by  alkalies,  acids,  alcohol,  ether,  and  chloroform.  On  the  contrary, 
it  resists  the  action  of  ammonia  or  alum  solution. 

As  the  visual  purple  is  easily  destroyed  by  light,  it  must  therefore  also  be 
regenerated  during  life.  Kuhne  has  also  found  that  the  retina  of  the  eye  of  the 
frog  becomes  bleached  when  exposed  for  a  long  time  to  strong  sunlight,  and  that 
its  color  gradually  returns  when  the  animal  is  placed  in  the  dark.  This  regenera- 
tion of  the  visual  purple  is  a  function  of  the  living  cells  in  the  layer  of  the  pigment 
epithelium  of  the  retina.  This  may  be  inferred  from  the  fact  that  a  detached 
piece  of  the  retina  which  has  been  bleached  by  light  may  have  its  visual  purple 
restored  if  it  is  carefully  laid  on  the  choroid  having  layers  of  the  pigment-epithe- 
lium attached.  The  regeneration  has,  it  seems,  nothing  to  do  with  the  dark 
pigment,  the  melanin  or  fuscin,  in  the  epithelium  cells.  A  partial  regeneration 
seems,  according  to  Kuhne,  to  be  possible  in  the  retina  which  has  been  completely 
removed.  On  account  of  this  property  of  the  visual  purple  of  being  bleached 
by  light  during  life  we  may,  as  Kuhne  has  shown,  under  special  conditions  and 
by  observing  special  precautions,  obtain  after  death,  by  the  action  of  intense 
light  or  more  continuous  light,  the  picture  of  bright  objects,  such  as  windows 
and  the  like — so-called  optograms. 

The  physiological  importance  of  visual  purple  is  unknown.  It  follows 
that  the  visual  purple  is  not  essential  to  sight,  since  it  is  absent  in  certain 
animals  and  also  in  the  cones. 

1  Centralbl.  f.  Physiol.,  9;  also  Maly's  Jahresber.,  25,  351. 


CRYSTALLINE   LENS.  617 

Visual  purple  must  always  be  prepared  exclusively  in  a  sodium  light.  It  is 
extracted  from  the  net  membrane  by  means  of  a  watery  solution  of  crystallized 
bile.  The  filtered  solution  is  evaporated  in  vacuo  or  dialyzed  until  the  visual 
purple  is  separated.  To  prepare  a  visual-purple  solution  perfectly  free  from 
hemoglobin,  the  solution  of  visual  purple  in  cholates  is  precipitated  by  saturating 
with  magnesium  sulphate,  washing  the  precipitate  with  a  saturated  solution  of 
magnesium  sulphate,  and  then  dissolving  in  water  by  the  aid  of  the  cholates  sim- 
ultaneously precipitated.1 

The  Pigments  of  the  Cones.  In  the  inner  segments  of  the  cones  of  birds,  rep- 
tiles, and  fishes  a  small  fat-globule  of  varying  color  is  found.  Kuhne  2  has 
isolated  from  this  fat  a  green,  a  yellow,  and  a  red  pigment  called  respectively 
chlorophan,  xanthophan,  and  rhodophan. 

The  dark  pigment  of  the  epithelium-cells  of  the  net  membrane,  which  was 
formerly  called  melanin,  but  has  since  been  named  fuscin  by  Kuhne  and  Mays,3 
contains  iron,  dissolves  in  concentrated  caustic  alkalies  or  concentrated  sul- 
phuric acid  on  warming,  but,  like  the  melanins  in  general,  has  been  little  studied. 
The  pigment  occurring  in  the  pigment-cells  of  the  choroid  will  be  discussed  with 
the  melanins  in  Chapter  XV. 

The  vitreous  humor  is  often  considered  as  a  variety  of  gelatinous 
tissue.  The  membrane  consists,  according  to  C.  Morner,  of  a  gelatin- 
forming  substance.  The  fluid  contains  a  little  proteid  and  a  mucoid, 
hyalomucoid,  which  was  first  shown  by  Morner,  and  which  is  precipitated 
by  acetic  acid.  This  contains  12.27  per  cent  N,  and  1.19  per  cent  S. 
Among  the  extractives  we  find  a  little  urea — according  to  Picard  5  p.  m., 
according  to  Rahlmann  0.64  p.  m.  Pautz4  found  besides  some  urea, 
paralactic  acid,  and,  in  confirmation  of  the  claims  of  Chabbas,  Jesner, 
and  Kuhn,  also  glucose  in  the  vitreous  humor  of  oxen.  The  reaction 
of  the  vitreous  humor  is  alkaline,  and  the  quantity  of  solids  amounts 
to  about  9-11  p.  m.  The  quantity  of  mineral  bodies  is  about  6-9  p.  m., 
and  the  proteins  0.7  p.  m.  In  regard  to  the  aqueous  humor  see  page 
361. 

The  Crystalline  Lens.  That  substance  which  forms  the  capsule  of 
the  lens  has  been  investigated  by  C.  Morner.  It  belongs,  according 
to  him,  to  a  special  group  of  proteins,  called  membranins.  The  mem- 
branin  bodies  are  insoluble  at  the  ordinary  temperature  in  water,  salt 
solutions,  dilute  acids,  and  alkalies,  and,  like  the  mucins,  yield  a  reducing 
substance  on  boiling  with  dilute  mineral  acids.  They  contain  lead- 
blackening  sulphur.  The  membranins  are  colored  a  very  beautiful  red 
by  Millon's  reagent,  but  give  no  characteristic  reaction  with  concentrated 
hydrochloric  acid  or  Adamkiewicz's  reagent.     They  are  dissolved  with 

1  Kuhne,  Zeitschr.  f.  Biologie,  32. 

2  Kuhne,  Die  nichtbestiindigen  Farben  der  Netzhaut,  Untersuch.  aus  dem  physiol. 
Institut  Heidelberg,  1,  341. 

3  Kuhne,  ibid.,  2,  324. 

4  Morner,  Zeitschr.  f.  physiol.  Chem.,  18;  Picard,  cited  from  Gamgee,  Physiol. 
Chem.,  1,  454;  Rahlmann,  Maly's  Jahresber.,  6;  Pautz,  Zeitschr.  f.  Biologie,  31.  A 
complete  review  of  the  literature  will  also  be  found  here. 


618  BRAIN  AND  NERVES. 

great  difficulty  by  pepsin-hydrochloric  acid  or  trypsin  solution,  but  are 
soluble  in  dilute  acids  and  alkalies  in  the  warmth.  Membranin  of  the 
capsule  of  the  lens  contains  14.10  per  cent  N  and  0.83  per  cent  S,  and  is 
a  little  less  soluble  than  that  from  Descemet's  membrane. 

The  principal  mass  of  the  solids  of  the  crystalline  lens  consists  of 
proteins,  whose  nature  has  been  investigated  by  C.  Morner.1  Some  of 
these  proteins  dissolve  in  dilute  salt  solution,  while  others  remain 
insoluble  in  this  solvent. 

The  Insoluble  Protein.  The  lens  fibers  consist  of  a  protein  sub- 
stance which  is  insoluble  in  water  and  in  salt  solution  and  to  which 
Morner  has  given  the  name  albumoid.  It  dissolves  readily  in  very  dilute 
acids  or  alkalies.  Its  solution  in  caustic  potash  of  0.1  per  cent  is  very 
similar  to  an  alkali-alb uminate  solution,  but  coagulates  at  about  50° 
C.  on  nearly  complete  neutralization  and  the  addition  of  8  per  cent  NaCl. 
Albumoid  has  the  following  composition:  C  53.12,  H  6.8,  N  16.62,  and 
S  0.79  per  cent.  The  lens  fibers  themselves  contain  16.61  per  cent  N 
and  0.77  per  cent  S.  The  inner  parts  of  the  lens  are  considerably  richer 
in  albumoid  than  the  outer.  The  quantity  of  albumoid  in  the  entire 
lens  amounts  on  an  average  to  about  48  per  cent  of  the  total  weight  of 
the  proteins  of  the  lens. 

The  Soluble  Protein  consists,  exclusive  of  a  very  Ftrall  quantity  of 
albumin,  of  two  globulins,  a-  and  fi-crystallin.  These  two  globulins  differ 
from  each  other  in  this  manner:  a-crystallin  contains  16.68  per  cent  N 
and  0.56  per  cent  S;  /3-crystallin,  on  the  contrary,  17.04  per  cent  N  and 
1.27  per  cent  S.  The  first  coagulates  at  about  72°  C.  and  the  other  at 
63°  C.  Besides  this,  /3-crystallin  is  precipitated  from  a  salt-free  solu- 
tion with  greater  difficulty  and  less  completely  by  acetic  acid  or  carbon 
dioxide.  These  globulins  are  not  precipitated  by  an  excess  of  NaCl  at 
either  the  ordinary  temperature  or  30°  C.  Magnesium  or  sodium  sul- 
phate in  substance  precipitates  both  globulins,  on  the  contrary,  at  30°  C. 
These  two  globulins  are  not  equally  divided  in  the  mass  of  the  lens.  The 
quantity  of  a-crystallin  diminishes  in  the  lens  from  without  inward; 
/3-crystallin,  on  the  contrary,  from  within  outward. 

A.  Jess2  has  found  that  the  different  proteins  of  the  crystalline  lens 
behave  differently  with  Arnold's  protein  reaction  with  sodium  nitro- 
prusside  (page  100).  The  albumoid  gives  negative  results  with  this 
reagent.  The  a-crystallin  gives  it  faintly,  while  the  /3-crystallin  gives  a 
strong  reaction.  The  absence  of  this  reaction,  as  observed  by  Weiss 
in  senile  cataract,  is  connected  with  the  fact  as  Jess  has  shown  by  his 
investigations  on   the   senile  cataract   in   oxen,  that  the   crystallin  con- 

1  Zeitschr.  f.  physiol.  Chem.,  18.     This  contains  also  the  pertinent  literature. 
2Zeit.srhr.  f.  Biol..  01. 


PROTEINS  OF  THE  LENS.  619 

taining  cysteine,  disappears  in  part  from  the  lens  and  is  partly  transformed 
into  albumoid.  The  relation  between  albumoid  and  crystallins  is  changed 
with  increasing  age,  so  that  the  albumoid  increases.  In  normal  lens 
the  relation  of  the  crystallins  to  the  albumoid  changes  correspondingly 
from  82:18  in  youth  to  41:59  in  old  age;  in  senile  cataract  the  relation 
can  be  changed  to  25 :  75.  The  amount  of  fat,  cholesterin  and  lecithin 
is  on  the  contrary  not  changed. 

The  average  results  of  four  analyses  made  by  Laptschinsky  *  of  the 
lens  of  oxen  are  here  given,  calculated  in  parts  per  1000: 

.  Proteins 349 . 3 

Lecithin 2.3 

Cholesterin 2.2 

Fat 2.9 

Soluble  salts 5.3 

Insoluble  salts 2.4 

In  cataract  the  amount  of  proteins  is  diminished  and  the  amount  of 
cholesterin  increased.     This  statement  requires  further  substantiation.2 

The  quantity  of  the  different  proteins  in  the  fresh  moist  lens  of  oxen 
is,  as  follows,  according  to  Morner: 

Albumoid  (lens  fibers) 170  p.  m. 

0-Crystallin 110    " 

a-Crystallin 68    " 

Albumin 2    " 

The  corneal  tissue  has  been  previously  considered  (page  550).  The 
sclerotic  has  not  been  closely  investigated,  and  the  choroid  coat  is  princi- 
pally of  interest  because  of  the  coloring-matter  (melanin)  it  contains 
(see  Chapter  XV). 

Tears  consist  of  a  water-clear,  alkaline  fluid  of  a  salty  taste.  Accord- 
ing to  the  analyses  of  Lerch3  they  contain  982  p.  m.  water,  18  p.  m.  solids 
with  5  p.  m.  albumin  and  13  p.  m.  NaCl. 

THE  FLUIDS   OF   THE   INNER  EAR. 

The  perilymph  and  endolymph  are  alkaline  fluids,  which,  besides 
salts,  contain — in  the  same  amounts  as  in  transudates — traces  of  protein, 
and  in  certain  animals  (codfish)  also  mucin.  The  quantity  of  mucin 
is  greater  in  the  perilymph  than  in  the  endolymph. 

Otoliths  contain  745-795  p.  m.  inorganic  substance,  which  consists 
chiefly  of  crystallized  calcium  carbonate.  The  organic  substance  is  very 
similar  to  mucin. 

1  Pfluger's  Arch.,  13. 

2  See  Gross,  Arch.  f.  Augenheilk.,  55  and  58. 

8  Cited  from  v.  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4  Aufl.,  401. 


CHAPTER  XII. 
ORGANS  OF  GENERATION. 

(a)  Male  Generative  Secretions. 

The  testes  have  been  little  investigated  chemically.  In  the  testes  of 
animals  we  find  protein  bodies  of  different  kinds — seralbumin,  alkali 
albuminate  (?),  and  an  albuminous  body  related  to  Rovida's  hyaline 
substance;  also  leucine,  tyrosine,  creatine,  purine  bases,  cholesterin,  lecithin, 
inosite,  and  fat.  In  regard  to  the  occurrence  of  glycogen  the  reports  are 
conflicting.  Dareste  1  found,  in  the  testes  of  birds,  starch-like  granules, 
which  were  colored  blue  with  difficulty  by  iodine. 

In  the  autolysis  of  the  testes  Levene  2  found  tyrosine,  alanine,  leucine,, 
aminovaleric  acid,  aminobutyric  acid,  a-proline,  phenylalanine,  aspartic  acid, 
glutamic  acid,  and  hypoxanthine.  Pyrimidine  and  hexone  bases  could  not  be 
detected. 

•  The  semen  as  ejected  is  a  white  or  whitish-yellow,  viscous,  sticky 
fluid  of  a  milky  appearance,  with  whitish,  non-transparent  lumps.  The 
milky  appearance  is  due  to  spermatozoa.  Semen  is  heavier  than  water, 
contains  proteins,  has  a  neutral  or  faintly  alkaline  reaction  and  a  peculiar 
specific  odor.  Soon  after  ejection  semen  becomes  gelatinous,  as  if  it 
were  coagulated,  but  afterward  becomes  more  fluid.  When  diluted 
with  water  white  flakes  or  shreds  separate  (Henle's  fibrin).  According 
to  the  analyses  of  Slowtzoff  3  human  semen  contains  on  an  average 
96.8  p.  m.  solids  with  9  p.  m.  inorganic  and  87.8  p.  m.  organic  substance. 
The  amount  of  protein  substances  was,  on  an  average,  22.6  p.  m.  and  1.69 
p.  m.  of  bodies  soluble  in  ether.  The  protein  substances  consist  of  nucleo- 
proteins,  traces  of  mucin,  albumin,  and  a  substance  similar  to  proteose 
f found  earlier  by  Posner).  According  to  Cavazzani  semen  contains 
relatively  considerable  nucleon,  more  than  any  organ,     v.  Hoffmann4 

1  Compt.  Rend.,  74. 

2  Amer.  Journ.  of  Physiol.,  11. 

J  Zeitschr.  f.  physiol.  Chem.,  35. 

*  Posner,  Berl.  klin.  Wochenschr.,  1888,  No.  21,  and  Centralbl.  f.  d.  med.  Wissensch., 
1890;  Cavazzani,  Biochem.  Centralbl.,  1,  502,  and  Centralbl.  f.  Physiol.,  19;  v.  Hoff- 
mann, cited  in  Bioch.  Centralbl.,  9,  206. 

620 


SEMEN.     SPERMINE.  021 

has  found  a  protamine  in  human  semen  which  yielded  arginine  and 
perhaps  also  lysine  on  cleavage.  The  mineral  bodies  consist  mainly  of  cal- 
cium phosphate  and  considerable  NaCl.  Potassium  occurs  only  in 
smaller  amounts. 

The  semen  in  the  vas  deferens  differs  chiefly  from  the  ejected  semen 
in  that  it  is  without  the  peculiar  odor.  This  last  depends  on  the  admixture 
with  the  secretion  of  the  prostate.  This  secretion,  according  to  Iversen, 
has  a  milky  appearance  and  ordinarily  an  alkaline  reaction,  very  rarely 
a  neutral  one,  and  contains  small  amounts  of  proteins,  especially  nucleo- 
proteins,  besides  a  substance  similar  to  fibrinogen  and  to  mucin  (Stern  '), 
and  mineral  bodies,  especially  NaCl.  Besides  this  it  contains  an  enzyme 
vesicidase  (see  below),  lecithin,  choline  (Stern),  and  a  crystalline  com- 
bination of  phosphoric  acid  with  a  base,  C2H5N.  This  combination  has 
been  called  Bottcher's  spermine  crystals,  and  it  is  claimed  that  the 
specific  odor  of  the  semen  is  due  to  a  partial  decomposition  of  these 
crystals. 

The  crystals  which  appear  on  slowly  evaporating  the  semen,  and 
which  are  also  observed  in  anatomical  preparations  kept  in  alcohol,  are 
not  identical  with  the  Charcot-Leyden  crystals  found  in  the  blood  and 
in  the  lymphatic  glands  in  leucaemia  (Th.  Cohn,  B.  Lewy2).  They  are, 
according  to  Schreiner,3  as  above  stated,  a  combination  of  phosphoric 
acid  with  a  I  ase,  spermine,  C2H5N,  which  he  discovered. 

Spermine.  Opinions  in  regard  to  the  nature  of  this  base  are  not  unanimous 
According  to  the  investigations  of  Ladenburg  and  Abel,  it  is  not  improbable 
that  spermine  is  identical  with  ethylenimine;  but  this  identity  is  disputed  by 
Majert  and  A.  Schmidt,  and  also  by  Poehl.  The  compound  of  spermine  with 
phosphoric  acid — Bottcher's  spermine  crystals — is  insoluble  in  alcohol,  ether, 
and  chloroform,  soluble  with  difficulty  in  cold  water,  but  more  readily  in  hot 
wqter,  and  easily  soluble  in  dilute  acids  or  alkalies,  also  alkali  carbonates  and 
ammonia.  The  base  is  precipitated  by  tannic  acid,  mercuric  chloride,  gold 
chloride,  platinic  chloride,  potassium-bismuth  iodide,  and  phosphotungstic  acid. 
Spermine  has  a  tonic  action,  and,  according  to  Poehl,4  it  has  a  marked  action  on 
the  oxidation  processes  of  the  animal  body. 

On  the  addition  of  a  solution  of  potassium  iodide  and  iodine  to  spermatozoa, 
characteristic  dark-brown  or  bluish-black  crystals  are  obtained — Florence's 
sperm  reaction,  which  is  considered  by  many  as  a  reaction  for  spermine.  Accord- 
ing to  Bocarius,5  this  reaction  is  due  to  choline. 

1  Iversen,  Xord.  med.  Ark.,  6;  also  Maly's  Jahresber.,  4,  358;  Stern,  Biochem. 
Centralbl.,  1,  74S. 

JTh.  Cohn,  Centralbl.  f.  allg^Path.  u.  path.  Anat.,  10  (1899), and  Zeitschr.f.Urolog., 
1908;  B.  Lewy,  Centralbl  f.  d.  med.  Wissensch.,  1899,  479. 

8  Annal.  d.  Chem.  u.  Pharni.,  194. 

4  Ladenburg  and  Abel,  Ber.  d.  deutsch.  chem.  Gesellsch.,  21;  Majert  and  A. 
Schmidt,  ibid.,  24;  Poehl,  Compt.  Rend.,  115,  Berlin,  klin.  Wochenschr.,  1891  and 
1893,  Deutsch.  med.  Wochenschr.,  1892  and  1895,  and  Zeitschr.  f.  klin.  Med.,  1894. 

6  In  regard  to  Florence's  sperm  reaction,  see  Posner,  Berl.  klin.  Wochenschr., 
1897,  and  Richter,  Wien.  klin.  Wochenschr.,  1897;  Bocarius,  Zeitschr.  f.  physioL 
Chem.,  34. 


622  ORGANS  OF  GENERATION. 

Camus  and  Gley  '  have  found  that  the  prostate  fluid  in  certain  rodents  has 
the  property  of  coagulating  the  contents  of  the  seminal  vesicles.  This  property 
is  due  to  a  special  ferment  substance  (vesiculase)  of  the  prostate  fluid. 

The  spermatozoa  show  a  great  resistance  to  chemical  reagents  in 
general.  They  do  not  dissolve  completely  in  concentrated  sulphuric 
acid,  nitric  acid,  acetic  acid,  or  in  boiling-hot  soda  solutions.  They 
are  soluble  in  a  boiling-hot  caustic-potash  solution.  They  resist  putre- 
faction, and  after  drying  they  may  be  obtained  again  in  their  original 
form  by  moistening  them  with  a  1-per  cent  common-salt  solution.  By 
careful  heating  and  burning  to  an  ash  the  shape  of  the  spermatozoa  may 
be  seen  in  the  ash.  The  quantity  of  ash  is  about  50  p.  m.  and  consists 
mainly  (three-quarters)  of  potassium  phosphate. 

The  spermatozoa  show  well-known  movements,  but  the  cause  of  this 
is  not  known.  These  movements  may  continue  for  a  very  long  time, 
as  under  some  conditions  they  may  be  observed  for  several  days  in  the 
body  after  death,  and  in  the  secretion  of  the  uterus  longer  than  a  week. 
Acid  liquids  stop  these  movements  immediately;  they  are  also  destroyed 
by  strong  alkalies,  especially  ammoniacal  liquids,  also  by  distilled  water, 
alcohol,  ether,  etc.  The  movements  continue  for  a  longer  time  in  faintly 
alkaline  liquids,  especially  in  alkaline  animal  secretions,  and  also  in 
properly  diluted  neutral  salt  solutions.2 

Spermatozoa  are  nucleus  formations  and  hence  are  rich  in  nucleic 
acid,  which  exists  in  the  heads.  The  tails  contain  protein,  and  are  besides 
this  rich  in  lecithin,  chclesterin,  and  fat,  which  bodies  occur  only  to  a 
small  extent  (if  at  all)  in  the  heads.  The  tails  seem  by  their  composi- 
tion to  be  closely  allied  to  the  non-medullated  nerves  or  the  axis-cylinders. 
In  the  various  kinds  of  animals  investigated,  the  head  contains  nucleic 
acid,  which  in  fishes  is  partly  combined  with  protamines  and  partly 
with  histones.  In  other  animals,  such  as  the  bull  and  boar,  protein- 
like  substances  occur  with  the  nucleic  acid,  but  no  protamine. 

Our  knowledge  of  the  chemical  composition  of  spermatozoa  has 
been  greatly  enhanced  by  the  important  investigations  of  Miescher3 
on  salmon  milt.  The  intermediate  fluid  of  the  spermatozoa  of  Rhine 
salmon  is  a  dilute  salt  solution  containing  1.3-1.9  p.  m.  organic  and 
6.5-7.6  p.  m.  inorganic  bodies.  The  last  consist  principally  of  sodium 
chloride  and  carbonate,  besides  some  potassium  chloride  and  sulphate. 
The  fluid  contains  only  traces  of  protein,  but  no  peptone.  The  tails  consist 
of  419  p.  m.  protein,  318.3  p.  m.  lecithin,  and  262.7  p.  m.  cholesterin  and 


1  Compt.  rend,  de  soc.  biolog.,  48,  49. 
1  See  G.  Gunther,  Pfliiger's  Arch.,  118. 

3  See  Miescher,  "  Die  histochemischen  und  physiologischen  Arbeiten  von  Friedrich 
Miescher,  gesammelt  und  herausgegeben  von  seinen  Freunden,"  Lefpzig,  1897. 


OVARIES.  623 

fat.  The  heads  extracted  with  alcohol-ether  contain  on  an  average 
960  p.  m.  protamine  nucleate,  which  nevertheless  is  not  uniform,  but  is 
so  divided  that  the  outer  layers  consist  of  basic  protamine  nucleate, 
while  the  inner  layers,  on  the  contrary,  consist  of  acid  protamine  nucleate. 
Besides  the  protamine  nucleate  there  are  present  in  the  heads,  although 
to  a  very  slight  extent,  organic  substances.  Of  these  we  must  mention 
a  nitrogenous  substance  containing  iron  which  gives  Millon's  reaction 
and  which  Miescher  calls  karyogen.  The  unripe  salmon  spermatozoa, 
while  developing,  also  contain  nucleic  acid,  but  no  protamine,  with  a 
protein  substance,  "  albuminose,"  which  probably  is  a  step  in  the  forma- 
tion of  protamine.  According  to  Kossel  and  Mathews,1  in  the  herring 
as  in  the  salmon,  the  heads  of  the  spermatozoa  consist  of  protamine 
nucleate  but  no  free  protein. 

The  chemical  investigations  on  the  spermatozoa  have  not  given 
us  any  information  as  to  the  condition  for  fertilization  and  the  develop- 
ment of  the  egg. 

Spermatin  is  a  name  which  has  been  given  to  a  constituent  similar  to  alkali 
albuminate,  but  it  has  not  been  closely  studied. 

Prostatic  concrements  are  of  two  kinds.  One  is  very  small,  generally  oval 
in  shape,  with  concentric  layers.  In  young  but  not  in  older  persons  they  are 
colored  blue  by  iodine  (Iversen  2).  The  other  kind  is  larger,  sometimes  the  size 
of  the  head  of  a  pin,  consisting  chiefly  of  calcium  phosphate  (about  700  p.m.),  with 
only  a  very  small  amount  (about  160  p.  m.)  of  organic  substance. 

(b)  Female  Generative  Organs. 

The  stroma  of  the  ovaries  is  of  little  interest  from  a  physiologico- 
chemical  standpoint,  and  the  most  important  constituents  of  the  ovaries, 
the  Graafian  follicles  with  the  ovum,  have  not  thus  far  been  the  subject 
of  a  careful  chemical  investigation.  The  fluid  in  the  follicles  (of  the 
cow)  does  not  contain,  as  has  been  stated,  the  peculiar  bodies,  paral- 
bumin or  metalbumin,  which  are  found  in  certain  pathological  •  ovarial 
fluids,  but  seems  to  be  a  serous  liquid.  The  corpora  lutea  are  colored 
yellow.  Earlier  investigators  (Piccolo  and  Lieben.  Kuhne  and  Ewald  3) 
have  found  a  crystalline  pigment  in  the  corpora  lutea.  In  recent 
investigations  Escher4  has  shown  that  this  substance  is  a  crystalline 
hydrocarbon  (C40-H50)  which  seems  to  be  identical  with  the  carotin  of 
the  carrot  and  green  leaves.  The  color  of  the  crystals  as  well  as  the  con- 
centrated solution  is  reddish-orange.  Carotin  differs  from  the  yellow 
pigment  of  the  yolk  of  the  egg,  the  lutein,  in  having  another  formula 

1  Zeitschr.  f.  physiol.  Chem.,  23. 

1  Nord.  med.  Ark.,  6. 

•  See  Chapter  V,  p.  301. 

♦Zeitschr.  f.  physiol.  Chem.,  83,  198  (1912). 


624  ORGANS  OF  GENERATION. 

(page  631)  and  being  soluble  with  difficulty  in  alcohol  and  readily  soluble 
in  petroleum  ether. 

The  cysts  often  occurring  in  the  ovaries  are  of  special  pathological 
interest,  and  these  may  have  essentially  different  contents,  depending 
upon  their  variety  and  origin. 

The  serous  cysts  (Hydrops  folliculorum  Graafii),  which  are 
formed  by  a  dilation  of  the  Graafian  follicles,  contain  a  serous  liquid 
which  has  a  specific  gravity  of  1.005-1.022.  A  specific  gravity  of  1.020 
is  less  frequent.  Generally  the  specific  gravity  is  lower,  1.005-1.014, 
with  10-40  p.  m.  solids.  As  far  as  is  known,  the  contents  of  these  cysts 
do  not  essentially  differ  from  other  serous  liquids. 

The  proliferous  cysts  (myxoid  cysts,  colloid  cysts),  which  are 
developed  from  Pfluger's  epithelium-tubes,  may  have  a  content  of  a 
decidedly  variable  composition. 

We  sometimes  find  in  small  cysts  a  semi-solid,  transparent,  or  some- 
what cloudy  or  opalescent  mass  which  appears  like  solidified  glue  or 
quivering  jelly,  and  which  has  been  called  colloid  because  of  its  physical 
properties.  In  other  cases  the  cysts  contain  a  thick,  tough  mass  which 
can  be  drawn  out  into  long  threads,  and  as  this  mass  in  the  different 
cysts  is  more  or  less  diluted  with  serous  liquids  their  contents  may  have 
a  variable  consistency.  In  still  other  cases  the  small  cysts  may  also 
contain  a  thin,  watery  fluid.  The  color  of  the  contents  is  also  variable. 
Sometimes  they  are  bluish-white,  opalescent,  and  again  they  are  yellow, 
yellowish-brown,  or  yellowish  with  a  shade  of  green.  They  are  often 
colored  more  or  less  chocolate-brown  or  red-brown,  due  to  the  decom- 
posed blood-coloring  matters.  The  reaction  is  alkaline  or  nearly  neutral. 
The  specific  gravity,  which  may  vary  considerably,  is  generally  1.015- 
1.030,  but  may  occasionally  be  1.005-1.010  or  1.050-1.055.  The  amount 
of  solids  is  very  variable.  In  rare  cases  it  amounts  to  only  10-20  p.  m. ; 
ordinarily  it  varies  from  50-70-100  p.  m.  In  a  few  instances  150-200 
p.  m.  solids  have  been  found. 

As  form-elements  one  finds  red  and  white  blood-corpuscles,  granular 
cells,  partly  fat-degenerated  epithelium  and  partly  large  so-called  Gluge's 
corpuscles,  fine  granular  masses,  epithelium-cells,  cholesterin  crystals,  and 
colloid  corpuscles — large,  circular,  highly  refractive  formations. 

Though  the  contents  of  the  proliferous  cyst  may  have  a  variable 
composition,  still  it  may  be  characterized  in  typical  cases  by  its  slimy 
or  ropy  consistency;  by  its  grayish-yellow,  chocolate-brown,  sometimes 
whitish-gray  color;  and  by  its  relatively  high  specific  gravity,  1.015- 
1.025.  Such  a  liquid  does  not  ordinarily  show  a  spontaneous  fibrin 
coagulation. 

We  consider  colloid,  metalbumin,  and  paralbumin  as  characteristic 
constituents  of  these  cysts. 


COLLOID.     PSEUDOMUCIN.  G25 

Colloid.  This  name  does  not  designate  any  particular  chemical 
substance,  but  is  given  to  the  contents  of  tumors  with  certain  physical 
properties  similar  to  gelatin  jelly.  Colloid  is  found  as  a  pathological 
product  in  several  organs. 

Colloid  is  a  gelatinous  mass,  insoluble  in  water  and  acetic  acid;  it  is 
dissolved  by  alkalies  and  gives  a  liquid  which  is  not  precipitated  by 
acetic  acid  or  by  acetic  acid  and  potassium  ferrocyanide.  According  to 
Pfannenstiel  '  such  a  colloid  is  designated  /3-pseudomucin.  Some- 
times a  colloid  is  found  which,  when  treated  with  a  very  dilute  alkali, 
gives  a  solution  similar  to  a  mucin  solution.  Colloid  is  very  closely 
related  to  mucin  and  is  considered  by  certain  investigators  as  a  modified 
mucin.  An  ovarial  colloid  analyzed  by  Panzer  contained  931  p.  m. 
water,  57  p.  m.  organic  substance,  and  12  p.  m.  ash.  The  elementary 
composition  was  C  47.27,  H  5.8G,  N  8.40,  S  0.79,  P  0.54,  and  ash  6.43 
per  cent.  A  colloid  found  by  Wurtz  2  in  the  lungs  contained  C  48.09, 
H  7.47,  X  7.00,  and  0(+S)  37.44  per  cent.  Colloids  of  different  origin 
seem  to  be  of  varying  composition. 

Mctolbumin.  This  name  Scherer3  gave  to  a  protein  substance 
found  by  him  in  an  ovarial  fluid.  The  metalbumin  was  considered  by 
Scherer  to  be  an  albuminous  body,  but  it  belongs  to  the  mucin  group, 
and  it  is  for  this  reason  called  pseudomucin  by  Hammarsten.4 

Pseudomucin.  This  body,  wrhich,  like  the  mucins,  gives  a  reducing 
substance  when  boiled  with  acids,  is  a  mucoid  of  the  following  com- 
position: C  49.75,  H  6.98,  N  10.28,  S  1.25,  O  31.74  per  cent  (Hammar- 
sten). With  water  pseudomucin  gives  a  slimy,  ropy  solution,  and  it  is 
this  substance  which  gives  the  fluid  contents  of  the  ovarial  cysts  their 
typical  ropy  property.  Its  solutions  do  not  coagulate  on  boiling,  but 
only  become  milky  or  opalescent.  Unlike  mucin,  pseudomucin  solutions 
are  not  precipitated  by  acetic  acid.  With  alcohol  they  give  a  coarse 
flocculent  or  thready  precipitate  which  is  soluble  even  after  having  been 
kept  under  water  or  alcohol,  for  a  long  time. 

Paralbumin  is  another  substance  discovered  by  Scherer,  which  occurs 
in  ovarial  liquids,  and  also  in  ascitic  fluids,  with  the  simultaneous  presence 
of  ovarial  cysts  and  rupture  of  the  same.  It  is  therefore  only  a  mixture 
of  pseudomucin  with  variable  amounts  of  protein,  and  the  reactions  of 
paralbumin  are  correspondingly  variable. 


1  Arch.  f.  Gynak.,  38. 

2  Panzer,  Zeitschr.  f.  physiol.  Chem.,  28;  Wiirtz,  see  Lebert,  Beitr.  zur  Kenntnis 
des  Gallertkrebses,  Virchow's  Arch.,  4. 

3  Yerh.  d.  physik.-med.  Gesellsch.  in  Wurzburg,  2,  and  Sitzungsber.  der  physik.- 
rr.ed.  Gesellsch.  in  Wurzburg  fur  1864-1865;  Wurzburg  med.  Zeitschr.,  7,  No.  6. 

4  Zeitschr.  f .  physiol.  Chem.,  6. 


626  ORGANS  OF  GENERATION. 

Mitjukoff  l  has  isolated  and  investigated  a  colloid  from  an  ovarial  cyst.  It 
had  the  following  composition:  C  51.76,  H  7.76,  N  10.7  S  1.09,  and  0  28.69  per 
cent,  and  differed  from  mucin  and  pseudomucin  by  reducing  Fehling's  solu- 
tion before  boiling  with  acid.  It  must  be  remarked  that  pseudomucin,  on  boiling 
sufficiently  long  with  alkali,  or  by  the  use  of  a  concentrated  solution  of  caustic 
alkali,  also  splits  and  causes  a  reduction.  This  reduction  is  nevertheless  weak 
as  compared  with  that  produced  after  boiling  with  an  acid.  The  body  isolated 
by  Mitjukoff  is  called  paramucin. 

The  pseudomucin  as  well  as  colloid  are  mucoid  substances,  and  the 
carbohydrate  obtained  from  them  is  glucosamine  (chitosairine),  as  espe- 
cially shown  by  Fr.  Muller,  Netjberg  and  Heymann.2  From  pseudo- 
mucin Zangerle3  obtained  30  per  cent  glucosamine,  and  Netjberg  and 
Heymann  have  shown  that  the  glucosamine  is  the  only  carbohydrate 
regularly  taking  part  in  the  structure  of  these  substances.  Still  there  are 
reports  as  to  the  occurrence  of  chondroitin-sulphuric  acid  (or  an  allied 
acid)  in  pseudomucin  or  colloid  (Panzer),  but  this  is  not  constant 
according  to  the  experience  of  Hammarsten. 

As  hydrolytic  cleavage  products  of  pseudomucin  Otori  obtained, 
besides  carbohydrate  derivatives  such  as  levulinic  acid  and  humus  sub- 
stances, leucine,  tyrosine,  glycocoll,  aspartic  acid,  glutamic  acid,  valeric 
acid,  arginine,  lysine,  and  guanidine.  The  quantity  of  guanidine,  it 
seems,  was  greater  than  that  which  could  be  derived  from  the  arginine, 
hence  this  body  probably  originated  from  another  complex.  Pregl4 
obtained  on  the  hydrolysis  of  a  colloid,  which  behaved  like  paramucin, 
no  glycocoll  and  only  traces  of  diamino  acids,  but  otherwise  the  same 
amino-acids  as  Otori  found,  besides  alanine,  proline,  phenylalanine  and 
tryptophane. 

The  detection  of  metalbumin  and  paralbumin  is  naturally  connected 
with  the  detection  of  pseudomucin.  A  typical  ovarial  fluid  containing 
pseudomucin  is,  as  a  rule,  sufficiently  characterized  by  its  physical  proper- 
ties, and  a  special  chemical  investigation  is  necessary  only  in  cases  where  a 
serous  fluid  contains  very  small  amounts  of  pseudomucin.  The  pro- 
cedure is  as  follows:  The  protein  is  removed  by  heating  to  boiling  with 
the  addition  of  acetic  acid;  the  filtrate  is  strongly  concentrated  and  pre- 
cipitated by  alcohol.  The  precipitate,  a  transformation  product  of 
pseudomucin,  is  carefully  washed,  with  alcohol  and  then  dissolved  in  water. 
A  part  of  this  solution  is  digested  with  saliva  at  the  temperature  of  the 
body  and  then  tested  for  glucose  (derived  from  glycogen  or  dextrin). 
If  glycogen  is  present,  it  will  be  converted  into  glucose  by  the  saliva; 
precipitate  again  with  alcohol  and  then  proceed  as  in  the  absence  of 

1  K.  Mitjukoff,  Arch.  f.  Gynakol.,  49. 

2  Muller,  Verh.  d.  Naturf.  Gesellsch.  in  Basel.  12,  part  2;  Neuberg  and  Heymaim; 
Hofmeister's  BeitriiKe,  2.     See  also  Leathes,  Arch.  f.  exp.  Path.  u.  Pharm.,  43. 

1  Mfinch.  med.  Wochenschr.,  1900. 

4  Otori,  Zeitschr.  f.  physiol.  Chem.,  42  and  43;  Pregl,  ibid.,  58. 


OVARIAL  CYSTS.  627 

glycogen.  In  this  last-mentioned  case,  first  add  acetic  acid  to  the  solu- 
tion of  the  alcohol  precipitate  in  water  so  as  to  precipitate  a/iy  existing 
mucin.  The  precipitate  produced  is  filtered  off,  the  filtrate  treated  with 
2  per  cent  HC1  and  wanned  on  the  water-bath  until  the  liquid  is  deep 
brown  in  color.  In  the  presence  of  pseudomucin  this  solution  gives 
Trommer's  test. 

The  other  protein  bodies  which  have  been  found  in  cystic  fluids  are 
serglobulin  and  seralbumin,  peptone  (?),  mucin,  and  mucin-peptone  (?). 
Fibrin  occurs  only  in  exceptional  cases.  The  quantity  of  mineral  bodies 
on  an  average  amounts  to  about  10  p.  m.  The  amount  of  extractive 
bodies  (cholesterin  and  urea)  and  fat  is  ordinarily  2-4  p.  m.  The  remaining 
solids,  which  constitute  the  chief  mass,  are  protein  bodies  and  pseudo- 
mucin. 

The  intraligamentary,  papillary  cysts  contain  a  yellow,  yellowish- 
green,  or  brownish-green,  liquid  which  contains  either  no  pseudomucin 
or  very  little.  The  specific  gravity  is  generally  rather  high,  1.032-1.036, 
with  90-100  p.  m.  solids.  The  principal  constituents  are  the  simple 
proteins  of  blood-serum. 

The  rare  tubo-ovarial  cysts  contain  as  a  rule  a  watery,  serous  fluid 
containing  no  pseudomucin. 

The  parovarial  cysts  or  the  cysts  of  the  ligamenta  lata  may  attain 
a  considerable  size.  In  general,  and  when  quite  typical,  the  contents  are 
watery,  mostly  very  pale-yellow-colored,  water-clear  or  only  slightly 
opalescent  liquids.  The  specific  gravity  is  low,  1.002-1.009,  and  the 
solids  only  amount  to  10-20  p.  m.  Pseudomucin  does  not  occur  as  a 
typical  constituent;  protein  is  sometimes  absent,  and  when  it  does  occur 
the  quantity  is  very  small.  The  principal  part  of  the  solids  consists  of 
salts  and  extractive  bodies.  In  exceptional  cases  the  fluid  may  be  rich 
in  protein  and  may  show  a  higher  specific  gravity. 

In  regard  to  the  quantitive  composition  of  the  fluid  from  ovarial 
cysts  we  refer  the  reader  to  the  work  of  Oerum.1 

E.  Ludwig  and  R.  v.  Zeynek  have  investigated  the  fat  from  dermoid  cysts. 
Besides  a  little  arachidic  acid,  they  found  oleic,  stearic,  palmitic,  and  myristic 
acids,  cetyl  alcohol,  and  a  cholesterin-like  substance.  In  regard  to  the  occurrence 
of  cetyl  alcohol  see  the  work  of  Ameseder,2  page  239. 

The  colloid  from  a  uterine  fibroma  analyzed  by  Stollmann3  contained  a 
pseudomucin  soluble  in  water,  and  a  colloid  (paramucin)  insoluble  in  water,  both 
of  which  behaved  differently  with  alcohol  as  compared  with  the  corresponding 
substances  from  ovarial  cysts. 

1  Kemiske  Studier  over  Ovariecystevaedsker,  etc.,  Koebenhavn,  1S84.  See  also 
Maly's  Jahresber.,  14,  450. 

2  Ludwig  and  v.  Zeynek,  Zeitschr.  f.  physiol.  Chem.,  23;  Ameseder,  ibid.,  52; 
Salkowski,  Bioch.  Zeitschr.,  32. 

1  Amer.  Gynecology,  1903. 


628  ORGANS  OF  GENERATION. 


The  Ovum. 

The  small  ova  of  man  and  mammals  eannot,  for  evident  reasons,  be 
the  subject  of  a  searching  chemical  investigation.  Up  to  the  present 
time  the  eggs  of  birds,  amphibians,  and  fishes  have  been  investigated, 
but  above  all  the  hen's  egg.  We  will  here  occupy  ourselves  with  the  con- 
stituents of  this  last. 

The  Yolk  of  the  Hen's  Egg.  In  the  so-called  white  yolk,  which 
forms  the  germ  with  a  process  reaching  to  the  center  of  the  yolk  (latebra), 
and  forming  a  layer  between  the  yolk  and  yolk-membrane,  there  occurs 
protein,  nuclein,  lecithin,  and  potassium  (Liebermann  l).  The  occur- 
rence of  glycogen  is  doubtful.  The  yolk-membrane  consists  of  an  albu- 
minoid similar  in  certain  respects  to  keratin  (Liebermann). 

The  principal  part  of  the  yolk — the  nutritive  yolk  or  yellow — is  a 
viscous,  non-transparent,  pale-yellow  or  orange-yellow  alkaline  emulsion 
of  a  mild  taste.  The  yolk  contains  vitellin,  lecithin,  cholesterin,  fat,  color- 
ing-matters, traces  of  neuridine  (Brieger  2),  purine  bases  (Mesernitzki3), 
glucose  in  very  small  quantities,  and  mineral  bodies.  The  occurrence  of 
cerebrin  and  of  granules  similar  to  starch  (Dareste  4)  has  not  been  posi- 
tively proved. 

Several  enzymes  have  been  found  in  the  yolk,  especially  a  diastatic 
enzyme  (Muller  and  Masuyama),  a  glycolytic  enzyme  (Stepanek) 
which  in  the  absence  of  air  brings  about  an  alcoholic  fermentation  of 
sugar  and  in  the  presence  of  air  forms  carbon  dioxide  and  lactic  acid, 
and  finally  a  proteolytic,  a  lipolytic,  and  a  chromolytic  (?)  enzyme 
(Wohlgemuth  5). 

Ovovitellin.  This  body,  which  is  often  considered  as  a  globulin 
is  in  reality  a  nucleoalbumin.  The  question  as  to  what  relation  other 
protein  substances,  which  are  related  to  ovovitellin,  like  the  aleuron 
grains  of  certain  seeds,  and  the  yolk  spherules  of  the  eggs  of  certain  fishes 
and  amphibians,  bear  to  this  substance  is  one  which  requires  further 
investigation. 

The  ovovitellin  which  has  been  prepared  from  the  yolk  of  eggs  is  not  a 
pure  protein  body,  but  always  contains  lecithin.  Hoppe-Seyler  found 
25  per  cent  lecithin  in  vitellin.  The  lecithin  may  be  removed  by  boiling 
alcohol,  but  the  vitellin  is  changed  thereby,  and  it  is  therefore  probable 


1  Pfluger's  Arch.,  43. 
1  Ueber  Ptomaine,  Berlin,  1885. 
a  Mesernitzki,  Bioehem.  Centralbl.,  1,  739. 
*  Compt.  Rend.,  72. 

6  Muller  and  Masuyama,  Zeitschr.  f.  Biologie,  39;  Stepanek,  Centralbl.  f.  Physiol., 
18,  188;  Wohlgemuth  in  Salkowski's  Festschrift  and  Zeitschr.  f.  physiol.  Chem.,  44. 


OVOVITELLIN.  629 

that  the  lecithin  is  chemically  ui.ited  with  the  vitellin  (Hoppe-Seyler  l). 
According  to  Osborne  and  Campbell,  the  so-called  ovovitellin  is  a  mix- 
ture of  various  vitellin-lecithin  combinations,  with  15  to  30  per  cent  of 
lecithin.  The  protein  substance  freed  from  lecithin  is  the  same  in  all 
these  compounds  and  has  the  following  composition:  C  51.24,  H  7.16, 
N  16.38,  S  1.04,  P  0.94,  O  23.24  pur  cent.  These  figures  differ  somewhat 
from  those  obtained  by  Gross  for  vitellin  prepared  by  another  method 
(precipitation  with  [NH4]2S04),  namely,  C  48.01,  H  6.35,  N  14.91-16.97, 
P  0.32-0.35,  S  0.88,  and  the  composition  of  ovovitellin  is  therefore  not 
positively  known.  Besides  the  vitellin  Gross  found  a  globulin  coagulat- 
ing at  76-77°  C.  in  a  solution  containing  salt,  and  Pllmmer2  found  a 
protein  which  he  calls  livetin  which  only  contained  0.1  per  cent  phos- 
phorus and  which  gave  more  monamino  acids  but  less  amide  and  diamino 
nitrogen  than  vitellin. 

On  the  pepsin  digestion  of  ovovitellin,  Osborne  and  Campbell 
obtained  a  pseudonuclein  with  varying  amounts  of  phosphorus,  2.52- 
4.19  per  cent.  Bunge3  prepared  a  pseudonuclein  by  digesting  the  yolk 
with  gastric  juice,  and  his  pseudonuclein, .  he  claims,  is  of  great  impor- 
tance in  the  formation  of  the  blood,  and  on  these  grounds  he  called  it 
hcematogen.  This  hsematogen  has  the  following  composition:  C  42.11, 
H  6.08,  N  14.73,  S  0.55,  P  5.19,  Fe  0.29,  and  O  31.05  per  cent.  The 
composition  of  this  substance  may  vary  considerably  even  on  using  the 
same  method  of  preparation. 

Vitellin  is  similar  to  the  globulins  in  that  it  is  insoluble  in  water,  but 
on  the  contrary  soluble  in  dilute  neutral-salt  solutions  (although  the  solu- 
tion is  not  quite  transparent).  It  is  also  soluble  in  hydrochloric  acid  of 
1  p.  m.  and  in  very  dilute  solutions  of  alkalies  or  alkali  carbonates.  It 
is  precipitated  from  its  salt  solution  by  diluting  with  water,  and  when 
allowed  to  stand  some  time  in  contact  with  water  the  vitellin  is  gradually 
changed,  forming  a  substance  more  like  the  albuminates.  The  coagu- 
lation temperature  for  the  solution  containing  salt  (NaCl)  lies  between 
70  and  75°  C,  or,  when  heated  very  rapidly,  at  about  80°  C.  Vitellin 
differs  from  the  globulins  in  yielding  pseudonuclein  by  peptic  digestion. 
It  is  not  always  completely  precipitated  by  NaCl  in  substance.  The 
ovovitellin  isolated  by  Gross  gave  Molisch's  reaction.  Neuberg4 
has  also  split  off  glucosamine  from  the  yolk  and  has  identified  it  as  nori- 

1  Med.  ehem.  Untersuch.,  216. 

2  Osborne  and  Campbell,  Connecticut  Agric.  Exp.  Station,  23d  Ann.  Report,  New 
Haven,  1900;  Gross,  Zur  Kenntn.  d.  Ovovitellin,  Inaug.-Diss.  Strassburg,  1S99; 
Plimmer,  Journ.  Chem.  Soc,  London,  93. 

3  Zeitschr.  f.  physiol.  Chem.,  9,  49.  See  also  Hugounenq  and  Morel,  Compt.  Rend., 
140  and  141. 

*  Ber.  d.  d.  chem.  Gesellsch.,  34. 


630  OPUAXS  OF  GENERATION. 

sosaccharic  acid.     It  is  difficult  to  state  whether  this  glucosamine  was 
derived    from  the  vitellin  or  from  some  other  constituent  of  the  yolk. 

The  principal  points  in  the  preparation  of  ovovitellin  are  as  follows: 
The  yolk  is  thoroughly  agitated  with  ether;  the  residue  is  dissolved  in 
a  10-per  cent  common-salt  solution,  filtered,  and  the  vitellin  precipitated 
by  adding  an  abundance  of  water.  The  vitellin  is  now  purified  by  repeat- 
edly redissolving  in  dilute  common-salt  solutions  and  precipitating  with 
water. 

Ichthulin,  which  occurs  in  the  eggs  of  the  carp  and  other  fishes  is,  accord- 
ing to  Kossel  and  Walter,  an  amorphous  modification  of  the  crystalline  body 
tchthidin,  which  occurs  in  the  eggs  of  the  carp.  Ichthulin  is  precipitated  on 
diluting  with  water.  It  was  formerly  considered  as  a  vitellin.  According  to 
Walter  it  yields  a  pseudonuclein  on  peptic  digestion;  and  this  pseudonuclein 
gives  a  reducing  carbohvdate  on  boiling  with  sulphuric  acid.  Ichthulin  has  the 
following  composition;  C  53.42,  H  7.63,  N  15.63,  0  22.19,  S  0.41,  P  0.43  percent. 
It  also  contains  iron.  The  ichthulin  investigated  from  codfish  eggs  by  Levene 
had  the  composition  C  52.44,  H  7.45,  N  15.96,  S  0.92,  P  0.65,  Fe+O  22.58 
per  cent,  and  yielded  no  reducing  substances  on  boiling  with  acids.  The  pure 
vitellin  isolated  by  Hammarsten  from  perch  eggs  had  a  similar  behavior  and 
was  very  readily  changed  by  a  little  hydrochloric  acid  so  that  it  was  converted 
into  a  typical  pseudonuclein.  The  codfish  ichthulin  yielded  a  pseudonucleic  acid 
with  10.34  per  cent  phosphorus,  but  this  acid  still  gave  the  protein  reactions. 
McClenden  1  has  prepared  a  vitellin  from  frogs'  eggs  which  he  calls  batrachiolin. 

The  yolk  also  contains  albumin,  besides  vitellin  and  the  above-men- 
tioned proteins. 

The  fat  of  the  yolk  of  the  eg?,  Liebermann2  claims,  is  a  mixture  of 
a  solid  and  a  liquid  fat.  The  solid  fat  consists  principally  of  tripalmitin 
with  some  tristearin.  On  the  saponification  of  the  egg-oil  Liebermann 
obtained  40  per  cent  oleic  acid,  -38.04  per  cent  palmitic  acid,  and  15.21 
per  cent  stearic  acid.  The  fat  of  the  yolk  of  the  egg  contains  less  carbon 
than  other  fats,  which  may  depend  upon  the  presence  of  monoglycerides 
and  diglycerides,  or  upon  a  quantity  of  fatty  acid  deficient  in  carbon 
(Liebermann).  The  composition  of  yolk  fat  is  dependent  upon  the 
food,  as  Henriques  and  Hansen3  have  shown  that  the  fat  of  the  food 
passes  into  the  egg. 

The  phosphatides  of  the  yolk  seem  to  be  of  various  kinds.  Thier- 
felder  and  Stern  have  found  three  different  phosphatides.  One  of 
these,  which  was  soluble  in  alcohol-ether,  behaved  like  lecithin.  The 
second  was  soluble  with  difficulty  in  alcohol,  but  readily  soluble  in  ether, 
contained  1.37  per  cent  N  and  3.96  per  cent  P.    The  third  was  a  diamino 

1  Walter,  Zeitsfhr.  f.  physiol.  Chem.,  15;  Levene,  ibid.,  32;  Hammarsten,  Skand. 
Arch.  f.  Physiol.,  17;  McClenden,  Amer.  Journ.  of  Physiol.  25;  see  also  Plimmer 
and  Scott,  Journ.  Chem.  Soc,  1)3. 

2PfluKer's  Arch.,  4:5. 

•Skand.  Arch.  f.  Physiol.,  14. 


LUTEIN.  631 

phosphatide,  soluble  with  difficulty  in  ether,  but  obtained  in  crystalline 
needles  from  hot  alcohol,  and  contained  2.77  per  cent  N  and  3.22  per  cent 
P,  and  had  a  melting-point  of  100-170°  C.  Frankel  and  Bolaffio  1 
also  found  a  substance  crystallizing  from  hot  alcohol  and  insoluble  in 
ether  with  2.78  per  cent  N  and  2.18  per  cent  P.  They  call  this  body 
neottin  and  claim  that  it  is  a  triamino-monophosphatide  having  the  formula 
C84H172N3PO15.  Barbieri  has  obtained  a  sulphurized  phosphatide 
called  ovin,  containing  1.35  per  cent  P,  3.06  per  cent  N  and  0.4  per  cent  S. 
The  relation  of  all  these  bodies  to  each  other  must  be  further  studied. 

Lutein.  With  the  name  lutein  we  in  the  past  have  included  several 
yellow  or  orange-red  amorphous  coloring-matters  which  occur  in  the 
yellow  of  the  egg,  and  in  several  other  places  in  the  animal  organism; 
for  instance,  in  the  blood-serum  and  serous  fluids,  fatty  tissues,  milk- 
fat,  corpora  lutea,  and  in  the  fat-globules  of  the  retina  as  well  as  in  dif- 
ferent plants  (Thudichum).  Among  these  bodies  belong  the  crys- 
talline substance  obtained  by  Escher  from  the  corpora  lutea  (page  623). 
It  was  difficultly  soluble  in  alcohol  but  readily  soluble  in  petroleum  ether 
and  showed  itself  isomeric  or  perhaps  identical  with  the  plant  pigment 
carotin  (CioHsc)  analyzed  by  Willstatter  and  Mieg.  The  lutein  of 
the  egg  yolk,  which  is  more  readily  soluble  in  alcohol  and  less  soluble  in 
petroleum  ether  than  carotin  has  also  been  obtained  by  Willstatter  and 
Escher  in  a  pure,  crystalline  form.  On  analysis  it  gave  the  formula 
C40H56O2.  As  shown  by  C.  A.  Schtjnck  the  yolk  lutein  stands  in 
close  relation  to  the  yellow  plant  pigment,  xanthophyll.  The  formula 
given  by  Willstatter  and  Escher  for  lutein  was  in  fact  the  same  as 
for  the  xanthophyll,  as  previously  found  by  Willstatter  and  Mieg. 
These  two  substances  are  also  similar  in  other  respects;  still  the  melting- 
points  of  the  two  are  different.  The  carotin  and  the  yolk  lutein  differ 
also  by  the  absorption  spectra,  which  is  different  in  different  solvents 
as  well  as  by  their  formulae  and  different  solubilities.2 

The  relation  of  the  other  substances  called  luteins  to  each  other  and 
to  the  yolk  lutein  is  unknown.  All  are  soluble  in  alcohol,  ether,  and  chloro- 
form. They  differ  from  the  bile-pigment,  bilirubin,  in  that  they  are  not 
separated  from  their  solution  in  chloroform  by  water  containing  alkali, 
and  also  in  that  they  do  not  give  the  characteristic  play  of  colors  with 
nitric  acid  containing  a  little  nitrous  acid,  but  give  a  transient  blue  color. 
The  luteins  withstand  the  action  of  alkalies  so  that  they  are  not  changed 
when  we  remove  the  fats  present  by  means  of  saponification. 

1  Thierfeldcr  and  Stern,  Zeitschr.  f.  physiol.  Chem.,  53;  Frankel  and  Bolaffio, 
Bioch.  Zeitschr.,  9;  Barbieri,  Compt.  Rend.,  145. 

2  Thudichum,  Centralbl.  f.  d.  ined.  Wiss.  1869;  Willstatter  and  Mieg.  Ann.  d. 
Chem.,  355  (1907);  Willstatter  and  Escher,  Zeitschr.  f.  physiol.  Chem.,  64  1909); 
76  (1911);  Schunck,  see  Chem.  Centralbl.,  1903. 


632  ORGANS  OF  GENERATION. 

Maly  '  found  two  pigments  free  from  iron  in  the  eggs  of  a  water-spider  (Maja 
squinado) — one  a  red  (riteUorubin)  and  the  other  a  yellow  pigment  (vitellolutein) . 
Both  of  these  pigments  are  colored  blue  by  nitric  acid  containing  nitrous  acid 
and  a  beautiful  green  by  concentrated  sulphuric  acid. 

The  mineral  bodies  of  the  yolk  of  the  egg  consist,  according  to  Poleck,2 
of  51.2-65.7  parts  soda,  80.5-89.3  potash,  122.1-132.8  lime,  20.7-21.1 
magnesia,  11.90-14.5  iron  oxide,  638.1-667.0  phosphoric  acid,  and  5.5- 
14.0  parts  silicic  acid  in  1000  parts  of  the  ash.  We  find  phosphoric  acid, 
and  lime  the  most  abundant,  and  then  potash,  which  is  somewhat  greater 
in  quantity  than  the  soda.  These  results  are  not,  however,  quite  cor- 
rect: first,  because  no  dissolved  phosphate  occurs  in  the  yolk  (Lieber- 
mann),  and  secondly,  in  burning,  phosphoric  and  sulphuric  acids  are 
produced,  and  these  drive  away  the  chlorine,  which  is  not  accounted 
for  in  the  above  analyses. 

The  yolk  of  the  hen's  egg  weighs  about  12-18  grams.  The  quan- 
tity of  water  and  solids  amounts,  according  to  Parke,3  to  471.9  p.  m. 
and  528.1  p.  m.  respectively.  Among  the  solids  he  found  156.3  p.  m. 
protein,  3.53  p.  m.  soluble  and  6.12  p.  m.  insoluble  salts.  The  quantity 
of  fat,  according  to  Parke,  is  228.4  p.  m.;  the  lecithin,  calculated  from 
the  amount  of  phosphorus  in  the  organic  substance  of  the  alcohol-ether 
extract,  was  107.2  p.  m.  and  the  cholesterin  17.5  p.  m. 

The  white  of  the  egg  is  a  faintly  yellow  albuminous  fluid  inclosed 
in  a  framework  of  thin  membranes;  and  this  fluid  is  in  itself  very  liquid, 
but  seems  viscous  because  of  the  presence  of  these  fine  membranes.  That 
substance  which  forms  the  membranes,  and  of  which  the  chalaza  con- 
sists, seems  to  be  a  body  closely  related  to  horn  substances  (Lieber- 
mann)  . 

The  white  of  egg  has  a  specific  gravity  of  1.038-1.045,  and  always 
has  an  alkaline  reaction  toward  litmus.  It  contains  850-880  p.  m.  water, 
100-130  p.  m.  protein  bodies,  and  7  p.  m.  salts.  Lehmann  found  a  fer- 
mentable variety  of  sugar  which  Salkowski  showed  was  glucose.  C.  Th. 
Morner  could  not  find  any  other  sugar  in  egg-white;  the  quantity  of 
glucose  as  found  by  Morner4  was  3-5  p.  m.  Besides  these  one  finds 
in  the  white  of  egg  traces  of  fats,  soaps,  lecithin  and  cholesterin. 

The  white  of  egg  of  the  Insessores  becomes  transparent  on  boiling  and  acts 
in  many  respects  like  alkali  albuminate.  This  albumin  Tarchanofp  6  called 
"  tatalbumin." 

1  Monatshefte  f.  Chem.,  2. 

2  Cited  from  v.  Gorup-Besanez,  Lehrbuch  d.  physiol.  Chem.,  4,  Aufl.,  740. 
1  Hoppe-Seyler,  Med.  chem.  Untersuch.,  Heft  2,  209. 

*  Lehmann,  Lehrb.  d.  physiol.  Chem.  2  Aufl.  1855,  Bd.  1,  s.  271;  Bd.  2,  s.  312. 
Salkowski,  Centralbl.  f.  d.  med.  Wiss.,  31  (1893);  Morner.  Zeitschr.  f.  physiol.  Chem; 
80  (1912). 

*  Pfliiger's  Arch.,  31,  33,  and  39. 


OVOGLOBULIN.     OVALBUMIN.  G33 

The  protein  substances  of  the  white  of  egg  behave  like  glycoproteins, 
as  they  all  yield  glucosamine.  For  the  globulin  and  albumin  it  has  not 
been  proved,  nor  is  it  probable?  that  the  glucosamine  belongs  to  the  pro- 
tein molecule  (sec  page  84).  According  to  the  solution  and  precipita- 
tion properties  they  arc  similar  to  the  globulins,  albumins  or  proteoses. 
The  representatives  of  the  first  two  groups,  are  ovoglobulin  and  ovalbumin. 
The  proteose-like  body  is  ovomucoid. 

Ovoglobulin  separates  in  part  on  diluting  the  egg-white  with  water. 
It  is  precipitated  upon  saturation  with  magnesium  sulphate,  or  upon 
one-half  saturation  with  ammonium  sulphate,  and  coagulates  at  about 
75°  C.  By  repeated  solution  in  water  and  precipitation  with  ammonium 
sulphate  a  part  of  the  globulin  becomes  insoluble  (Langstein).  This 
also  occurs  on  precipitation  by  diluting  with  water  or  by  dialysis,  and 
it  is  quite  possible  that  the  globulin  is  a  mixture.  That  portion  which 
readily  becomes  insoluble  seems  to  be  identical  with  Eichholz's  gly- 
coprotein or  Osborne  and  Campbell's  ovomucin.  Langstein  obtained 
11  per  cent  of  glucosamine  from  the  soluble  ovoglobulin.  The  total 
quantity  of  globulins,  according  to  Dillner,  is  about  6.7  per  cent  of 
the  total  protein  substances,  and  this  corresponds  with  the  recent  deter- 
minations of  Osborne  and  Campbell.  In  regard  to  the  probable  occur- 
rence of  several  globulins  in  the  white  of  the  egg  there  are  the  determina- 
tions of  Corin  and  Berard  as  well  as  of  Langstein,1  but  they  have 
not  led  to  any  positive  conclusions. 

Ovalbumin.  The  so-called  albumin  of  the  egg-white  is  undoubtedly 
a  mixture  of  at  least  two  albumin-like  proteins.  Opinions  differ  con- 
siderably in  regard  to  the  number  of  these  proteins  (Bondzynski  and 
Zoja,  Gautier,  Bechamp,  Corin  and  Berard,  Panormoff,  and  others). 
Since  Hofmeister  has  been  able  to  prepare  ovalbumin  in  a  crystalline 
form,  and  since  Hopkins  and  Pinkus  2  have  shown  that  not  more  than 
one-half  of  the  ovalbumin  can  be  obtained  in  such  a  form,  Osborne  and 
Campbell  have  isolated  two  different  ovalbumins  or  principal  fractions; 
the  crystallizable  they  call  ovalbumin  and  the  non-cry stallizable  con- 
albumin.  The  two  fractions  have  only  a  slight  variation  in  elementary 
composition;  the  conalbumin  coagulates  between  50-60°  C,  nearer  to 
60°  C,  and  the  ovalbumin  at  64°  C  or  at  a  higher  temperature.  There 
are   no   conclusive   investigations  as  to   whether   the   non-crystallizal  le 

1  Langstein,  Hofmeister's  Beitrage,  1;  Eichholz,  Journ.  of  Physiol.,  23;  Osborne 
and  Campbell,  Connecticut  Agric.  Exp.  Station.,  23d  Ann.  Report,  New  Haven,  1900; 
Dillner,  Maly's  Jahresber.,  15;    Corin  and  Berard,  ibid.,  18. 

2  Hofmeister,  Zeitschr.  f.  physiol.  Chem.,  14,  16,  and  24,  Gabriel,  ibid.,  15;  Bond- 
zynski and  Zoja,  ibid.,  19;  Gautier,  Bull.  Soc.  chim.,  14;  Bechamp,  ibid.,  21;  Corin 
and  Berard,  1.  c.;  Hopkins  and  Pinkus,  Ber.  d.  d.  ehem.  Gesellsch.,  31,  and  Journ.  of 
Physiol.,  23;  Osborne  and  Campbell,  1.  c.;  Panormoff,  Maly's  Jahresber.,  27  and  28. 


634  ORGANS  OF  GENERATION. 

conalbumin  is  a  mixture  or  not,  and  the  question  concerning  the  unity 
of  the  crystallizable  ovalbumin  is  also  disputed.  According  to  Bond- 
zynski  and  Zoja,  crystallizable  ovalbumin  is  a  mixture  of  several  albumins 
having  somewhat  different  coagulation  temperatures,  solubilities,  and 
specific  rotations,  while  Hofmeister  and  Langstein  on  the  contrary 
believe  that  crystallizable  ovalbumin  is  a  unit.  The  reports  as  to  the 
specific  rotation  of  the  different  fractions  unfortunately  differ,  and  the 
elementary  analyses  have  also  given  no  positive  results,  as  a  variation 
of  1.2-1.7  per  cent  has  been  observed  in  the  quantity  of  sulphur.  Accord- 
ing to  the  consistent  analyses  of  Osborne  and  Campbell  and  of  Lang- 
stein, the  conalbumin  contains  about  1.7  per  cent  sulphur  and  about 
16  per  cent  nitrogen,  while  the  ovalbumin  contains  on  an  average  about 
15.3  per  cent  nitrogen.  Langstein1  obtained  10-11  per  cent  glucosa- 
mine from  ovalbumin  and  about  9  per  cent  from  conalbumin.  The 
ovalbumin,  like  the  conalbumin,  has  the  properties  of  the  albumins  in 
general,  but  differs  from  seralbumin  in  that  the  specific  rotation 
is  lower.  It  is  quickly  made  insoluble  by  alcohol  and  is  precipitated 
by  a  sufficient  quantity  of  HC1,  but  dissolves  in  an  excess  of  acid  with 
greater  difficulty  than  the  seralbumin.  The  products  isolated  by 
Abderhalden  and  Pregl  2  on  the  hydrolysis  of  ovalbumin  do  not  show 
anything  of  special  interest. 

As  in  the  past  certain  doubts  have  existed  as  to  the  purity  and  chem- 
ical unity  of  the  ovalbumins,  or  also  of  the  crystalline  ovalbumin,  so  now 
this  doubt  has  become  still  stronger  since  ovalbumin  has  been  pre- 
pared partly  free  from  phosphorus  and  partly  with  a  variable  phos- 
phorus content  of  0.1-3.06  per  cent   (Kaas,  Willcock  and  Hardy3). 

In  preparing  .crystalline  ovalbumin,  mix,  according  to  Hofmeister, 
the  beaten  white  of  egg  free  from  foam  with  an  equal  volume  of  a  saturated 
ammonium-sulphate  solution,  filter  off  the  globulin,  and  allow  the  nitrate 
to  evaporate  slowly  in  thin  layers  at  the  temperature  of  the  room.  After 
a  time  the  masses  which  separate  out  are  dissolved  in  water,  treated 
with  ammonium  sulphate-solution  until  they  begin  to  get  cloudy,  and 
are  allowed  to  stand.  After  repeated  recrystallization  the  mass  is  either 
treated  with  alcohol,  which  makes  the  crystals  insoluble,  or  they  are 
dissolved  in  water  and  purified  by  dialysis.  From  these  solutions  the 
proteid  does  not  crystallize  again  on  spontaneous  evaporation.  (See  also 
page  633,  footnote  2,  for  the  Hopkins  and  Pinkus  method.)  Will- 
cock4  has  recently  found  that*  magnesium  sulphate  can  also  be  used  in 
the  crystallization  of  ovalbumin. 

1  Zeitsrhr.  f.  physiol.  Chem.,  31. 
-  Ibid.,  4«. 

3  Kaas,  Monatsh.  f.  Chem.,  27;  Willcock  and  Hardy,  cited  from  Chem.  Centralbl., 
1907,  2,  821. 

4  Journ.  of  Physiol.,  37. 


OVOMUCOID.  635 

Conalbumin  can  ho  removed  from  the  filtrate,  after  the  complete 
crystallization  of  the  ovalbumin,  by  removing  the  sulphate  by  means  of 
dialysis  and  coagulating  by  heat. 

Gautier  '  found  a  fibrinogen-like  substance  in  the  white  of  egg,  which  was 
changed  into  a  fibrin-like  body  by  the  action  of  a  ferment. 

Ovomucoid.  This  substance,  first  observed  by  Neumeister  and 
considered  by  him  as  a  pseudopeptone,  and  then  later  studied  by  Salkow- 
ski,  is,  according  to  C.  Th.  Morner,2  a  mucoid  with  12.65  per  cent 
nitrogen  and  2.20  per  cent  sulphur.  Ovomucoid  exists  in  hens'  eggs 
to  the  extent  of  about  12  per  cent  of  the  total  solids. 

A  solution  of  ovomucoid  is  not  precipitated  by  mineral  acids  nor  by 
organic  acids,  with  the  exception  of  phosphotungstic  acid  and  tannic 
acid.  It  is  not  precipitated  by  metallic  salts,  but  basic  lead  acetate  and 
ammonia  render  it  insoluble.  Ovomucoid  is  thrown  down  by  alcohol, 
but  sodium  chloride,  sodium  sulphate,  and  magnesium  sulphate  give 
no  precipitates  cither  at  the  ordinary  temperature  or  when  the  salts  are 
added  to  saturation  at  30°  C.  Its  solutions  are  not  precipitated  by  an 
equal  volume  of  a  saturated  solution  of  ammonium  sulphate,  but  are 
precipitated  on  adding  more  salt  thereto.  The  substance  is  not  pre- 
cipitated on  boiling,  but  the  part  which  has  become  insoluble  in  cold 
water  and  which  has  been  dried,  is  dissolved  by  boiling  water.  Zanetti 
has  prepared  glucosamine  on  splitting  ovomucoid  with  concentrated 
hydrochloric  acid,  and  Seemann  found  that  the  quantity  of  glucosamine 
in  ovomucoid  was  34.9  per  cent.3 

Ovomucoid  may  be  prepared  by  removing  all  the  proteins  by  boil- 
ing with  the  addition  of  acetic  acid,  and  then  concentrating  the  filtrate 
and  precipitating  with  alcohol.  The  substance  is  purified  by  repeated 
solution  in  water  and  precipitation  with  alcohol. 

Paxormow  believes  that  the  eggs  of  other  birds,  such  as  the  pigeon  and  duck, 
contain  a  special  protein  in  the  egg-white,  which  is  not  identical  with  that  of  the 
hen's  egg.  Worms  4  has  prepared  a  crystalline  albumin  from  the  white  of  the 
turkey  eggs  which  contained  15.37  per  cent  N,  1.6  per  cent  S  and  had  a  specific 
rotation  of  (o;)d  =  —34.9°. 

The  mineral  bodies  of  the  white  of  egg  have  been  analyzed  by 
Poleck  and  Weber.5     They  found  in   1000  parts  of  the  ash:  276.6- 

iCompt.  Rend.,  135. 

2  R.  Neumeister,  Zeitschr.  f.  Biologic,  27;  Salkowski.  Centralbl.  f.  d.  med.  Wis- 
senseh.,  1893,  513  and  706;  C.  Morner,  Zeitsch.  f.  physiol.  Chem.,  18  and  80.  See 
also  Langstein,  Hofmeister's  Beitrage,  3  (literature). 

3  Zanetti,  Chem.  Centralbl.,  1898,  1;  Seemann,  cited  from  Langstein,  Ergebnisse 
der  Physiol.,  1,  Abt.  1,  86. 

4  Panormow,  see  Bioch.  Centralbl.,  5;  Worms,  cited  from  Chem.  Centralbl.,  1906, 
2,  1508. 

5  Cited  from  Hoppe-Seyler,  Physiol.  Chem.,  778. 


636  ORGANS  OF  GENERATION. 

284.5  grams  potash,  235.6-329.3  soda,  17.4-29.0  lime,  17-31.7  magnesia, 
4.4-5.5  iron  oxide,  238.4-285.6  chlorine,  31.6-48.3  phosphoric  acid  (P205), 
13.2-26.3  sulphuric  acid,  2.8-20.4  silicic  acid,  and  96.7-116.0  grams  carbon 
dioxide.  Traces  of  fluorine  have  also  been  found  (Nickles  1).  The 
white  of  egg  contains,  as  compared  with  the  yolk,  a  greater  amount  of 
chlorine  and  alkalies  and  a  smaller  amount  of  lime,  phosphoric  acid,  and 
iron. 

The  Shell-membrane  and  the  Egg-shell.  The  shell-membrane  con- 
sists, as  above  stated  (page  112),  of  a  keratin  substance.  The  shell  con- 
tains very  little  organic  substance,  36-65  p.  m.  The  principal  mass,  more 
than  900  p.  m.,  consists  of  calcium  carbonate;  besides  this  there  are  very 
small  amounts  of  magnesium  carbonate  and  earthy  phosphates. 

The  diverse  coloring  of  birds'  eggs  is  due  to  several  different  coloring-matters. 
Among  these  we  find  a  red  or  reddish-brown  pigment  called  "  oorodein  "  by 
Sorby,2  which  is  perhaps  identical  with  hsematoporphyrin.  The  green  or  blue 
coloring-matter,  Sorby's  oocyan,  seems,  according  to  Liebermann  3  and  Kruken- 
berg,4  to  be  partly  biliverdin  and  partly  a  blue  derivative  of  the  bile-pigments. 

The  eggs  of  birds  have  a  space  at  their  blunt  end  filled  with  gas;  this 
gas  contains  on  an  average  18.0-19.9  per  cent  oxygen  (Hufner5). 

The  weight  of  a  hen's  egg  varies  between  40-60  grams  and  may  some- 
times reach  70  grams.  The  shell  and  shell-membrane  together,  when 
carefully  cleaned,  but  still  in  the  moist  state,  weigh  5-8  grams.  The 
yolk  weighs  12-18  and  the  white  23-34  grams,  or  about  double.  The 
entire  egg  contains  2.8-7.5,  or  average  4.6,  milligrams  of  iron  oxide,  and 
the  quantity  of  iron  can  be  increased  by  food  rich  in  iron  (Hartung  6) . 

The  white  of  the  egg  of  cartilaginous  and  bony  fishes  contains  only  traces  of 
true  albumin,  but  consists,  at  least  in  many  fishes,  of  mucin  substance;  and  the 
cover  of  the  frog's  egg  also  consists,  according  to  Giacosa,  of  mucin.  The  eggs 
of  the  river-perch  contain,  Hammarsten  7  claims,  mucin  in  the  envelope  in  the 
unripe  state  and  only  mucinogen  in  the  ripe  state.  The  crystalline  formations 
(yolk-spherules,  or  dotterpldttchen)  which  have  been  observed  in  the  egg  of  the 
tortoise,  frog,  ray,  shark,  and  other  fishes,  and  which  are  described  by  Valen- 
ciennes and  Fremy  under  the  names  emydin,  ichthin,  ichthidin,  and  ichthulin, 
seem,  as  above  stated  in  connection  with  ichthulin,  to  consist  mainly  of  phos- 
phoglycoproteins.  The  klupeovin  obtained  by  Hugounenq  8  from  the  herrings' 
eggs  and  from  which  he  obtained  the  three  so-called  hexone  bases  and  abundant 

1  Compt.  Rend.,  43. 

2  Cited  from  Krukenberg,  Verb.  d.  phy.s.-cbem.  Gessellsch.  in  Wurzburg,  17. 
8  Ber.  d.  deutsch.  chem.  Gesellsch.,  11. 

•lie. 

6  Arch.  f.  (Anat.  u).  Physiol.,  1892. 
»Zeitechr.  f.  Biol.,  43. 

7Giacos;i,  Zeitschr.  f.  physiol.  Chem.,  7;  Hammarsten,  Skand.  Arch.  f.  Physiol.,  17. 
8  Valenciennes    and    Fremy.    eited    from    Hoppe-Seyler,    Physiol.    Chem.,    p.    77; 
Hugounenq,  Bull.  soc.  chim.  (3),  33,  and  Compt.  Rend.,  143. 


THE  EGG.  G37 

monamino-acids,  especially  leucine,  hut  not  glycocoll  or  glutamic  acid,  is  to 
all  appearances  not  a  unit  body.  The  eggs  of  the  river-crab  and  the  lobster 
contain  the  same  pigment  as  the  shell  of  the  animal.  This  pigment,  called 
cyanocrystallin,  becomes  red  on  boiling  in  water. 

C.  Morner  l  has  isolated  a  substance  which  he  calls  pcrcnglobulin,  from  the 
unripe  eggs  of  the  river-perch.  It  is  a  globulin  and  has  a  strong  astringent  taste. 
Especially  striking  is  its  property  of  precipitating  certain  glycoproteins,  such  as 
ovomucoid  and  ovarial  mucoids,  and  polysaccharides,  such  as  glycogen,  gum, 
tragacanth  and  starch-paste,  and  of  being  precipitated  by  them.  Percaglobulin 
could  not  be  obtained  by  Morner  from  the  eggs  of  the  sea-bass. 

In  fossil  eggs  (of  apetnodytes,  pelecanus,  and  hall,eus)  in  old  guano  deposits, 
a  yellowish-white,  silky,  laminated  compound  has  been  found  which  is  called 
guanovulit,  (NH4)2S04-r-2K2S04+3KHS04-f-4H20,  and  which  is  easily  soluble  in 
water,  but  is  insoluble  in  alcohol  and  ether. 

Those  eggs  which  develop  outside  of  the  mother-organism  must  con- 
tain all  the  elements  necessary  for  the  young  animals.  One  finds,  there- 
fore, in  the  yolk  and  white  of  the  egg  an  abundant  quantity  of  protein 
bodies  of  different  kinds,  and  especially  phosphorized  proteins  in  the 
yolk.  Further,  we  also  find  abundance  of  phosphatides  in  the  yolk, 
which  seem  to  occur  habitually  in  all  developing  cells.  Kato  and  Bleib- 
treu  2  found  glycogen  in  the  eggs  of  the  frog  which  during  the  spawning 
season  increased  at  the  cost  of  the  liver  glycogen.  Besides  this  the  egg 
is  very  rich  in  fat,  which  doubtless  is  important  as  a  source  of  supply 
for  nourishment  and  in  maintaining  respiration  for  the  embryo.  The 
cholesterin  or  at  least  the  lutein  can  hardly  have  a  direct  influence  on 
the  development  of  the  embryo.  The  egg  also  seems  to  contain  the 
mineral  bodies  necessary  for  the  development  of  the  young  animal. 
The  lack  of  phosphoric  acid  is  compensated  by  an  abundant  amount  of 
phosphorized  organic  substance,  and  the  nucleoalbumin  containing 
iron,  from  which  the  haematogen  (see  page  629)  is  formed,  is  doubtless,  as 
Bunge  claims,  of  great  importance  in  the  formation  of  the  haemoglobin 
containing  iron.  The  silicic  acid,  necessary  for  the  development  of  the 
feathers,  is  also  found  in  the  egg. 

During  the  period  of  incubation  the  egg  loses  weight,  due  chiefly  to 
loss  of  water.  The  quantity  of  solids,  especially  the  fat  and  the  proteins, 
diminishes,  and  the  egg  gives  off  carbon  dioxide,  but  Tangl  disproves 
the  older  claim  of  Liebermann  3  that  nitrogen  or  a  nitrogenous  substance 
is  given  off.  On  the  contrary  a  corresponding  absorption  of  oxygen 
takes  place,  and  it  is  found  that  during  incubation  a  respiratory  exchange 
of  gases  occurs. 

As  Bohr  and  Hasselbalch  have  shown  by  exact  investigations,  the 
elimination  of  carbon  dioxide  is  very  small  in  the  first  days  of  incuba- 

1  Zeitschr.  f.  physiol.  Chem.,  40  and  58. 

»Kato,  Pfliiger's  Arch.  132;  Bleibtreu,  ibid.,  132  (1010). 

*  Tangl  and  v.  Mituch,  Pfluger's  Arch.,  121 ;  Liebermann,  ibid.,  43. 


638  ORGANS  OF  GENERATION. 

tion;  on  the  fourth  day  the  carbon-dioxide  production  gradually  increases, 
and  after  the  ninth  day  it  augments  in  the  same  proportion  as  the  weight 
of  the  foetus.  Calculated  upon  1  kilogram  weight  for  one  hour  it  is,, 
from  the  ninth  day  on,  about  the  same  as  in  the  full-grown  hen.  Hassel- 
balch  l  has  also  shown  that  the  fertilized  hen's  egg  not  only  gives  off 
nitrogen  the  first  five  or  six  hours  of  incubation,  but  also  some  oxygen, 
and  that  we  are  here  dealing  with  an  oxygen  production  which  runs 
parallel  with  the  cell-division.  It  is  not  known  whether  this  oxygen 
formation  connected  with  the  life  of  the  cell  is  a  fermentative  or  a  so- 
called  vital  process. 

While  the  quantity  of  dry  substance  in  the  egg  during  this  period 
always  decreases,  the  quantity  of  mineral  bodies,  protein,  and  fat  always 
increases  in  the  embryo.  The  increase  in  the  amount  of  fat  in  the 
embryo  depends,  in  great  part  upon  a  taking  up  of  the  nutritive  yolk 
in  the  abdominal  cavity.  Plimmer  and  Scott  2  have  observed  in  the  incu- 
bation of  the  hen's  egg,  that  a  rapid  diminution  of  phosphorized  substances 
soluble  in  ether  takes  place,  while  at  the  same  time  an  increase  in  the 
inorganic  phosphorus  is  found  in  the  chick. 

The  weight  of  the  shell  and  the  quantity  of  lime-salts  contained  therein 
do  not  remain  unchanged,  according,  to  the  recent  investigations  of 
Tangl.3  The  egg-shell  (lime  shell,  and  shell-membrane)  of  a  hen's  egg 
weighing  60  grams  loses  (calculated  on  the  dry)  during  incubation  about 
0.4  gram,  of  which  0.15  gram  is  calcium  and  0.2  gram  is  organic  substance. 

A  very  complete  and  careful  chemical  investigation  on  the  develop- 
ment of  the  embryo  of  the  hen  has  been  made  by  Liebermann.4  From 
his  researches  we  may  quote  the  following:  In  the  earlier  stages  of  the 
development,  tissues  very  rich  in  water  are  formed,  but  upon  the  con- 
tinuation of  the  development  the  quantity  of  water  decreases.  The 
absolute  quantity  of  the  bodies  soluble  in  water  increases  with  the  develop- 
ment, while  their  relative  quantity,  as  compared  with  the  other  solids, 
continually  decreases.  The  quantity  of  the  bodies  soluble  in  alcohol 
quickly  increases.  A  specially  important  increase  is  noticed  in  the  fat, 
whose  quantity  is  not  very  great  even  on  the  fourteenth  day,  but  after 
that  it  becomes  considerable.  The  quantity  of  protein  bodies  and  albu- 
minoids soluble  in  water  grows  continually  and  regularly  in  such  a  way 
that  their  absolute  quantity  increases,  while  their  relative  quantity 
remains    nearly    unchanged.     Liebermann    found    no    gelatin    in    the 


1  Bohr    and    Hasselbaleh,    Maly's    Jahresber.,    2i);  Hasselbalch,    Skand.    Arch.    f. 
Physiol.,  13. 

2Journ.  of  Physiol.,  .'{S. 

a  Tangl  with  Hammerschlag,  Pfliiger's  Arch..  121. 

*  1.  c. 


DEVELOPMENT  OF  THE  CHICK    EMBRYO.  639 

embryo  of  the  hen.  The  embryo  doea  not  contain  any  gelatin-forming 
substance  until  the  tenth  day,  and  from  the  fourteenth  da}'  on  it  contains 
a  body  which,  when  boiled  with  water,  gives  a  BUDStance  similar  to  chon- 
drin.  A  body  similar  to  mucin  occurs  in  the  embryo  when  about  six 
days  old,  but  then  disappears.  The  quantity  of  haemoglobin  shows  a 
continual  increase  compared  with  the  weight  of  the  body.  Liebermann 
found  that  the  relation  of  the  haemoglobin  to  the  body  weight  was  1:728 
on  the  eleventh  day  and  1:421  on  the  twenty-first  day. 

By  means  of  Berthelot's  thermometric  methods  Tangl  x  has 
determined  the  chemical  energy  present  at  the  beginning  and  end  of 
the  development  of  the  embryo  of  the  sparrow's  and  hen's  eggs.  The 
difference  was  considered  as  work  of  development.  He  found  that  the 
chemical  energy  necessary  for  the  development  of  each  gram  of  ripe  hen's 
embryo  (Plymouth)  was  equal  to  0.805  Cal.  This  energy  originated 
chiefly  from  the  fat.  Of  the  total  chemical  energy  utilized,  about  70 
per  cent  was  used  for  the  embryo  and  about  30  per  cent  remained  in  the 
yolk.  Of  the  utilized  energy  about  two-thirds  was  used  in  the  con- 
struction of  the  embryo  and  about  one-third  transformed  into  other 
forms  of  energy  as  work  of  development. 

By  their  investigations  on  the  development  of  the  trout  egg,  Tangl 
and  Farkas  2  have  found  that  the  loss  in  weight  of  each  egg  which  had 
an  average  weight  of  88  milligrams  was  4.9  milligrams  during  the  42 
days  of  incubation,  of  which  4.11  milligrams  was  water  and  0.722  milli- 
gram dry  substance  with  0.367  milligram  C.  The  eggs  lose  no  nitro- 
gen and  no  fat.  The  fat  content  increases  a  little,  and  indeed,  as  these 
authors  believe,  at  the  expense  of  the  proteins.  The  chemical  energy 
used  during  development  was  6.68  gram-calories. 

The  highly  interesting  investigations  made  by  Loeb  upon  the  fer- 
tilization of  the  eggs  of  lower  sea-animals  will  be  discussed  in  this  con- 
nection. According  to  these  experiments  after  the  fertilization  of  the 
egg  by  means  of  a  sort  of  cytolysis  small  drops  of  a  colloid  substance 
form  on  the  surface  of  the  egg.  These  drops  enlarge  in  volume  and 
conglomerate  to  a  continuous  mass,  while  its  surface  hardens  to  a  tight, 
continuous  membrane — the  fertilization  membrane.  The  process  of 
membrane  formation  is  in  fact  the  essential  step  in  the  fertilization. 
Besides,  by  spermatozoa,  the  membrane  formation  is  caused  by  different 
actions.  For  many  eggs  all  that  is  necessary  is  the  artificial  calling 
forth  of  the  processes  for  the  membrane  formation  in  order  that  the 
egg  shall  develop  to  normal  larvae  (for  example  the  eggs  of  the  star 
fish  and  of  certain  worms).  In  other  cases,  for  example  the  sea-urchin, 
Strongylocentrotus,  a  second  action  is  necessary  for  the  production  of 

1  Pfluger's  Arch.,  93  ani  121.  !  Ibid.,  104. 


640  ORGANS  OF  GENERATION. 

normal  larva?.     The  principal  points  in  the  treatment  of  such  eggs  are 
the  following. 

The  formation  of  the  fertilization  membrane  can  be  brought  about 
by  placing  the  eggs  in  sea  water  which  has  been  faintly  acidified  with 
a  fatty  acid,  for  example  with  butyric  acid,  and  after  If  to  2  minutes 
placed  again  in  sea-water.  The  formation  of  the  membrane  now  takes 
place.  The  oxy acids  and  especially  the  inorganic  acids  are  less  active, 
than  the  fatty  acids.  The  H-ions  are  without  effect  in  this  acid  action 
and  Loeb  explains  the  action  by  the  introduction  of  the  undissociated 
molecules  into  the  egg.  Parallel  with  the  membrane  formation  chemical 
processes  begin,  among  which  we  must  especialty  mention  oxidations. 
These  processes,  if  they  proceed  undisturbed,  especially  at  15°  or  above, 
lead  quickly  to  the  death  of  the  egg.  This  can,  nevertheless,  be  prevented 
if  the  oxidation  processes  are  inhibited  40-60  minutes  after  the  mem- 
brane formation  by  removing  the  oxygen  or  by  the  addition  of  some 
potassium  cyanide.  In  this  process  probably  certain  injurious  substances 
for  the  egg  are  destroyed.  If  eggs  treated  in  this  way  are  placed  in 
sea-water  after  2-3  hours  they  develop  in  a  normal  manner. 

The  membrane  formation  can  also  be  brought  about  in  other  ways 
besides  by  the  action  of  acids,  for  example  by  treating  the  egg  with  saponin, 
solanin,  digitalin,  soaps  and  fat  dissolving  substances  such  as  amylene, 
benzene,  toluene,  chloroform,  ether  and  alcohol.  The  sea-urchin  egg  is 
also  excited  to  membrane  formation  by  the  serum  of  certain  animals. 
Alkalies  and  elevation  of  temperature  can  also  cause  the  formation  of 
membrane. 

On  the  other  hand  the  chemical  processes,  which,  when  not  prevented, 
lead  to  the  death  of  the  egg,  can  also  be  inhibited  by  placing  the  eggs  in 
a  hypertonic  solution  (50  ccl  sea-water  and  8  cc.  2.5  normal  NaCl) 
about  one  hour  after  the  artificial  membrane  has  been  formed  and  then 
after  20-50  minutes  placing  them  in  sea-water  again. 

According  to  Loeb  the  artificial  fertilization  of  the  sea-urchin's  egg 
depends  upon  two  special  actions,  of  which  the  first  brings  about  the  for- 
mation of  membrane  with  oxidation  processes  by  means  of  cytolysis 
while  the  second  gives  the  direction  of  these  oxidation  processes  necessary 
for  the  maintenance  of  life. 

The  non-fertilized,  ripe  egg,  as  the  investigations  of  Loeb  on  star- 
fish have  shown,  dies  in  4-6  hours  at  sufficiently  high  temperatures.  The 
death  of  the  egg  can,  nevertheless,  be  prevented  if  oxygen  is  removed 
from  the  egg  or  the  oxidation  inhibited  by  the  addition  of  traces  of  potas- 
sium cyanide.  If  the  ripe  egg  is  fertilized  by  spermatozoa  then  it  remains 
alive  although  the  process  of  fertilization,  as  Warburg  l  found,  causes 

1  Zeitschr.  f.  physiol.  Chem.,  57,  60,  66. 


PLACENTA.  64L 

a  considerable  rise  in  the  oxidation.  For  this  reason  Loeb  believes  that 
the  spermatozoa  save  fche  life  of  the  egg  by  bringing  membrane  forming 
substances  to  the  egg,  but  also  other  substances,  which  remove  or  make 
inert  a  harmful  substance  or  condition  complex  of  the  unfertilized  egg, 
so  that  even  now  the  increased  oxidation  cannot  have  any  harmful  effect.1 

The  enzymes  of  the  sea-urchin  suffer  an  increase  in  natural  as  well 
as  in  artificial  fertilization  as  Jacoby2  has  shown  that  glycyltryptophane 
is  split  after  fertilization  but  not  before. 

The  placenta  has  recently  been  the  subject  of  several  investigations. 
This  tissue  contains  a  protein  which  coagulates  at  60-65°  C.  (Bottazzi 
and  Delfino)  whose  relation  to  the  nucleoprotein,  found  by  others,  is 
not  clear.  The  protein  found  by  Savare  contained  0.45  per  cent  phos- 
phorus. The  nucleic  acid  studied  by  Kikkoji,3  which  is  very  similar  to 
the  thymus  nucleic  acid,  originates  from  this  nucleoprotein.  Glycogen 
occurs  regularly  in  the  placenta,  and  Moscati  believes  the  human  pla- 
centa contains  5  p.  m.  glycogen.  After  removal  the  glycogen  diminishes, 
and  after  24  hours  it  has  disappeared.  According  to  Lochhead  and 
Cramer4  the  quantity  of  glycogen  in  the  placenta  is  not  increased  by 
food  rich  in  carbohydrate.  In  the  fcetus  (rabbits)  the  above  authors 
found  that  the  placenta  is  a  storage  organ  for  glycogen  until  the  second 
half  of  the  gestation  period,  when  the  liver  begins  to  functionate  in  this 
direction.  From  this  time  on  the  quantity  of  glycogen  in  the  placenta 
diminishes. 

Enzymes  of  various  kinds,  proteolytic  as  well  as  lipolytic  (mono- 
butyrase),  amylases  and  oxidases  have  been  found  in  the  placenta.5 
In  the  edges  of  the  placenta  of  the  bitch  and  of  cats,  an  orange-colored, 
crystalline  pigment  (bilirubin)  and  a  green,  amorphous  pigment,  whose 
relation  to  biliverdin  is  not  clear,  have  been  found.6 

From  the  cotyledons  of  the  placenta  in  ruminants  a  white  or  faintly  rose-colored 
creamy  fluid,  the  uterine  milk,  can  be  obtained  by  pressure.     It  is  alkaline  in 

1  A  complete  review  of  the  investigations  of  Loeb  and  his  collaborators,  with  the 
literature  can  be  found  in  Vorlesungen  iiber  die  Dynamik  der  Lebenserscheinungen, 
Leipzig,  1906,  s.  239.  See  also  Uber  den  chemischen  Charakter  des  Befruchtungsvor- 
ganges,  Leipzig,  1908;  Zeitschr.  f.  physik.  Chem.  70,  220  (1910),  Arch.  f.  Entwickelungs- 
mech.,  31,  658  (1910). 

2Bioch.  Zeitschr.,  26,  333  (1910). 

3  Bottazzi  and  Dcltino,  Centralbl.  f.  Physiol.,  18, 114;  Savare,  Hofmeister's,  Beitrage, 
11;  Kikkoji,  Zeitschr.  f.  physiol.  Chem.,  53. 

4  Moscati,  Zeitschr,  f.  physiol.  Chem.,  53;  Lochhead  and  Cramer,  Proc.  Roy.  Soc, 
80  B.  (1908). 

6Ascoli,  Centralbl.  f.  Physiol.,  16;  Raineri,  Bioch.  Centralbl..  4,  428;  Bergell  and 
Liepmann,  Munch,  med.  Wochenschr.,  1905;  Savare,  Hofmeister's  Beitrage,  9;  Bergell 
and  Falk,  Munch,  med.  Wochenschr.,  55. 

6  See  Etti,  Maly's  Jahresber.,  2,  287,  and  Preyer,  Die  Blutkristalle,  Jena,  1871. 


642  ORGANS  OF  GENERATION. 

reaction,  but  quickly  becomes  acid.  Its  specific  gravity  is  1.033-1.040.  It  con- 
tains as  form-elements  fat-globules,  small  granules,  and  epithelium-cells.  There 
have  been  found  81.2-120.9  p.  m.  solids,  61.2-105.6  p.  m.  protein,  about  10  p.  m. 
fat,  and  3.7-8.2  p.  m.  ash  in  the  uterine  milk. 

The  fluid  occurring  in  the  so-called  grape-mole  (Mola  racemosa)  has  a  low 
specific  gravity,  1.009-1.012,  and  contains  19.4-26.3  p.  m.  solids  with  9-10  p.  m. 
protein  bodies  and  6-7  p.  m.  ash. 

The  amniotic  fluid  in  women  is  thin,  wrhitish,  or  pale  yellow;  some- 
times it  is  somewhat  yellowish-brown  and  cloudy.  AVhite  flakes  separate. 
The  form-elements  are  mucus-corpuscles,  epithelium-cells,  fat-drops,  and 
lanugo  hair.  The  odor  is  stale,  the  reaction  neutral  or  faintly  alkaline. 
The  specific  gravity  is  1:002-1.028. 

The  amniotic  fluid  contains  the  constituents  of  ordinary  transudates. 
The  amount  of  solids  at  birth  is  scarcely  20  p.  m.  In  the  earlier  stages  of 
pregnancy  the  fluid  contains  more  solids,  especially  proteins.  Among 
the  protein  bodies,  Weyl  found  one  substance  similar  to  vitellin,  and  with 
great  probability  also  seralbumin,  besides  small  quantities  of  mucin. 
Enzymes  of  various  kinds  (pepsin,  diastase,  thrombin,  lipase)  occur, 
according  to  Bondi.  Sugar  is  regularly  found  in  the  amniotic  fluid  of 
cows,  but  not  in  human  beings.  In  the  ox,  pig,  and  goat  Gurber  and 
Grunbaum  also  found  fructose.  The  human  amniotic  fluid  also  contains 
some  urea,  uric  acid,  allantoin  and  creatinine  (Amberg  and  Rowntree). 
The  quantity  of  these  may  be  increased  in  hydramnion  (Prochownick, 
Harnack),  which  depends  on  an  increased  secretion  by  the  kidneys  and 
skin  of  the  foetus.  Lactates  are  doubtful  constituents  of  the  amniotic 
fluid.  The  quantity  of  urea  in  the  amniotic  fluid,  is,  according  to  Pro- 
chownick, 0.16  p.  m.  In  the  fluid  in  hydramnion  Prochownick  and 
Harnack  found,  respectively,  0.34  and  0.48  p.  m.  urea.  The  principal 
mass  of  the  solids  consists  of  salts.  The  quantity  of  chlorides  (NaCl)  is 
5.7-6.6  p.  m.  The  molecular  concentration  of  the  amniotic  fluid  is  some- 
what lower  than  that  of  the  blood,  which  is  no  doubt  due  to  a  dilution 
by  the  fcetal  urine  (Zangemeister  and  Meissl  1). 

iWeyl,  Arch.  f.  (Anat.  u.)  Physiol.,  187G;  Bondi,  Centralbl.  f.  Gynakol.,  1903; 
Prochownick,  Arch.  f.  Gynak.,  11,  also  Maly's  Jahresber.,  7,  155;  Harnack,  Berlin, 
klin.  Wochenschr.,  1888,  No.  41;  Zangemeister  and  Meissl,  Munch,  med.  Wochenschr. 
1903;  Gurber  and  Grunbaum,  ibid.,  1904;  Amberg  and  Rowntree,  cited  from  Bioch. 
Centralbl.,  10,  237. 


CHAPTER  XIII. 
MILK. 

The  chemical  constituents  of  the  mammary  glands  have  been  little 
studied.  The  cells  are  rich  in  protein  and  nucleoproteins.  Among  the 
latter  we  have  one  that  yields  pentose  and  guanine,  on  boiling  with  dilute 
mineral  acids,  but  no  other  purine  base.  This  compound  protein,  inves- 
tigated by  Odenius,  contains  as  an  average  the  following:  17.28  per 
cent  N,  .0.89  per  cent  S,  and  0.277  per  cent  P.  Besides  this  compound 
proteid  we  have  at  least  one  other,  as  Mandel  and  Levene  and  Loebisch  l 
have  isolated  a  nucleic  acid  from  the  mammary  gland,  which,  like  the 
thymonucleic  acids,  yielded  adenine,  guanine,  thymine,  and  cytosine. 
This  nucleic  acid  also  gave  the  pentose  reactions  and  yielded  an  abundance 
of  levulinic  acid.  Besides  this  nucleic  acid,  Mandel  and  Levene  isolated 
from  the  glands  a  glucothionic  acid  with  2.65  per  cent  S  and  4.38  per 
cent  N.  Among  the  cleavage  products  of  the  nucleoprotein  Mandel  2 
obtained  no  glycocoll,  and  the  products  of  hydrolysis  show  a  great  cor- 
respondence with  those  of  casein.  We  cannot  state  what  relation  the 
above-mentioned  nucleic  acids  and  the  glucothionic  acid  bear  to  the 
not  well-known  constituent  of  the  glands  fcund  by  Bert  and  by  Thier- 
felder  and  which  yields  a  reducing  substance  when  boiled  with  dilute 
acids. 

It  is  to  be  expected  that  these  bodies  are  steps  in  the  formation  of 
milk-sugar;  still  we  have  no  point  of  support  for  such  an  assumption, 
and  recent  investigations  seem  to  indicate  that  the  milk-sugar  is  produced 
in  the  glands  by  a  transformation  of  the  sugar  of  the  blood.  Fat  seems, 
at  least  in  the  secreting  glands,  to  be  a  never-failing  constituent  of  the 
cells,  and  this  fat  may  be  observed  in  the  protoplasm  as  large  or  small 
globules  similar  to  milk-globules.  The  extractive  bodies  of  the  mam- 
mary glands  have  been  little  investigated,  but  among  them  are  found 
considerable  amounts  of  purine  bases.  The  mammary  glands  also 
contain    enzymes,    among     which    we    especially    mention  :     catalase, 

Odenius,  Maly's  Jahresber.,  30;  Mandel  and  Levene,  Zeitschr.  f.  physiol.  Chem., 
46;  Loebisch,  Hofineister'sBeitriige,  8. 

*  Mandel  and  Levene,  Zeitschr.  f.  physiol.  Chem.,  45,  Mandel,  Bioch.  Zeitschr  ,  23 

643 


644  MILK. 

peroxidase  and  a  proteolytic  enzyme  which,  according  to  Hildebrandt,1 
occurs  to  a  much  greater  extent  in  the  active  gland  as  compared  with 
the  inactive  one. 

As  human  milk  and  the  milk  of  animals  are  essentially  of  the  same 
constitution,  it  seems  best  to  speak  first  of  the  one  most  thoroughly 
investigated,  namely,  cow's  milk,  and  then  of  the  essential  properties 
of  the  remaining  important  kinds  of  milk.2 

Cow's  Milk. 

Cow's  milk,  like  every  other  kind,  forms  an  emulsion  which  consists 
of  very  finely  divided  fat  suspended  in  a  solution  consisting  principally 
of  protein  bodies,  milk-sugar,  and  salts.  Milk  is  non-transparent,  white, 
whitish-yellow,  or  in  thin  layers  somewhat  bluish-white,  of  a  faint,  insipid 
odor  and  mild,  faintly  sweetish  taste.  The  specific  gravity  is  1.028 
to  1.0345  at  15°  C.  The  freezing-point  is  -0.54-0.59°  C.,  average 
—  0.563°  C,  and  the  molecular  concentration  0.298. 

The  reaction  of  perfectly  fresh  milk  is  generally  amphoteric  toward 
litmus.  The  extent  of  the  acid  and  alkaline  part  of  this  amphoteric 
reac:ion  has  been  determined  by  different  investigators,  especially 
Thorner,  Sebelien,  and  Cotjrant.3  The  results  differ  with  the  indi- 
cators used,  and  moreover  the  milk  from  different  animals,  as  well  as 
that  from  the  same  animal  at  different  times  during  the  lactation  period, 
varies  slightly.  Courant  determined  the  alkaline  part  by  N/10  sul- 
phuric acid,  using  blue  lacmoid  as  indicator,  and  the  acid  part  by  N/10 
caustic  soda,  using  phenolphthalein  as  indicator.  He  found,  as  an  average 
for  the  first  and  last  portions  of  the  milking  of  twenty  cows,  that  100 
cc.  milk  had  the  same  alkaline  reaction  toward  blue  lacmoid  as  41  cc. 
N/10  caustic  soda,  and  the  same  acid  reaction  toward  phenolphthalein 
as  19.5  cc.  N/10  sulphuric  acid.  The  actual  reaction  of  cow's  milk, 
which  follows  from  the  electrometric  estimation,  is,  on  the  contrary, 
Foa4  claims,  nearly  neutral,  like  the  reaction  of  animal  fluids  and 
tissues  in  general. 

Milk  gradually  changes  when  exposed  to  the  air,  and  its  reaction 
becomes  more  and  more  acid.  This  depends  on  a  gradual  transforma- 
tion of  the  milk-sugar  into  lactic  acid,  caused  by  micro-organisms. 

'Bert,  Compt.  Rend.,  98;  Thierfelder,  Pfliiger's  Arch.,  34,  and  Maly's  Jahresber., 
13;  Hildebrandt,  Hofmeister's  Beitrage,  5. 

*  A  very  complete  reference  to  the  literature  on  milk  may  be  found  in  Raudnitz's 
"Die  Bestandteile  der  Milch,"  in  Er{r''bnisse  der  Physiol.,  2,  Abt.  1.  The  literature 
of  the  last  few  years  may  be  found  in  the  references  by  Raudnitz,  Monatsschrift  f. 
Kinderheilkunde. 

'  Thorner,   Maly's  Jahresber.,   22;    Sebelien,   ibid.,  Courant;  Pfliiger's  Arch.,   50. 

*  Compt.  rend.  soc.  biolog.  (58),  59,  51. 


COW'S  MILK.  645 

Perfectly  fresh  amphoteric  milk  does  not  coagulate  on  boiling,  but 
forms  a  pellicle  consisting  of  coagulated  casein  and  lime-salts,  which 
rapidly  re-forms  after  being  removed.  After  a  sufficiently  strong  spon- 
taneous formation  of  acid  it  coagulates  on  boiling,  and  lastly,  when  the 
formation  of  lactic  acid  is  sufficient,  it  coagulates  spontaneously  at  the 
ordinary  temperature,  forming  a  solid  mass.  It  may  also  happen,  espe- 
cially in  the  warmth,  that  the  casein-clot  contracts  and  a  yellowish  or 
yellowish-green  acid  liquid  (acid  whey)  separates. 

Milk  may  undergo  various  fermentations.  Lactic-acid  fermentation,  brought 
about  by  Huppe's  lactie-acid  bacillus  and  also  other  varieties,  takes  first  place. 
In  the  spontaneous  souring  of  milk  we  generally  consider  the  formation  of  lactic 
acid  as  the  most  essential  product,  but  a  formation  of  succinic  acid  may  also  take 
place,  and  in  certain  bacterial  decompositions  of  milk,  succinic  acid  and  no  lactic 
acid  is  formed.  The  materials  from  which  these  two  acids  are  formed  are  lactose 
and  lactophosphocarnic  acid.  Besides  the  lactic  acids,  the  optically  inactive 
as  well  as  the  dcxtro  and  levo  acids,  and  succinic  acid,  volatile  fatty  acids,  such 
as  acetic  acid,  butyric  acid,  and  others,  may  be  formed  in  the  bacterial  decompo- 
sition of  milk. 

Milk  sometimes  undergoes  a  peculiar  kind  of  coagulation,  being  converted 
into  a  thick,  ropy,  slimy  mass  (thick  milk).  This  conversion  depends  upon  a 
peculiar  change  in  which  the  milk-sugar  is  made  to  undergo  a  slimy  transforma- 
tion. This  transformation,  which  requires  further  investigation,  is  caused  by 
special  micro-organisms. 

If  the  milk  is  sterilized  by  heating,  and  contact  with  micro-organisms 
prevented,  the  formation  of  lactic  acid  may  be  entirely  stopped.  The 
production  of  acid  may  also  be  prevented,  at  least  for  sometime,  by  many 
antiseptics,  such  as  salicylic  acid,  thymol,  boric  acid,  and  other  bodies. 

If  freshly  drawn  amphoteric  milk  is  treated  with  rennet,  it  coagulates 
quickly,  especially  at  the  temperature  of  the  body,  to  a  solid  mass  (curd) 
from  which  a  yellowish  fluid  (sweet  whey)  is  gradually  pressed  out.  This 
coagulation  occurs  without  any  change  in  the  reaction  of  the  milk,  and 
therefore  it  is  distinct  from  the  acid  coagulation. 

In  cow's  milk  we  find  as  form-elements  a  few  colostrum  corpuscles 
{see  Colostrum)  and  a  few  pale  nucleated  cells.  The  number  of  these 
form-elements  is  very  small  compared  with  the  immense  amount  of  the 
most  essential  form-constituents,  the  milk-globules. 

The  Milk-globules.  These  consist  of  extremely  small  drops  of  fat 
whose  number  is,  according  to  Woll,1  1.06-5.75  millions  in  1  c.mm., 
and  whose  diameter  is  0.0024-0.0046  mm.  and  0.0037  mm.  as  an  average 
for  different  kinds  of  animals.  It  is  unquestionable  that  the  milk-globules 
contain  fat,  and  we  consider  it  as  positive  that  all  the  milk-fat  exists  in 
them.  Another  disputed  question  is  whether  the  milk-globules  consist 
entirely  of  fat  or  whether  they  also  contain  protein. 

^n  the  Conditions  Influencing  the  Number  and  Size  of  Fat-globules  in  Cow's 
Milk,  Wisconsin  Exp.  Station,  6,  1892. 


646  MILK. 

The  observations  of  Ascherson1  show  that  drops  of  fat,  when  dropped  in  an 
alkaline  protein  solution,  are  covered  with  a  fine  albuminous  coat,  a  so-called  haptogen- 
membrane.  As  milk  on  shaking  with  ether  does  not  give  up  its  fat,  or  only  very 
slowly  in  the  presence  of  a  great  excess  of  ether,  and  as  this  takes  place  very  readily 
after  the  addition  of  acids  or  alkalies,  which  dissolve  proteins,  it  was  formerly 
thought  that  the  fat-globules  of  the  milk  were  enveloped  in  a  protein  coat.  A 
true  membrane  has  not  been  detected;  and  since,  when  no  means  of  dissolving 
the  protein  is  resorted  to — for  example,  when  the  milk  is  precipitated  by  carbon 
dioxide  after  the  addition  of  very  little  acetic  acid,  or  when  it  is  coagulated  by 
rennet — the  fat  can  be  very  easily  extracted  by  ether,  the  theory  of  a  special  albu- 
minous membrane  for  the  fat-globule  has  been  generally  abandoned.  The  observa- 
tions of  Quincke  2  on  the  behavior  of  the  fat-globules  in  an  emulsion  prepared 
with  gum  have  led,  at  the  present  time,  to  the  conclusion  that  each  fat-globule 
in  the  milk  is  surrounded  by  a  stratum  of  casein  solution  held  by  molecular  attrac- 
tion, and  this  prevents  the  globules  from  uniting  with  each  other.  Everything 
that  changes  the  physical  condition  of  the  casein  in  the  milk  or  precipitates  it 
must  necessarily  help  the  solution  of  the  fat  in  ether,  and  it  is  in  this  way  that  the 
alkalies,  acids,  and  rennet  act. 

V.  Storch  has  shown,  in  opposition  to  these  views,  that  the  milk- 
globules  are  surrounded  by  a  membrane  of  a  special  slimy  substance. 
This  substance  is  very  insoluble,  contains  14.2-14.79  per  cent  nitrogen, 
and  yields  a  sugar,  or  at  least  a  reducing  substance,  on  boiling  with 
hydrochloric  acid.  It  is  neither  casein  nor  lactalbumin,  but  it  seems  to 
all  appearances  to  be  identical  with  the  so-called  "  stroma  substance  " 
detected  by  Radenhausen  and  Danilewsky.  Storch  was  able  to 
show,  by  staining  the  fat-globules  with  certain  dyes,  that  this  substance 
enveloped  them  like  a  membrane.  Recently  Voltz  has  given  further 
proofs  of  the  view  that  the  fat-globules  probably  have  a  membrane, 
which  in  his  opinion  is  a  very  labile  formation  of  variable  composition, 
and  Bauer  has  also  given  further  proofs  for  the  assumption  of  a  mem- 
brane. Droop-Richmond  and  Bonnema,3  on  the  other  hand,  present 
several  deductions  conflicting  with  Storch's  theory.  If  Storch's  observa- 
tion that  the  purified  fat-globules  contain  a  special  protein  substance 
differing  from  the  dissolved  proteins  of  the  milk  is  correct,  then  the 
assumption  as  to  a  special  body  forming  a  membrane  or  stroma  of  the 
fat-globules  becomes  very  probable.  The  correctness  of  Storch's  view 
has  been  substantiated  very  recently  by  Abderhalden  and  Voltz.4 
On  the  acid  hydrolysis  of  the  fat-globules  they  obtained  glycocoll,  which 
is  absent  in  the  casein  as  well  as  in  the  lactalbumin,  and  this  shows  that  the 


1  Arch.  f.  Anat.  u.  Physiol.,  1840. 

*  Pfluger's  Arch.,  19. 

1  V.  Storch,  see  Maly's  Jahresber.,  27;  Radenhausen  and  Danilewsky,  Forschungen 
auf  dem  Gebiete  der  Viehhaltung  (Bremen,  1880),  Heft  9;  Voltz,  Pfluger's  Arch.,  102; 
Bauer,  Bioch.  Zeitecbf.  32;  Droop-Richmond,  see  Chem.  CentralbL,  1094,  2,  356;. 
Bonnema,  ibid.,  1243. 

4  Zeitschr.  f.  physiol.  Chem.,  59. 


MILK   FAT.     CASEIN.  647 

fat-globules  at  least  cannot  contain  these  two  proteins  alone.  They 
must  contain  another  protein,  and  it  is  still  a  question  whether  besides  this 
they  also  contain  casein  and  lactalbumin. 

The  milk-fat  which  is  obtained  under  the  name  of  butter  consists 
mainly  of  olcin  and  palmitin.  Besides  these  it  contains,  as  triglycerides, 
myristic  acid,  stearic  acid,  small  amounts  of  lauric  acid,  arachidic  acid, 
and  dioxystearic  acid,,  besides  butyric  acid  and  caproic  acid,  traces  of 
caprylic  acid  and  capric  acid.  Riegel  claims  that  triglycerides  of  vola- 
tile fatty  acids  do  not  occur,  but  rather  mixed  triglycerides  of  volatile 
and  non-volatile  fatty  acids.  Milk-fat  also  contains  small  quantities 
of  phosphatides  (lecithin),  and  cholesterin  and  a  yellow  coloring-matter. 
The  quantity  of  volatile  fatty  acids  in  butter  is,  according  to  Duclaux, 
on  an  average  about  70  p.m.,  of  which  37-51  p.m.  is  butyric  acid  and 
30-33  p.  m.  is  caproic  acid.  The  non-volatile  fat  consists  of  iV- &  olein 
and  the  remainder  is  principally  palmitin.  The  composition  of  butter 
is  not  constant,  but  varies  considerably  under  different  circumstances.1 
The  question  whether  the  small  fat-globules  have  a  different  composition 
from  the  large  ones  is  still  disputed. 

The  milk-plasma,  or  that  fluid  in  which  the  fat-globules  are  suspended, 
contains  several  different  proteins,  the  statements  as  to  the  number  and 
nature  of  which  are  somewhat  at  variance.  The  three  following,  casein, 
lactalbumin,  and  lactoglobulin,  have  been  most  closely  studied  and  are 
well  characterized.  The  milk-plasma  contains  at  least  two  carbohy- 
drates, of  which  the  one,  lactose,  is  of  great  importance.  It  also  contains 
extractive  bodies,  traces  of  urea,  creatine,  creatinine,  orotic  acid,  hypoxan- 
thine  (?),  cholesterin,  citric  acid  (Soxhlet  and  Henkel2),  and  lastly  also 
mineral  bodies  and  gases. 

Casein.  This  protein  substance,  which  thus  far  has  been  detected 
positively  only  in  milk,  belongs  to  the  nucleoalbumins,  and  differs  from 
the  albuminates  chiefly  by  its  content  of  phosphorus  and  by  its  behavior, 
with  the  rennet  enzyme.  Casein  from  cow's  milk  has  about  the  follow- 
ing composition:  C  53.0,  H  7.0,  N  15.7,  S  0.8,  P  0.85,  and  O  22.65  per 
cent.  Its  specific  rotation  is,  according  to  Hoppe-Seyler,  rather  variable; 
in  neutral  solution  it  is  (a)D=— 80°;  its  faintly  alkaline  solution  has  a 
stronger  rotation,  namely,  — 97.8  to  —111.8°,  in  a  solution  of  N/10-N/5 


'Riegel,  Maly's  Jahresber.,  34;  Duclaux,  Compt.  Rend.,  104.  Various  statement, 
as  to  the  composition  of  milk-fat  can  be  found  in  Koefoed,  Bull.  d.  l'Acad.  Roys 
Danoise,  1891,  and  Wanklyn,  Chemical  News,  63;  Browne,  Chem.  Centralbl.,  1899, 
2,  883.  In  regard  to  the  elementary  composition  of  milk-fat  see  Fleischmann  and 
Warmbold,  Zeitschr.,  f.  Biol.,  50. 

2  Cited  from  Soldner,  Die  Salze  der  Milch,  etc.,  Landwirthsch.  Versuchsstation, 
35,  Separatabzug,  18. 


648  MILK. 

NaOH  (Long  l).  The  question  whether  the  casein  from  different  kinds 
of  milk  is  identical  or  whether  there  are  several  caseins  cannot  be  decided 
by  the  elementary  analysis.  According  to  Tangl  and  Csokas,  2  mare's 
and  ass's  casein  seem  to  be  somewhat  richer  in  nitrogen  (16.44  and  16.28 
per  cent,  respectively)  but  poorer  in  sulphur  (0.528  and  0.588  per  cent) 
and  carbon  (52.36  and  52.27  per  cent)  than  the  casein  from  cud  chewers. 
The  ass's  casein  was  richer  in  phosphorus  (1.057  per  cent)  than  the  mare's 
or  cow's  casein  (both  with  0.887  per  cent). 

Casein  when  dry  appears  like  a  fine  white  powder,  which  has  no 
measurable  solubility  in  pure  water  (Laqueur  and  Sackur).  Casein 
is  only  very  slightly  soluble  in  the  ordinary  neutral-salt  solutions.  Accord- 
ing to  Arthus  it  dissolves  rather  easily  in  a  1-per  cent  solution  of  sodium 
fluoride,  ammonium  or  potassium  oxalate.  Robertson  thinks  that  it 
is  more  soluble  in  potassium  cyanide  and  the  alkali  salts  of  certain  vola- 
tile fatty  acids  such  as  butyric  acid  and  valeric  acid,  than  in  solutions 
of  the  ordinary  neutral  salts.  It  is  at  least  a  tetrabasic  acid,  whose 
equivalent  weight  is  1135,  according  to  Laqueur  and  Sackur,  and  1250 
according  to  Robertson.  The  statements  as  to  the  molecular  weight 
are  disputed  (Laqueur  and  Sackur,  L.  and  D.  van  Slyke3). 

It  dissolves  readily  in  water  with  the  aid  of  alkali  or  alkaline  earths, 
also  calcium  carbonate,  from  which  it  expels  carbon  dioxide  ana  it 
thus  forms  caseinates  of  variable  composition.  If  casein  is  dissolved 
in  lime-water  and  the  solution  carefully  treated  with  very  dilute  phos- 
phoric acid  until  it  is  neutral  in  reaction  (to  litmus),  the  casein  appears 
to  remain  in  solution,  but  is  probably  only  swollen  as  in  milk,  and  the 
liquid  contains  at  the  same  time  a  large  quantity  of  calcium  phosphate 
without  any  precipitate  or  any  suspended  particles  being  visible.  The 
casein  solutions  containing  lime  are  opalescent,  and  have  on  warming 
the  appearance  of  milk  deficient  in  fat  (which  is  also  true  for  the  salts 
of  casein  with  the  alkaline  earths).  Therefore  it  is  not  impossible  that 
the  white  color  of  the  milk  is  due  partly  to  the  casein  and  calcium  phos- 
phate. Soldner  and  others  have  prepared  two  calcium  compounds 
of  casein  with  1.5  p.  c.  CaO  (the  neutral  caseinate  according  to  Soldner) 
and  2.4  p.  c.  CaO  (the  basic  caseinate).  The  first  is  neutral  to  litmus 
while  the  other  is  neutral  to  phenolphthalein. 

According  to  Robertson  4  the  alkali  equivalent  of  casein  at  neutrality 
toward  litmus  =53  XlO-5  equivalent-grm.-mol.  per  gram  and  at  neutrality  toward 

1  Hoppe-Seyler,  Handb.  d.  physiol.  u.  pathol.  chem.  Analyse,  8.  Aufl.,  489;  Long, 
Journ.  Amer.  Chem.  Soc,  27. 

2  Pfluger'a  An.-h.,  121. 

3  Laqueur  and  Sackur,  Hofmeister's  Beitrage,  3;  M.  Arthus,  Theses  presentees 
a  la  faculty  des  sciences  de  Paris,  1893;  Robertson,  Journ.  of  biol.  Chem.,  2;  L.  an  1 
D.  van  Slyke,  Amer.  Chem.  Journ.,  38. 

4  See  Ergebnis.  d.  Physiol.  10  and  Journ.  of  physical  Chem.,  13. 


CASEIN.  649 

phenolphthalein  =  S0xl0-5  equivalent-gnu. -mol.  per  gram.  On  BaturatioD  (with 
monacidic  liases)  the  alkali  equivalent  is  =  llxi0~5  grm.-mol.  per  gram.  Od 
saturating  (with  monobasic  acids)  the  acid  equivalent  is=32X10-5  grm.-mol. 
per  gram. 

Besides  the  rather  earlier  investigations  on  the  salts  of  casein  by  Sold- 
ner,  Courant,  Rohmanx.  Laqieur,  Raudnitz  !  and  others  we  have 
the  recent  observations  and  theoretical  discussion  of  Robertson2  on  the 
composition,  nature  and  dissociation  of  the  caseinates.  We  can  here 
only  refer  to  this  and  the  earlier  investigations. 

Casein  solutions  do  not  coagulate  on  boiling,  but  solutions  of  casein- 
lime  are  covered,  like  milk,  with  a  pellicle.  They  are  precipitated  by 
very  little  acid,  but  the  presence  of  neutral  salts  retards  the  precipitation. 
A  casein  solution  containing  salt  or  ordinary  milk  requires,  therefore, 
more  acid  for  precipitation  than  a  salt-free  solution  of  casein  of  the  same 
concentration.  The  precipitated  casein  dissolves  very  easily  again  in 
a  small  excess  of  hydrochloric  acid,  but  less  readily  in  an  excess  of  acetic 
acid.  The  combination  between  casein  and  acid,  like  other  protein 
and  acid  compounds,  is  precipitated  by  neutral  salts.  These  acid  solu- 
tions are  precipitated  by  mineral  acids  in  excess.3  Casein  is  precipitated 
from  neutral  solutions  or  from  milk  by  common  salt  containing  calcium, 
or  magnesium  sulphate  in  substance,  without  changing  its  properties.4 
Metallic  salts,  such  as  alum,  zinc  sulphate  and  copper  sulphate,  com- 
ply precipitate  the  casein  from  neutral  solutions. 

On  drying  at  100°  C,  casein,  according  to  Laqieur  and  Sackur,  decomposes 
and  splits  into  two  bodies.  One  of  these,  called  caseid.  is  insoluble  in  dilute  alkalies, 
while  the  other,  the  isocasein,  is  soluble  therein.  The  isocasein  is  a  stronger  acid 
and  has  other  precipitation  limits  and  a  rather  lower  equivalent  weight  than  the 
casein. 

The  property  which  is  the  most  characteristic  of  casein  is  that  it 
coagulates  with  rennet  in  the  presence  of  a  sufficiently  large  amount 
of  lime-salts.  In  solutions  free  from  lime-salts  the  casein  does  not  coagu- 
late with  rennet,  but  it  is  changed  so  that  the  solution  (even  if  the  enzymes 
are  destroyed  by  heating)  yields  a  coagulated  mass,  ha  ring  the  properties 
of  a  curd,  if  lime-salts  are  added.     The  rennet  enzyme,  rennin,  has  there 

1  Soldner,  Die  Salze  der  Milch,  etc.,  and  Maly's  Jahresber.,  25;  Courant,  1.  c. ; 
Rohmann,  Berlin,  klin.  Wochenschr.,  1895;  Laqueur,  L  c;  and  Hofmeister's  Beitrage, 
7;   Raudnitz,  Ergebn.  d.  Physiol.,  2,  Abt.  1. 

2  Journ.  of  physical  Chem.,  11  and  12;  Journ.  of  biol.  Chem.,  5. 

3  In  regard  to  the  acid  combinations  of  casein  and  the  ability  to  take  up  acid,  see 
Laxa.  Milch  win  hsoh.  Centralbl.,  1905;  Long,  Journ.  Amer.  Chem.  Soc,  29;  L.  and 
D.  van  Slyke,  Amer.  Chem.  Journ.,  38;   Robertson,  Journ.  of  biol.  Chem.,  4. 

4  See  the  works  of  Hammarsten  and  Schmidt-Nielsen,  Hammarsten's  Festschrift, 
1906. 


650  MILK. 

fore  an  action  on  casein  even  in  the  absence  of  lime-salts.  These  last 
are  only  necesary  for  the  coagulation  or  the  separation  of  the  curd, 
and  the  process  of  coagulation  is  hence  a  two-phase  process.  The  first 
phase  is  the  transformation  of  the  casein  by  the  rennin,  the  second  is 
the  visible  coagulation  caused  by  the  lime-salts.  This  fact,  which  was 
first  proved  by  Hammarsten,  was  later  confirmed  by  Arthus  and  Pages 
and  recently  closely  studied  by  Fuld,  Spiro,  and  Laqueur  and  others.1 

The  curd  formed  on  the  coagulation  of  milk  contains  large  quantities  of 
calcium  phosphate.  According  to  Soxhlet  and  Soldner,  the  soluble  lime-salts 
are  of  essential  importance  only  in  coagulation,  while  the  calcium  phosphate 
is  without  importance.  Cocrant  believes  that  the  calcium-casein  on  coagula- 
tion may  earn'  down  with  it,  if  the  solution  contains  dicalcium  phosphate,  a  part 
of  this  as  tricalcium  phosphate,  leaving  mono-calcium  phosphate  in  the  solution. 
A  solution  of  calcium  casein  is  not  coagulated  by  rennin  alone  but  only  when  soluble 
lime-salts  are  added.  Contrary  to  the  generally  accepted  view  that  the  soluble 
lime-salts  are  of  importance  in  the  coagulation,  van  Dam  2  claims  that  it  is  the 
quantity  of  lime  combined  with  the  casein  which  is  of  importance  in  the  coagula- 
tion process.  The  role  of  the  lime-salts  in  coagulation  is  not  clear,  and  this  fol- 
lows from  the  chemical  procedure  in  rennin  coagulation. 

If  one  makes  use  of  a  pure  solution  of  casein  and  as  pure  rennin  as 
possible,  then  after  coagulation  it  is  always  found  that  the  filtrate  con- 
tains very  small  amounts  of  a  protein,  the  whey  protein,  which  is  probably 
formed  in  the  coagulation.  This  behavior,  which  was  first  shown  by 
Hammarsten,  has  been  substantiated  by  many  others  and  recently  by 
Fuld,  Spiro  and  Schmidt-Nielsen.  Whey  protein  is  generally  con- 
sidered as  a  proteose  substance,  and  Koster3  found  13.2  per  cent  nitro- 
gen therein.  In  correspondence  with  these  observations  casein  coagula- 
tion with  rennin  is  considered  as  a  cleavage  process,  in  which  the  principal 
mass  of  the  casein,  sometimes  more  than  90  per  cent,  is  split  off  as  para- 
casein* a  body  closely  related  to  casein,  and  in  the  presence  of  sufficient 

1  See  Maly's  Jahresber.,  2  and  4;  also  Hammarsten,  Zur  Kenntniss  des  Kaseins  und 
der  Wirkung  des  Labfermentes,  Nova  Acta  Reg.  Soc.  Scient.  Upsala,  1877,  Fest- 
schrift; Zeitschr.  f.  physiol.  Chem.,  22;  Arthus  et  Pages,  Arch,  de  Physiol.  (5),  2, 
and  M6m.  soc.  biol.,  43;  Fuld,  Hofmeister's  Beitrage,  2,  and  Ergebnisse  der  Physiol., 
1,  Abt.  1,  where  a  good  review  of  the  literature  may  be  found,  Spiro,  Hofmeister's 
Beitrage,  6  and  7,  with  Reichel,  ibid.,  7  and  8;  Laqueur,  ibid.,  7. 

2  Zeitschr.  f.  physiol.  Chem.,  58. 

'Hammarsten,  1.  c;  Fuld.  Bioch.  Zeitschr.,  4,  and  Hofmeister's  Beitrage,  10; 
Spiro,  Hofmeister's  Beitrage,  8;  Schmidt-Nielsen,  Hammarsten's  Festschrift,  1906; 
Koster,  see  Maly's  Jahresber.,  11,  14. 

*  It  has  been  proposed  to  designate  the  ordinary  casein  as  caseinogen  and  the  curd 
as  casein.  Although  such  a  proposition  is  theoretically  correct,  it  leads  in  practice 
to  confusion.  On  this  account  the  author  calls  the  curd  paracasein,  according  to 
Schulze  and  Rose  (Landwirthsch.  V'ersuchsstat.,  31).  A  summary  of  the  literature  on 
the  casein  coagulation  may  he  found  in  E.  Fuld,  Ergebnisse  der  Physiol.,  1;  Raudnitz, 
ibid..  2;  and  Laqueur,  Biochem.  Centralbl.,  4,  344. 


CASEIN.  651 

amounts  of  lime-salts  the  paraeasein-lime  precipitates  out  while  the 
proteose-like  substance  (whey  protein)  remains  in  solution.  In  the 
coagulation  in  an  acid  medium  the  conditions  are  entirely  different  and 
proteoses  and  peptones  are  hereby  formed  to  a  considerable  extent. 

The  paracasein  is  very  similar  to  casein,  but  cannot  be  recoagulated 
by  rennin.  A  solution  of  alkali-paracaseinate  is  much  more  readily 
precipitated  by  CaCb  than  an  alkali-caseinate  solution  of  the  same  con- 
centration, and  the  precipitation  limits  for  saturated  ammonium-sul- 
phate solution,  the  upper  as  well  as  the  lower  limit,  lie,  according  to 
Laqueur,  lower  with  paracasein  than  with  casein.  The  internal  friction 
of  paracasein  solutions  is  also,  in  his  opinion,  less  than  that  of  casein 
solutions  and  indeed  even  to  20  per  cent. 

By  continued  action  of  rennin  upon  paracasein  a  further  transformation 
has  been  found  in  many  cases  (Petky,  Slowtzoff,  v.  Hekwerden  l).  This 
is  explained  by  the  presence  of  another  proteolytic  enzyme  in  the  (impure)  rennin 
preparation.  This  assumption  seems  to  be  plausible,  and  we  are  here  probably 
dealing  only  with  a  secondary  process  which  has  nothing  whatever  to  do  with  the 
true  formation  of  paracasein.  Whey  protein  is  also  formed  after  the  very  short 
action  of  rennin,  and  the  continued  cleavage  occurs  with  varying  .speed.  Thus 
Schmidt-Nielsen  found  that  the  quantity  of  whey  protein  was  even  3  per  cent 
of  the  casein  nitrogen  after  the  action  of  rennet  for  15  minutes,  and  only  4.25 
per  cent  after  6  hours'  action.  These  and  other  recent  investigations  favor 
the  assumption  that  the  casein  coagulation  by  rennet  is  a  hydrolytic  cleavage, 
but  the  conditions  are  not  so  clear  that  this  can  be  considered  as  proved.2 

Frtsh,  unchanged  milk  does  not,  as  is  known,  coagulate  on  boiling;  but  in 
not  too  rapid  action  of  rennin  a  state  may  be  observed  in  which  the  milk  coagu- 
lates on  heating  (metacasein  reaction).  A  solution  of  paracasein  lactate,  accord- 
ing to  Laxa,3  coagulates  with  rennin  the  same  as  a  solution  of  casein  lactate, 
which  indicates,  he  believes,  that  the  paracasein  is  transformed  into  casein  again 
by  the  lactic  acid.  But  as  a  precipitation  of  the  paracasein  from  the  acid  solu- 
tion is  perhaps  a  pepsin  action,  the  transformation  of  the  paracasein  into  casein 
by  the  lactic  acid  must  not  be  considered  as  proved. 

In  the  digestion  of  casein  with  pepsin-hydrochloric  acid  primarily  a 
phosphorized  proteose  is  formed,  from  which  then  the  pseudonuclein  is 
split  off  (Salkowski).  The  quantity  thus  split  off  is  variable,  as  shown 
by  the  researches  of  Salkowski,  Hahn,  Moraczewski,  Sebelien, 
and  Zaitschek.4  The  amount  of  phosphorus  in  the  pseudonucleins 
obtained  also  varies  considerably.  Salkowski  considers  that  the  quan- 
tity of  pseudonuclein  split  off  is  dependent  upon  the  relation  between 
the  casein  and  the  digestion  fluid,  e.g.,  the  quantity  of  the  pseudonu- 

^etry,  Hofmeister's  Beitriige,  8;  Slowtzoff,  ibid.,  9;  v.  Herwerden,  Zeitschr.  f. 
physiol.  Chem.,  52;  \V.  van  Dam,  ibid.,  61. 

2  See  also  Werncken,  Zeitschr.,  f.  Biol.,  52. 

3  Laxa,  1.  c. 

4  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  27;  Salkowski  and  Hahn,  Pflii^er's  Arch., 
59;  Salkowski,  ibid.,  63;  v.  Moraczewski,  Zeitschr.  f.  physiol.  Chem.,  20;  Sebelien 
ibid.,  20;  Zaitschek,  Pfluger's  Arch.,  104. 


652  MILK. 

cleins  diminishes  as  the  pepsin-hydrochloric  acid  increases.  In  the 
presence  of  500  grams  of  pepsin-hydrochloric  acid  to  1  gram  of  casein, 
Salkowski  digested  the  latter  completely  without  obtaining  any 
pseudonuclein. 

In  peptic  as  well  as  tryptic  digestion  a  part  of  the  organic  phosphorus 
is  split  off  as  orthophosphoric  acid,  the  quantity  increasing  as  the"  diges- 
tion progresses.  Another  part  of  the  phosphorus  is  retained  in  organic 
combination  in  the  proteoses  as  well  as  in  the  true  peptones  (Salkowski, 
Biffi,  Alexander,  Aders-Plimmer  and  Bayliss  '). 

From  the  products  of  peptic  digestion  of  casein,  after  the  separation  of  the 
pseudonuclein,  Salkowski  2  has  isolated  an  acid  rich  in  phosphorus.  He  con- 
siders this  a  paranucleic  acid.  This  acid  which  gives  the  biuret  test  and  a 
faint  xanthoproteic  reaction,  contains  4.05-4.31  per  cent  phosphorus.  A  still 
richer  product  in  phosphorus,  with  6.9  per  cent  P,  has  been  isolated  by  Reh 
from  the  peptic  digestive  products  of  casein.  He  calls  this  body  polypeptid 
phosphoric  acid.  This  product,  which  also  gives  the  above-mentioned  protein  re- 
actions, and  is  not  comparable  with  the  nucleic  acids,  is  characterized  by  a  remark- 
ably high  content  of  amino-nitrogen,  namely,  23. S  per  cent.  Among  the  products 
obtained  by  Reh,  Dietrich3  found  a  mixture  of  at  least  four  different  lime-salts 
of  a  peptone  character,  and  which  he  considers  as  polypeptide-like  combination 
with  P205,  caseonphosphoric  acids.  The  amount  of  phosphorus  was,  respectively, 
10.0,  4.1,  3.84  and  3.88  per  cent. 

Casein  may  be  prepared  in  the  following  way:  The  milk  is  diluted 
with  4  vols,  of  water  and  the  mixture  treated  with  acetic  acid  to  0.75- 
1  p.  m.  Casein  thus  obtained  is  purified  by  repeatedly  dissolving  in  water 
with  the  aid  of  the  smallest  quantity  of  alkali  possible,  by  filtering  and 
reprecipitating  with  acetic  acid  and  thoroughly  washing  with  water. 
Most  of  the  milk-fat  is  retained  by  the  filter  on  the  first  filtration,  and  the 
casein  contaminated  with  traces  of  fat  is  purified  by  treating  with  alcohol 
and  ether. 

Lactoglobulin  was  obtained  by  Sebelien  from  cow's  milk  by  saturating 
it  with  NaCl  in  substance  (which  precipitated  the  casein)  and  saturating 
the  filtrate  with  magnesium  sulphate.  As  far  as  it  has  been  investigated 
it  had  the  properties  of  serglobulin;  the  globulin  isolated  by  Tiemann 4 
from  colostrum  had,  nevertheless,  a  markedly  low  content  of  carbon, 
namely,  49.83  per  cent. 

Lactalbumin  was  first  prepared  in  a  pure  state  from  milk  by  Sebelien. 
He  gives  its  composition  as,  C  52.19,  H  7.18,  N  15.77,  S  1.73,  O  23.13 
per  cent.     Lactalbumin  has  the  properties  of  the  albumins,  and  Wich- 


1  Salkowski,  1.  c;  Biffi,  Virchow's  Arch.,  152;  Alexander,  Zeitschr.  f.  physiol. 
Chem.,  25;  Plimmer  and  Bayliss,  Journ.  of  Physiol.,  33;  See  also  Kuttner,  Pfluger'a 
Arch.  129. 

2  Zeitschr.  f..  physiol.  Chem.,  32. 

1  Reh.  Hofmeister's  Beitrage  11;  Dietrich,  Bioch.  Zeitschr.  22. 
4  Zeitschr.  f.  physiol.  Chem.,  25. 


LACTALBUMIN.     ENZYMES.  653 

mann  found  that  it  crystallizes  in  forms  similar  to  ser-  or  ovalbumin. 
It  coagulates,  depending  on  the  concentration  and  the  amount  of  salt 
in  solution,  at  72-84°  C.  It  is  similar  to  seralbumin,  but  differs  from 
it  in  having  a  considerably  lower  specific  rotatory  power:  (a)D=—  37°. 
According  to  Fasal  1  it  is  especially  rich  in  tryptophane,  namely,  3.07 
per  cent. 

The  principle  of  the  preparation  of  lactalbumin  is  the  same  as  for  the 
preparation  of  seralbumin  from  serum.  The  casein  and  the  globulin 
are  removed  by  MgSC>4  in  substance,  and  the  filtrate  treated  as  previously 
stated  (page  263). 

The  occurrence  of  other  proteins,  such  as  proteoses  and  peptones,  in  milk  has 
not  been  positively  proved.  These  bodies  are  easily  produced  as  laboratory 
products  from  the  other  proteins  of  the  milk.  Such  a  laboratory  product  is 
Millox's  and  Comatlle's  lactoprotein,  which  is  a  mixture  of  a  little  casein  with 
changed  albumin,  and  proteose  *  which  is  formed  by  chemical  action.  In  regard 
to  opalisin,  see  Human  Milk,  p.  G62. 

Milk  also  contains,  Siegfried  3  claims,  a  nucleon  related  to  phos- 
phocarnic  acid,  which  yields  fermentation  lactic  acid  (instead  of  para- 
lactic  acid)  and  a  special  carnic  acid,  orylic  acid  (instead  of  muscle  carnic 
acid),  as  cleavage  products.  Lactophosphocarnic  acid  may  be  precipitated 
as  an  iron  compound  from  the  milk  freed  from  casein  and  coagulable 
proteins  as  well  as  from  earthy  phosphates. 

Milk  also  contains  enzymes  of  various  kinds.  Of  these  we  must  men- 
tion catalases,  peroxidases,  and  reductases,  but  the  statements  as  to  their 
occurrence  in  the  milk  from  different  animals  as  well  as  the  question 
how  much  of  their  action  is  due  to  micro-organisms  are  conflicting. 
Among  these  enzyme  actions  a  special  interest  has  been  given  to  the 
Schardinger  reaction,  which  consists  in  the  fact  that  milk  at  70°  C. 
in  the  presence  of  formaldehyde  or  acetaldehyde  reduces  certain  dyes, 
such  as  methylene  blue,  to  leucobases.  An  amylolytic  enzyme  which 
converts  starch  into  maltose  occurs,  especially,  in  human  milk,  while 
it  is  absent  in  cow's  milk  or  occurs  only  to  a  slight  extent.  A  fermenta- 
tion enzyme  which  in  the  absence  of  micro-organisms  decompose^  the 
lactose  into  lactic  acid,  alcohol,  and  CO2,  occurs,  according  to  Stoklasa4 
and  his  co-workers,  in  cow's  milk  as  well  as  in  human  milk.  Human 
milk,  as  well  as  cow's  milk,  contains  a  lipase  which  has  the  property 
at  least  of  acting  upon  monobutyrin.  Babcock  and  Russel  have 
found  in  these  two  kinds  of  milk,  as  well  as  certain  others,  a  proteolytic 

^ebelien,  Zeitschr.,  f.  physiol.  Chem.,  9;  Wichmann,  ibid.,  27;  Fasal,  Bioch. 
Zeitschr.,  44. 

*  See  Hammarsten,  Mary's  Jahresber.,  6,  13. 
8  Zeitschr.  f.  physiol.  Chem.,  21  and  22. 
♦See  Chem.  Centralbl.,  1905,  1,  107. 


654  MILK. 

enzyme  which  they  call  galactase,  which  is  allied  to  trypsin,  but  differs 
therefrom  in  that  it  develops  ammonia  from  milk  even  in  the  early  stages 
of  digestion.  The  occurrence  of  such  an  enzyme  is  denied  by  Zaitschek 
and  v.  Szontagh,  but  on  the  other  hand  Vandevelde,  de  Waele,  and 
Sugg  *  confirm  the  occurrence  of  a  proteolytic  enzyme  in  milk. 

Orotic  acid,  C5HnN,04.2H20,  is  the  name  given  by  Biscaro  and  Belloni  l 
to  a  new  constituent  of  milk  which  they  have  discovered.  This  acid,  which  can 
be  precipitated  by  basic  lead  acetate  from  whey  free  from  protein,  is  slightly 
soluble  in  water,"  crystalline,  and  gives  several  crystalline  salts.  The  mono- 
methyl  and  ethyl  esters  of  this  acid  are  also  known.  It  yields  urea  on  treatment 
with  potassium  permanganate. 

Lactose,  milk-sugar,  Ci2H220n+H<20.  This  sugar,  on  hydrolysis, 
can  be  split  into  two  hexoses,  glucose  and  galactose.  It  yields  mucic  acid 
besides  other  organic  acids,  by  the  action  of  dilute  nitric  acid.  Levulinic 
acid  is  formed,  besides  formic  acid  and  humin  substances,  by  the  stronger 
action  of  acids.  By  the  action  of  alkalies,  among  other  products  we 
find  lactic  acid  and  pyrocatechin. 

Milk-sugar  occurs,  as  a  rule,  only  in  milk,  but  it  has  also  been  found 
in  the  urine  of  pregnant  women,  on  stagnation  of  milk,  as  well  as  in  the 
urine  after  partaking  of  large  quantities  of  the  same  sugar. 

Lactose  occurs  ordinarily  as  colorless  rhombic  crystals  with  1  mole- 
cule of  water  of  crystallization,  which  is  driven  off  by  slowly  heating  to 
100°  C,  but  more  easily  at  130-140°  C.  On  quickly  boiling  down  a  milk- 
sugar  solution,  anhydrous  milk-sugar  separates  out.  Milk-sugar  dissolves 
in  6  parts  cold  or  in  2.5  parts  boiling  water;  it  has  a  faintly  sweetish 
taste.  It  does  not  dissolve  in  ether  or  absolute  alcohol.  Its  solutions 
are  dextrogyrate.  The  rotatory  power,  which  on  heating  the  solution  to 
100°  C.  becomes  constant,  is  («);>= +52.5°.  Milk-sugar  combines  with 
bases;  the  alkali  combinations  are  insoluble  in  alcohol. 

Milk-sugar  is  not  fermentable  with  pure  yeast.  It  undergoes,  on  the 
contrary,  alcoholic  fermentation  by  the  action  of  certain  schizomycetes, 
and  E.  Fischer3  found  that  the  milk-sugar  is  first  split  into  glucose 
and  galactose  by  an  enzyme,  lactase,  existing  in  the  yeast.  The  prep- 
aration of  milk-wine,  "  kumyss,"  from  mare's  milk  and  "  kephir  "  and 
"  yoghurt  "  from  cow's  milk  is  based  upon  this  fact.  Other  micro-organ- 
isms also  take  part  in  this  change,  causing  a  lactic-acid  fermentation  of 
the  milk-sugar. 


1  Babcock  and  Russel,  Centralbl.  f.  Bakt.  u.  Parisitenkunde  (II),  6,  and  Maly's 
Jahresber.,  31;  Zaitsrhek  and  v.  Szontagh,  Pfliiger's  Arch.,  104;  Vandevelde,  de 
Waele,   and   Sugg,    Hofmeister's  Beitrage,   5. 

*  See  Chem.  Centralbl.,  1905,  2,  63. 

*  Ber.  d.  d.  Chem.  Gesellsch.,  27. 


LACTOSE.  655 

Lactose  responds  to  the  reactions  of  glucose,  such  as  Moore's,1 
Trommer's  and  Rubner's,  and  the  bismuth  test.  It  also  reduces  mer- 
curic oxide  in  alkaline  solutions.  After  warming  with  phenylhydrazine 
acetate  it  gives  on  cooling  a  yellow  crystalline  precipitate  of  phenyl 
lactosazone,  C24H32N4O9.  It  differs  from  cane-sugar  by  giving  positive 
reactions  with  Moobe's  or  Trommer's  and  the  bismuth  test,  and  also  in 
that  it  does  not  darken  when  heated  to  100°  C  with  anhydrous  oxalic- 
acid.  It  differs  from  glucose  and  maltose  by  its  solubility  and  crystalline 
form,  but  especially,  by  its  not  fermenting  with  yeast,  and  by  yielding 
mucie  acid  with  nitric  acid. 

The  osazone  obtained  with  phenylhydrazine  acetate,  which  melts  at 
200°  C,  differs  from  the  other  osazones  by  being  inactive  when  0.2  gram 
is  dissolved  in  4  cc.  of  pyridine  and  G  cc.  of  absolute  alcohol  and  viewed 
through  a  layer  10  centimeters  long  (Xeuberg2). 

For  the  preparation  of  milk-sugar  we  make  use  of  the  by-product 
in  the  preparation  of  cheese,  the  sweet  whey.  The  protein  is  removed 
by  coagulation  with  heat,  and  the  filtrate  evaporated  to  a  syrup.  The 
crystals  which  separate  after  a  certain  time  are  recrystallized  from  water 
after  decolorizing  with  animal  charcoal.  A  pure  preparation  may  be 
obtained  from  the  commercial  milk-sugar  by  repeated  recrystallization. 
The  quantitative  estimation  of  milk-sugar  may  be  performed  either  by 
the  polaristrobometer  or  by  means  of  titration  with  Fehling's  solution. 
Ten  cc.  of  Fehling's  solution  are  reduced  by  0.0G7G  gram  of  milk-sugar 
in  0.5-1.5  per  cent  solution  after  boiling  for  six  minutes.  (In  regard  to 
Fehling's  solution  and  the  titration  of  sugar  see  larger  hand-books.) 

From  the  non-correspondence  between  the  quantity  of  sugar  in  the 
milk  as  determined  by  polarization  and  gravimetrically,  when  the  polar- 
ization results  are  always  higher,  Sebelien  3  has  concluded  that  the 
milk  must  contain  a  second  reducing  substance  which  polarizes  stronger 
than  lactose.  This  substance  is  probably  a  pentose  and  occurs  to  a  very 
slight  extent  in  ordinary  milk,  0.25-0.3")  p.  m.  (Sebelien  and  Sunde), 
and  more  in  colostrum,  0.5  p.  m. 

Ritthausen  found  another  carbohydrate  in  milk  which  is  soluble  in  water, 
non-crystallizable,  which  has  a  faint  reducing  action,  and  which  yields,  on  boiling 
with  an  acid,  a  body  having  a  greater  reducing  power.  Becha.mp  4  considers 
this  as  dextrin. 

1  The  well-known  beautiful  red  color,  which  milk  produces  after  the  addition  of 
alkali,  at  the  room  temperature  and  to  which  attention  has  been  called  recently  by 
Gautier,  Morel,  and  Monod  (Compt.  rend.  soc.  biol.,  60  and  C»2),  and  Kriiger  (Zeitschr. 
f.  Physiol.  Chem.,  50)  is  a  Moore's  reaction  modified  by  the  presence  of  protein  and 
perhaps  also  other  milk  constituents. 

2  Ber.  (1.  d.  Chem.  Gesellsch.,  32. 

'Sebelien,  Hammarsten's  Festschrift,  1906;  with  Sunde,  Zeitschr.  f.  angew. 
Chem.,  21. 

4  Ritthausen,  Journ.  f.  prakt.  Chem.  (N.  F.),  15;   Beehamp,  Bull.  Soc.  Chim.  (3),  6. 


656  MILK. 

The  mineral  bodies  of  milk  will  be  treated  in  connection  with  its  quan- 
titative composition. 

The  methods  for  the  quantitative  analysis  of  milk  are  very  numerous, 
and  a*  all  cannot  be  treated  here,  we  will  give  the  principal  points  of  a 
few  of  the  methods  considered  most  trustworthy  and  most  frequently 
employed. 

In  determining  the  solids  a  carefully  weighed  quantity  of  milk  is  mixed  with 
an  equal  weight  of  heated  quartz  sand,  fine  glass  powder,  or  asbestos.  The 
evaporation  is  first  done  on  the  water-bath  and  finished  in  a  current  of  carbon 
dioxide  or  hydrogen  not  above  100°  C. 

The  mineral  bodies  are  determined  by  incinerating  the  milk,  using  the  pre- 
cautions mentioned  in  the  text-books.  The  results  obtained  for  the  phosphoric 
acid  are  incorrect  on  account  of  the  burning  of  phosphorized  bodies,  such  as 
casein  and  lecithin.  We  must,  therefore,  according  to  Soldner,  subtract  in  round 
numbers  25  per  cent  from  the  total  phosphoric  acid  found  in  the  milk.  The 
quantity  of  sulphate  in  the  ash  also  depends  on  the  combustion  of  the  proteins. 

In  the  determination  of  the  total  amount  of  proteins  Ritthausen's  method 
is  employed,  namely,  the  precipitation  of  the  milk  with  copper  sulphate  according 
to  the  modification  suggested  by  Munk.1  He  precipitates  all  the  proteins  by 
means  of  cupric  l)3rdroxide  at  boiling  heat,  and  determines  the  nitrogen  in  the 
precipitate  by  means  of  Kjeldahl's  method.  This  modification  gives  more 
exact  results. 

According  to  Sebelien's  method,  three  to  four  grams  of  milk  are  diluted 
with  an  equal  volume  of  water,  a  little  common-salt  solution  added,  and  the 
proteins  precipitated  with  an  excess  of  tannic  acid.  The  precipitate  is  washed 
with  cold  water,  and  then  the  quantity  of  nitrogen  determined  by  Kjeldahl's 
method.  The  total  nitrogen  found  when  multiplied  by  6.37  (casein  and  lactal- 
bumin  contain  both  15.7  per  cent  nitrogen)  gives  the  total  quantity  of  proteins. 
This  method,  which  is  readily  performed,  gives  very  good  results.  I.  Munk 
used  this  method  in  the  analysis  of  woman's  milk.  In  this  case  the  quantity 
of  nitrogen  found  must  be  multiplied  by  6.34.  G.  Simon  2  found  that  the 
precipitation  with  tannic  acid,  also  with  phosphotungstic  acid,  is  the  simplest 
and  most  accurate.  The  objection  to  this  and  other  methods  in  which  the  pro- 
teins are  precipitated  is  that  perhaps  other  bodies  (extractives)  may  be  carried 
down  at  the  same  time  (Camerer  and  Soldner  3).  It  is  not  known  to  what 
extent  this  takes  place. 

A  part  of  the  nitrogen  in  the  milk  exists  as  extractives,  and  this  nitrogen  is 
calculated  as  the  difference  between  the  total  nitrogen  and  the  protein  nitrogen. 
According  to  Mink's  analyses  about  r$  of  the  total  nitrogen  belongs  to  the  extract- 
ives in  cow's  milk.  Camerer  and  Soldner  determine  the  nitrogen  in  the  filtrate 
from  the  tannic-acid  precipitate  by  Kjeldahl's  method,  and  also  according  to 
Hufner's  method  (hypobromite).  In  this  way  they  found  18  milligrams  of 
nitrogen  according  to  Hufner  (urea,  etc.)  in  100  grams  of  cow's  milk. 

To  determine  the  casein  and  albumin  separately  we  may  make  use  of  the 
method  first  suggested  by  Hoppe-Seyler  and  Tolmatscheff,4  in  which  the  casein 
is  precipitated  by  magnesium  sulphate.  According  to  Sebelien  the  milk  is  diluted 
with  its  own  volume  of  a  saturated  magnesium-sulphate  solution,  then  saturated 
with  the  salt  in  substance,  and  the  precipitate  then  filtered  and  washed  with  a 
saturated  magnesium-sulphate  solution.     The  nitrogen  is  determined  in  the  pre- 

1  Ritthausen,  Journ.  f.  prakt.  Chem.  (N.  F.),  15;   I.  Munk,  Virchow's  Arch.,  134. 

'  Sebelien,  Zeitschr.  f.  physiol,  Chem.,  13;  Simon,  ibid.,  33. 

1  Zeitschr.  f.  Biologie,  33  and  36. 

4  Hoppe-Seyler,  Med.  chem.  Untersuch.,  272. 


MILK  ANALYSIS.  657 

cipitate  by  Kjeldahl's  method,  and  the  quantity  of  casein  (+globulin)  drtormined 
by  multiplying  the  result  by  0.37.  The  quantity  of  lactalbumin  may  be  calculated 
as  the  difference  between  the  casein  and  the  total  proteins  found.  The  lactal- 
bumin may  also  be  precipitated  by  tannic  acid  from  the  filtrate  from  the  casein 
precipitate  containing  MgS04,  after  diluting  with  water,  the  nitrogen  determined 
by  Kjeldahl's  method  and  the  result  multiplied  by  0.37. 

Schlossmann  •  suggests  an  alum  solution,  which  precipitates  the  casein, 
in  order  to  separate  the  casein  from  the  other  proteins,  and  the  albumin  is  then 
precipitated  from  the  filtrate  by  tannic  acid.  The  nitrogen  in  the  precipitate 
is  determined  by  the  Kjeldahl  method.  This  method  has  recently  been  tested 
by  Simon  and  he  recommends  it  highly. 

The  fat  is  gravimetrically  determined  by  thoroughly  extracting  the  dried 
milk  with  ether,  evaporating  the  ether  from  the  extract,  and  weighing  the  residue. 
The  fat  may  be  determined  by  aerometric  means  by  adding  alkali  to  the  milk, 
shaking  with  ether,  and  determining  the  specific  gravity  of  the  fat  solution  by  means 
of  Soxhlet's  apparatus.  In  determining  the  amount  of  fat  in  a  large  number  of 
samples  the  lactocrit  of  De  Laval  may  be  used  with  success.  There  are  numer- 
ous other  methods  for  estimating  milk-fat,  but  they  cannot  be  considered  here. 

In  determining  the  milk-sugar  the  proteins  are  first  removed.  For  this  pur- 
pose we  precipitate  either  with  alcohol,  which  must  he  evaporated  from  the  filtrate, 
or  by  diluting  with  water,  and  removing  the  casein  by  the  addition  of  a  little  acid, 
and  the  lactalbumin  by  coagulation  at  boiling  heat.  The  sugar  is  determined  by 
titration  with  Fehling's  or  Knapp's  solution  (see  Chapter  XIV).  The  principle 
of  the  titration  is  the  same  as  for  the  titration  of  sugar  in  the  urine;  10  cc.  of 
Fehling's  solution  correspond  to  0.0070  gram  of  milk-sugar;  10  cc.  of  Knapp's 
solution  correspond  to  0.0311-0.0310  gram  of  milk-sugar,  when  the  saccharine 
liquid  contains  about  |-1  per  cent  of  sugar.  In  regard  to  the  modus  operandi 
of  the  titration  we  must  refer  the  reader  to  more  extensive  works. 

Instead  of  these  volumetric  determinations  other  methods  of  estimation,  such 
as  Allihn's  method,  the  polariscope  method,  and  others,  may  be  used.  In  calcu- 
lating the  analysis  or  in  determining  the  solids  it  is  of  importance  to  remember, 
as  suggested  by  Camerer  and  Soldner,  that  the  milk-sugar  in  the  residue  is 
anhydrous.  Many  other  methods  for  determining  the  milk-sugar  have  been 
suggested  and  recommended. 

The  quantitative  composition  of  cow's  milk  is  naturally  very  variable. 
The  average  obtained  by  Konig  2  is  as  follows  in  1000  parts: 

Water.  Solids.  Casein.  Albumin.  Fats.  Sugar.  Salts. 

871.7  128.3  30.2  5.3  36.9  48.8  7.1 


35.5 

The  quantity  of  mineral  bodies  in  1000  parts  of  cow's  milk  is,  accord- 
ing to  the  analyses  of  Soldner,  as  follows:  K2O  1.72,  Na20  0.51,  CaO 
1.98,  MgO  0.20,  P2O5  1.82  (after  correction  for  the  pseudonuclein) , 
C1J1.98  grams.  Bunge  found  0.0035  gram  Fe203,  and  Edelstein  and 
Csonka3  found  0.0007-0.001  gm.  Fe20?.  According  to  Soldner  the 
K,  Na,  and  CI  are  found  in  the  same  quantities  in  whole  milk  as  in  milk- 
serum.     Of  the  total  phosphoric  acid  36-56  per  cent,  and  of  the  lime 

1  Zeitschr.  f.  physiol.  Chem.,  22. 

2  Chemie  der  menschlichen  Nahrungs-  und  Genussmittel,  4.  Aufl. 

3  Bunge,  Zeitschr.  f.  Biol.  10;  Edelstein  and  Csonka,  Bioch.  Zeitschr.,  38. 


658  MILK. 

53-72  per  cent  is  not  in  simple  solution.  A  part  of  this  lime  is  combined 
with  the  casein;  the  remainder  is  found  united  with  the  phosphoric  acid 
as  a  mixture  of  dicalcium  and  tricalcium  phosphates  which  is  kept  disr 
solved  or  suspended  by  the  casein.  Rona  and  Michaelis  x  found  that 
about  40-50  per  cent  of  the  total  quantity  of  lime  was  diffusable ;  accord- 
ing to  them  nearly  one-half  of  the  calcium  is  contained  in  the  milk  as  a 
non-dissociable  casein  compound,  while  the  milk  only  contains  the  very 
smallest  amounts  of  suspended  calcium  phosphate. 

The  bases  are  in  excess  of  the  mineral  acids  in  the  milk-serum. 
The  excess  of  the  first  is  combined  with  organic  acids,  which  correspond 
to  2.5  p.  m.  citric  acid  (Soldner). 

The  gases  of  the  milk  consist  mainly  of  CO2,  besides  a  little  N  and 
traces  of  0.  Pfluger  2  found  10  vols,  per  cent  CO2  and  0.6  vol.  per  cent 
N  calculated  at  0°  C.  and  760  mm.  pressure. 

The  variation  in  the  composition  of  cow's  milk  depends  on  several 
circumstances. 

The  colostrum,  or  the  milk  which  is  secreted  before  calving  and  in 
the  first  few  days  after,  is  yellowish,  sometimes  alkaline,  but  often  acid, 
of  higher  specific  gravity,  1.046-1.080,  and  richer  in  solids  than  ordinary 
milk.  The  colostrum  contains,  besides  fat-globules,  an  abundance  of 
colostrum-corpuscles — nucleated  granular  cells  0.005-0.025  mm.  in  di- 
ameter with  abundant  fat-granules  and  fat-globules.  The  fat  of  colos- 
trum has  a  somewhat  higher  melting-point  and  is  poorer  in  volatile  fatty 
acids  than  the  fat  from  ordinary  milk  (Nilson  3).  The  iodine  equivalent 
of  the  colostrum-fat  is  higher  than  that  of  milk-fat.  The  quantity  of 
cholesterin  and  lecithin  is  generally  greater.  The  most  apparent  dif- 
ference between  it  and  ordinary  milk  is  that  colostrum  coagulates  on  heat- 
ing to  boiling  because  of  the  absolutely  and  relatively  greater  quantities 
of  globulin  and  albumin  that  it  contains.4  The  composition  of  colostrum 
varies  considerably.  Konig  gives  as  average  the  following  figures  in 
1000  parts: 


Water. 

Solids. 

Casein. 

Albumin  and  Globulin. 

Fat. 

Sugar. 

Salts. 

740.7 

253.3 

40.4 

130.0 

35.9 

20.7 

15.0 

The  influence  which  food  exercises  upon  the  composition  of  milk  will 
be  discussed  in  connection  with  the  chemistry  of  the  milk  secretion. 


'Bioch.  Zeitschr.,  21. 

2Pfliiger'sArch.,  2. 

*  See  Maly's  Jahresber.,  21.  See  also  Engel  and  Bode,  Zeitschr.  f.  physiol.  Chem., 
74. 

♦See  Sebelien,  Maly's  Jahresber.,  18,  and  Tiemann,  Zeitschr.  f.  physiol.  Chem., 
25.     See  also  Simon,  ibid.,  33;  Winterstein  and  Strickler,  ibid.,  47. 


MILK  OF  OTHER  ANIMALS.  059 

In  the  following  table  is  given  the  average  composition  of  skimmed  milk  and 
certain  other  preparations  of  milk: 

Water  f'r.itc-ins.  Fat.  Sugar.   Lactic  Acid.  Salts. 

Skimmed  milk 906.6         31.1  7.4         47:,                       7    | 

Cream 655.1          :ti   1  267.5        35.2                      6   I 

Buttermilk <»<>->  7         40.6  9.3         37  3         3  4         6  7 

Whey 032  4           8.5  2.3         47.0         3.3         6.5 

Kumyss,  kephir  and  yoghurt  are  obtained,  as  above  stated,  by  the  alcoholic 
and  laetic-acid  fermentation  of  the  milk-sugar,  the  first  from  mare's  milk  and 
the  other  from  cow's  milk.  Large  quantities  of  carbon  dioxide  are  formed  thereby, 
and  besides  this  the  protein  bodies  of  the  milk  are  partly  converted  into  proteoses 
and  peptones,  which  increase  the  digestibility.  The  quantity  of  lactic  acid  in 
these  preparations  may  be  about  10-20  p.  m.  The  quantity  of  alcohol  varies 
from  10  to  35  p.  m. 

Milk  of  Other  Animals.  Goat's  milk  has  a  more  yellowish  color  and  a 
more  specific  odor  than  cow's  milk.  The  coagulum  obtained  by  acid  or  rennet 
is  more  solid  and  is  harder  than  that  from  cow's  milk.  Sheep's  milk  is  similar 
to  goat's  milk,  but  has  a  higher  specific  gravity  and  contains  a  greater  amount 
of  solids. 

Mare's  milk  is  alkaline  and  contains  a  casein  which  is  not  precipitated,  by 
acids,  in  lumps  or  solid  masses,  but,  like  the  casein  from  woman's  milk,  in  fine 
flakes.  This  casein  is  only  incompletely  precipitated  by  rennet,  and  it  is  very 
similar  also  in  other  respects  to  the  casein  of  human  milk.  In  Beil's  «  opinion 
the  casein  from  mare's  and  cow's  milk  is  the  same,  and  the  different  behavior 
of  the  two  varieties  of  milk  is  due  to  varying  amounts  of  salts  and  to  a  different 
relation  between  the  casein  and  the  albumin.  This  does  not  agree  with  the 
analyses  of  casein  by  Tangl  and  Csoa'ks  given  above  nor  with  the  investiga- 
tions of  Zaitschek  and  v.  Szontagh,  who  find  that  the  casein  from  mare's  milk, 
like  that  from  human  and  ass's  milk,  is  digested  by  pepsin-hydrochloric  acid 
without  leaving  a  residue.  According  to  Engel  and  Dennemark  2  the  colostrum 
from  the  mare  differs  from  that  from  the  ass  by  being  richer  in  casein  than  the  milk. 
The  milk  of  the  ass  is  claimed  by  earlier  authorities  to  be  similar  to  human  milk' 
but  Schlossmann  finds  it  considerably  poorer  in  fat.  The  researches  of  Ellen- 
berger  give  similar  results,  and  show  great  similarity  between  ass's  milk  and 
human  milk.  The  average  results  were  15  p.  m.  protein  with  5.3  p.  m.  albumin 
and  9.4  p.  m.  casein.  This  latter,  like  human  casein,  does  not  yield  any  pseudo- 
nuclein  on  pepsin  digestion,  which  agrees  well  with  the  above-mentioned  investiga- 
tions of  Zaitschek.  The  quantity  of  nucleon  was  about  the  same  as  in  woman's 
milk.  The  quantity  of  fat  was  15  p.  m.,  and  the  sugar  was  50-60  p.  m.  Reindeer 
milk  is  characterized,  according  to  Werenskiold,3  by  being  very  rich  in  fat, 
144.6-197.3  p.  m.,  and  casein,  80.6-  86.9  p.  m. 

The  milk  of  carnivora  (the  bitch  and  cat)  is  acid  in  reaction  and  very  rich 
in  solids.  The  composition  of  the  milk  of  these  animals  varies  with  the  com- 
position of  the  food. 

To  illustrate  the  composition  of  the  milk  of  other  animals  the  following  figures, 
the  compilation  of  Konig,  are  given.  As  the  milk  of  each  kind  of  animal  may 
have  a  variable  composition,  these  figures  should  only  be  considered  as  examples 
of  the  composition  of  milk  of  various  kinds:4 

1  Studein  iiber  die  Eiweissstoffe  des  Kumys  und  Kefirs,  St.  Petersburg,  1S86  (Ricker). 

2  Zeitschr.  f.  physiol.  Chem.,  76. 

5  Zaitschek,  1.  c;  Schlossmann,  Zeitschr.  f.  physiol.  Chem.,  22;  Ellenberger,  Arch. 
f.  (Anat.  u.)  Physiol.,  1S99  and  1902;  Werenskiold,  Maly's  Jahresber.,  25. 

4  Details  in  regard  to  the  milk  of  different  animals  may  be  found  in  Proscher, 
Zeitschr.  f.  physiol.  Chem.,  24;  Abderhalden,  ibid.,  27.  In  regard  to  pig  milk,  see 
Zuntz  and  Ostertag,  Landw.  Jahresb.,  37. 


Solids 

Proteins. 

Fat. 

Sugar. 

Salts. 

245.6 

99.1 

95.7 

31.9 

7.3 

183.7 

90.8 

33.3 

49.1 

5.8 

130.9 

36.9 

40.9 

44.5 

8.6 

165.0 

57.4 

61.4 

39.6 

6.6 

128.3 

35.5 

36.9 

4S.8 

7.1 

99.4 

18.9 

10.9 

66.5 

3.1 

100.0 

21.0 

13.0 

63.0 

3.0 

176.3 

60.9 

64.4 

40.4 

10.6 

321.5 

30.9 

195.7 

88.5 

6.5 

513.3 

437.6 

4.6 

302.0 

luman 

94.3 
Milk. 

194.0 

9.9 

660  MILK. 


Milk  of  the  Water. 

Dog 754.4 

Cat 816.3 

Goat 869 . 1 

Sheep 835.0 

Cow 871.7 

Horse 900 . 6 

Ass 900.0 

Pig 823.7 

Elephant 678.5 

Dolphin 486 . 7 

Whale1 698.0 


Woman's  milk  is  amphoteric  in  reaction.  According  to  Cotjrant 
its  reaction  is  relatively  more  alkaline  than  cow's  milk,  but  it  has,  never- 
theless, a  lower  absolute  reaction  for  alkalinity  as  well  as  for  acidity.  He 
found  between  the  tenth  day  and  the  fourteenth  month  after  confinement 
practically  constant  results.  The  alkalinity,  as  well  as  the  acidity,  was 
a  little  lower  than  in  childbed.  One  hundred  cc.  of  the  milk  had  the 
same  average  alkalinity  as  10.8  cc.  N/10  caustic  soda,  and  the  same 
acidity  as  3.6  cc.  N/10  acid.  The  relation  between  the  alkalinity  and  the 
acidity  in  woman's  milk  was  as  3:1,  and  in  cow's  milk  as  2.1:1.  The 
actual  reaction  determined  electrometrically  is,  according,  to  FoA,2  still 
nearly  neutral,  like  the  other  kinds  of  milk.  Allaria  has  also  arrived 
at  similar  results,  according  to  whom  the  tendency  of  human  milk  toward 
alkaline  reaction  even  in  the  most  prominent  cases  never  corresponds 

N 

to  a NaOH  solution. 

1,000,000 

Human  milk  also  contains  fewer  fat-globules  than  cow's  milk,  but 
they  are  larger  in  size.  The  specific  gravity  of  woman's  milk  varies 
between  1.026  and  1.036,  generally  between  1.028  and  1.034.  It  is  highest 
in  well-fed  and  lowest  in  poorly-fed  women.  The  freezing-point  is  lowered 
on  an  average  0.589°  C,  according  to  Winter  and  Parmentier3  con- 
stant at  0.55°,  and  the  molecular  concentration  is  0.318. 

The  fat  of  woman's  milk  has  been  investigated  by  Ruppel.  It  forms 
a  yellowish-white  mass,  similar  to* ordinary  butter,  having  a  specific  gravity 
of  0.966  at  15°.  It  melts  at  34.0°  C.  and  solidifies  at  20.2°  C.  The  fol- 
lowing fatty  acids  can  be  obtained  from  the  fat,  namely,  butyric,  caproic, 
capric,  myristic,  palmitic,  stearic,  and  oleic  acids.  The  fat  from  woman's 
milk  is,  according  to  Ruppel  and  Laves,4  relatively  poor  in  volatile 
fatty  acids.     The  non-volatile  fatty  acids  consist  of  one-half  oleic  acid, 

1  Scheibe,  cited  in  Bioch.  Centralbl.,  7,  553. 

2Cornpt.  rend.  soc.  biol.  58;  Allaria,  Maly's  Jahresb.,  39,  242. 

'  .See  Maly's  Jahresber.,  34. 

*  Ruppel,  Zeitschr.  f.  Biologie,  31;  Laves,  Zeitschr.  f.  physiol.  Chem.,  19. 


HUMAN   MILK.  661 

while  among  the  solid  fatty  acids  myristic  and  palmitic  acids  are  found 
to  a  greater  extent  than  stearic  acid. 

The  essential  qualitative  difference  between  woman's  and  cow's  milk 
seems  to  lie  in  the  proteins  or  in  the  more  accurately  determined  casein. 
A  number  of  both  the  earlier  and  more  recent  investigators  '  claim  that 
the  casein  from  woman's  milk  has  other  properties  than  that  from  cow's 
milk.  The  essential  differences  are  the  following:  The  casein  from 
woman's  milk  is  precipitated  with  greater  difficulty  with  acids  or  salts. 
It  does  not  coagulate  uniformly  in  the  milk  after  the  addition  of  rennet, 
which  depends,  essentially,  upon  the  low  amount  of  lime-salts  and  casein 
contained  in  the  milk.2  It  may  be  precipitated  by  gastric  juice,  but 
dissolves  completely  and  easily  in  an  excess  of  gastric  juice;  the  casein  pre- 
cipitate produced  by  an  acid  is  more  easily  soluble  in  an  excess  of  the  acid; 
and  lastly,  the  clot  formed  from  the  casein  of  woman's  milk  does  not 
appear  in  such  large  and  coarse  masses  as  in  the  casein  from  cow's 
milk,  but  is  more  loose  and  flocculent.  This  last-mentioned  fact  is  of 
great  importance,  since  it  explains  the  generally  admitted  fact  of  the 
easy  digestibility  of  the  casein  from  woman's  milk. 

The  question  as  to  whether  the  above-mentioned  variations  depend 
on  a  decided  difference  in  the  two  caseins,  or  only  on  an  unequal  relation 
between  the  casein  and  the  salts  in  the  two  kinds  of  milk,  or  upon  other 
circumstances,  has  not  as  yet  been  decided.  According  to  Szontagh 
and  Zaitschek  and  also  Wr6blewsky,  the  casein  from  human  milk 
does  not  yield  any  pseudonuclein  on  peptic  digestion,  and  hence  it  cannot 
be  a  nucleoalbumin.  According  to  Kobrak,  woman's  casein  yields 
some  pseudonuclein,  and  with  repeated  solution  in  alkali  and  precipitation 
by  an  acid  it  becomes  more  and  more  like  cow's  casein.  He  therefore 
suggests  the  possibility  that  woman's  casein  is  a  compound  between  a 
nucleoalbumin  and  a  basic  protein.  Wroblewsky  found  the  follow- 
ing for  the  composition  of  casein  from  woman's  milk:  C  52.24,  H  7.32, 
N  14.97,  P  0.6S,  S  1.117  per  cent.  Langstein  and  Bergell  obtained 
much  lower  figures  for  N,  S  and  especially  P,  namely,  14.34,  0.85  and  0.27 
per  cent,  respectively.  According  to  Langstein  and  Edelstein  the 
phosphorus  content  is  only  0.22-0.29  per  cent.  On  hydrolysis  Abder- 
halden  and  Langstein3  could  not  find  any  difference  between  cow 
and  human  casein. 

1  See  Biedert,  Untersuchungen  iiber  die  chemischen  Unterschiede  der  Menschen- 
und  Kuhmilch  (Stuttgart),  1884;  Langgaard,  Virchow's  Arch.,  64;  Makris,  Studien 
iiber  die  Eiweisskorper  der  Frauen-  und  Kuhmilch.  [naug.-Diss.  Strassburg,  1876. 

2  See  among  others  Bienenfeld,  Bioch.  Zeitschr.,  7,  an  1  Full  and  Wohlgemuth, 
ibid.,  8. 

s  Szontagh,  Maly's  Jahresber.,  22;  Zaitschek,  1.  c. ;  Wr6blewsky,  Beitrage  zur 
Kenntniss  des  Frauenkaseins,  Inaug.-Diss.  Bern.  1894,  and  Ein  neuer  eiweissartiger 


662  MILK. 

Woman's  milk  also  contains  lactalbumin,  besides  the  casein,  and  a  protein 
substance,  very  rich  in  sulphur  (4.7  per  cent)  and  relatively  poor  in  carbon,  which 
Wroblewsky  calls  opalisin.  The  statements  as  to  the  occurrence  of  proteoses  and 
peptones  are  conflicting  as  in  many  other  cases.  No  positive  proof  as  to  the 
occurrence  of  proteoses  and  peptones  in  fresh  milk  has  been  given. 

Because  of  the  properties  and  low  amount  of  casein  in  human  milk 
it  is  often  difficult  to  precipitate  it,  with  acid,  and  to  prepare  it,  but 
this  can  easily  be  accomplished  by  dialysis.  A  number  of  methods 
have  been  suggested  for  the  preparation  of  human  casein.  Fuld  and 
Wohlgemuth  recommend  the  freezing  of  the  milk  previous  to  pre- 
cipitation, so  that  the  casein  masses  become  larger  to  a  certain  extent 
and  the  precipitation  becomes  easier.  Engel  :  recommends  dilution 
with  water  to  5  volumes,  and  the  addition  of  60-80  cc.  N/10  acetic  acid 
for  each  100  cc.  milk.  The  mixture  is  first  cooled  for  2-3  hours  and  then, 
after  shaking,  warmed  on  the  water-bath  to  40°  for  a  few  minutes. 

Even  after  those  differences  are  eliminated  which  depend  on  the  imper- 
fect analytical  methods  emploj'ed,  the  quantitative  composition  of  woman's 
milk  is  variable  to  such  an  extent  that  it  is  impossible  to  give  any  average 
results.  The  numerous  analyses,  especially  those  made  on  a  large  number 
of  samples  by  Pfeiffer,  Adriance,  Camerer  and  Soldner,2  have  posi- 
tively shown  that  woman's  milk  is  essentially  poorer  in  proteins  but 
richer  in  sugar  than  cow's  milk.  The  quantity  of  protein  varies  between 
10-20  p.  m.,  often  amounting  to  only  15-17  p.  m.  or  less,  and  is  dependent 
upon  the  length  of  lactation  (see  below).  The  quantity  of  fat  also  varies 
considerably,  but  ordinarily  amounts  to  30-40  p.  m.  The  quantity  of 
sugar  should  not  be  below  50  p.  m.,  but  may  rise  to  even  80  p.  m.  About 
60  p.  m.  may  be  considered  as  an  average,  but  it  should  be  borne  in  mind 
that  the  quantity  of  sugar  is  also  dependent  upon  the  length  of  lactation, 
as  it  increases  with  duration.  The  amount  of  mineral  bodies  varies 
between  2  and  4  p.  m. 

The  division  of  the  total  nitrogen  in  human  milk  is,  according  to  A. 
Frehn,3  very  variable.  As  approximate  average  figures  we  can  say 
that  40-45  per  cent  of  the  total  nitrogen  is  casein,  35-40  per  cent  remain- 

Bestandteil  der  Milch,  Anzeiger  der  Akad.  d.  Wiss.  in  Krakau,  1898;  Kobrak,  Pfluger's 
Arch.,  80;  Langstein  and  Bergell,  cited  in  Bioch.  Centralbl.,  8,  323;  Langstein  and 
Edelstein,  Maly's  Jahresber,  40,  254;  Abderhalden  and  Langstein.  Zeitschr.,  f.  physiol. 
Chem..  06. 

1  Fuld  and  Wohlgemuth,  Bioch.  Zeitschr.,  5;  Engel,  ibid.,  14. 

2  Pfeiffer,  Jahrb.  f.  Kinderheilkundc,  20,  also  Maly's  Jahresber.,  13;  V.  Adriance 
and  J.  Adriance,  A  Clinical  Report  of  the  Chemical  Examination,  etc.,  Archives  of 
Pediatrics,  1897;  Camerer  and  Soldner,  Zeitschr.  f.  Biologie,  33  and  36.  In  regard 
to  the  composition  of  Woman's  milk,  see  also  Biel,  Maly's  Jahresber.,  4;  Christenn, 
ibid.,  7;  Mendes  de  Leon,  ibid.,  12;  Cerber,  Bull.  soc.  chim.,  23;  Tolmatscheff, 
Hoppe-Seyler'fl  Med. -chem.  Untersuch.,  272. 

Zeitschr.  f.  physiol.  Chem.,  65;  see  also  Engel  and  Frehn,  Maly's  Jahresber.,  40. 


HUMAN  MILK.  663 

ing  proteins  and  about  20  per  cent  for  rest  nitrogen.     The  principal  part 
of  the  rest  nitrogen  is  considered  as  urea. 

From  a  quantitative  standpoint,  the  most  essential  differences  between 
woman's  and  cow's  milk  are  the  following:  As  compared  with  the  quan- 
tity of  albumin,  the  quantity  of  casein  is  not  only  absolutely  but  also 
relatively  smaller  in  woman's  milk  than  in  cow's  milk,  while  the  latter  is 
poorer  in  milk-sugar.  Human  milk  is  richer  in  lecithin,  at  least  relatively 
to  the  amount  of  protein.  Burow  found  0.49-0.58  p.  m.  lecithin  in  cow's 
milk  and  0.58  p.  m.  in  woman's  milk,  which  corresponds  to  1.40  per  cent 
for  the  first  milk  and  3.05  per  cent  for  the  second,  calculated  on  the  per- 
centage of  protein.  Nerking  and  Haensel  found  as  average  for  lecithin 
in  cow's  milk  0.63  p.  m.  and  in  woman's  milk  0.50  p.  m.  Glikin  found 
0.765  p.  m.  lecithin  (phosphatides)  as  average  for  cow's  milk  and  1.329 
p.  m.  for  human  milk.  Koch  found  that  both  human  milk  and  cow's 
milk  contain  lecithin  as  well  as  cephalin.  The  total  quantity  of  both 
bodies  in  human  milk  was  0.78  p.  m.  and  in  cow's  milk  0.72-0.86  p.  m. 
The  quantity  of  nucleon  is  greater  in  woman's  milk.  Wittmaack  claims 
that  cow's  milk  contains  0.566  p.  m.  nucleon,  and  woman's  milk  1.24 
p.  m.,  and  according  to  Valenti  the  quantity  of  nucleon  in  human  milk 
is  indeed  still  higher.  Siegfried  finds  that  the  nucleon  phosphorus 
amounts  to  6.0  per  cent  of  the  total  phosphorus  in  cow's  milk  and  41.5 
per  cent  in  woman's  milk,  and  also  that  in  human  milk  the  phosphorus 
is  almost  all  in  organic  combination.  This  does  not  agree  with  the  results 
of  Sikes  who  found  on  an  average  of  only  42  per  cent  of  the  total  P2O5 
in  organic  combination.  Because  of  the  large  amount  of  casein  (and 
calcium  phosphate)  cow's  milk  is  much  richer  in  phosphorus  than  human 
milk.  The  relation  PoOs:N,  according  to  Schlossmann,1  is  equal  to 
1:5.4  in  human  milk  and  1:2.7  in  cow's  milk.  Woman's  milk  is  poorer 
in  mineral  bodies,  especially  lime,  and  it  contains  only  one-sixth  of  the 
quantity  of  lime  as  compared  with  cow's  milk.  The  mineral  constituents 
of  human  milk  are  better  assimilated  by  the  organism  of  the  nursing 
child  than  those  of  cow's  milk.  Human  milk  is  also  claimed  to  be  poorer 
in  citric  acid  (Scheibe2),  although  this  is  not  an  essential  difference. 

Another  difference  between  woman's  milk  and  other  varieties  of  milk  is 
Umikoff's  reaction,  which  seems  to  depend  upon  the  quantitative  composition, 
especially  the  relation  between  the  milk-sugar,  citric  acid,  lime,  and  iron  (Sieber  3). 
This  reaction  consists  in  treating  5  cc.  of  woman's  milk  with  2.5  cc.  ammonia 

1  Burow,  Zeitschr.  f.  physiol.  Chem.,  30;  Koch,  ibid.,  47;  Wittmaack,  ibid.,  22; 
Siegfried,  ibid.,  22;  Nerking  and  Haensel,  Bioch.  Zeitschr.,  13;  Glikin,  ibid.,  21;  Valenti, 
Biochem.  Centralbl.,  4;  Schlossmann,  Arch.  f.  Kinderheilkunde,  40;  Sikes,  Journ. 
of  Physiol.,  34. 

2  Maly's  Jahresber.,  21. 
'Zeitschr.  f.  physiol.  Chem.,  30. 


664  MILK. 

(10  per  cent)  and  heating  to  60°  C.  for  15  —20  minutes,  when  the  mixture  becomes 
violet-red.     Cow's  milk  gives  a  yellowish-brown  color  when  thus  treated. 

According  to  Rubner  woman's  milk  contains  about  3  p.  m.  soaps,  but  this 
could  not  be  substantiated  by  Camerer  and  Soldner.  They  conclude  that 
woman's  milk  contains  no  soaps,  or  at  least  only  very  small  amounts.  They  also 
found  the  quantity  of  urea  nitrogen  in  woman's  milk  to  be  0.11-0.12  p.  m., 
although  Schondorff  l  found  nearly  twice  this  amount,  namely,  0.23  p.  m. 

In  regard  to  the  quantity  of  mineral  bodies  in  woman's  milk  we  have 
the  analyses  of  several  investigators,  especially  of  Btjnge  (analyses  A 
and  B)  and  of  Soldner  and  Camerer  (analysis  C).2  Btjnge  analyzed  the 
milk  of  a  woman,  fourteen  days  after  delivery,  whose  diet  contained 
very  little  common  salt  for  four  days  previous  to  the  analysis  {A),  and  again 
three  days  later  after  a  daily  addition  of  30  grams  of  NaCl  to  the  food 
(B).     The  figures  are  in  1000  parts  of  the  milk- 

ABC 

K,0 0.780  0.703  0.884 

Na,0 0.232  0.257  0.357 

CaO 0.328  0.343  0.378 

MgO 0.064  0.065  0.053 

Fe>03 0.004  0.006  0.002 

P206 0.473  0.469  0.310 

CI 0.438  0.445  0.591 

The  relation  of  the  two  bodies  potassium  and  sodium  to  each  other 
may,  Btjnge  believes,  vary  considerably  (1.3-4.4  equivalents  of  potash 
to  1  of  soda).  By  the  addition  of  salt  to  the  food,  the  quantity  of 
sodium  and  chlorine  in  the  milk  increases,  while  the  quantity  of  potas- 
sium decreases.  De  Lange  found  more  Na  than  K  in  the  milk  at  the 
beginning  of  lactation.  Jolles  and  Friedjung  found  on  an  average 
5.9  milligrams  of  iron  per  liter  of  woman's  milk.  Camerer  and  Soldner  3 
find  about  the  same  amount,  namely,  10-20  milligrams  Fe203  =  3.5-7 
milligrams  iron  in  1000  grams  human  milk. 

The  gases  of  woman's  milk  have  been  investigated  by  Kulz  4  He 
found  1.07  -1.44  cc.  of  oxygen,  2.35-2.87  cc.  of  carbon  dioxide,  and  3.37- 
3.81  cc.  of  nitrogen  in  100  cc.  of  milk. 

The  proper  treatment  of  cow's  milk  by  diluting  it  with  water  and  by 
certain  additions  in  order  to  render  it  a  proper  substitute  for  woman's 
milk  in  the  nourishment  of  children  cannot  be  determined  before  the 
difference  in  the  protein  bodies  of  these  two  kinds  of  milk  has  been  com- 
pletely studied. 

Rubner,  Zeitschr.  f.  Biologie,  36;  Camerer  and  Soldner,  ibid.,  39;  Schondorff, 
Pfliiger's  Arch.,  81. 

2  Bunge,  Zeitschr.  f .  Biologie,  10;  Camerer  and  Soldner,  ibid.,  39  and  44. 

3  De  Lange,  Maly's  Jahresber.,  27;  Jolles  and  Friedjung,  Arch.  f.  exp.  Path.  u„ 
Pharm.,  4(5;  Camerer  and  Soldner,  Zeitschr.  f.  Biologie,  46. 

4  Zeitschr.  f.  Biologie,  32. 


HUMAN  COLOSTRUM.  GG5 

The  colostrum  has  a  higher  specific  gravity,  1.040-1.060,  a  greater 
quantity  of  coagulablo  proteins,  and  a  deeper  yellow  color  than  ordinary 
woman's  milk.  Even  a  few  days  after  delivery  the  color  becomes  Leefl 
yellow,  the  quantity  of  albumin  less,  and  the  Dumber  of  colostrum-cor- 
puscles diminishes. 

We  have  the  older  analyses  of  Clemm  1  and  the  recent  investigations 
of  Pfeiffer,  V.  and  J.  Adriance,  Camerer  and  Soldner  on  the  changes 
in  the  composition  of  milk  after  delivery.  It  follows,  as  a  unanimous 
result  from  these  investigations,  that  the  quantity  of  protein,  which 
amounts  to  more  the  first  two  days,  sometimes  to  more  than  30  p.  m. 
at  first,  rather  qu'ckly  and  then  more  generally  diminishes  as  long  as  the 
lactation  continues,  so  that  in  the  third  week  it  equals  about  10-18  p.  m. 
Like  the  protein  substances,  the  mineral  bodies  also  gradually  decrease. 
The  quantity  of  fat  shows  no  regular  or  constant  variation  during  lacta- 
tion, while  the  lactose,  especially  according  to  the  observations  of  V. 
and  J.  Adriance  (120  analyses),  increases  rather  quickly  the  first  days 
and  then  only  slowly  until  the  end  of  lactation.  The  analyses  of  Pfeiffer, 
(  amerer  and  Soldner  also  show  an  increase  in  the  quantity  of  milk-sugar. 

The  two  mammary  glands  of  the  same  woman  may  yield  somewhat  different 
milk,  as  shown  by  Sourdat  and  later  by  Brunner.2  Likewise  the  different 
portions  of  milk  from  the  same  milking  may  have  varying  composition.  The 
first  portions  are  always  poorer  in  fat. 

According  to  l'Heritier  and  to  Vernois  and  Becquerel,  the  milk  of  blondes 
contains  less  casein  than  that  of  brunettes,  a  difference  which  Tolmatscheff  3 
could  not  substantiate.  Women  of  delicate  constitutions  yield  a  milk  richer  in 
solids,  especially  in  casein,  than  women  with  strong  constitutions  (V.  and  B.). 

According  to  Vernois  and  Becquerel,  the  age  of  the  woman  has  an  effect  on 
the  composition  of  the  milk,  so  that  we  find  a  greater  quantity  of  proteins  and 
fat  in  women  15-20  years  old  and  a  smaller  quantity  of  sugar.  The  smallest 
quantity  of  proteins  and  the  greatest  quantity  of  sugar  are  found  at  20  or  from 
25  to  30  years  of  age.  Vernois  and  Becquerel,  consider  that  the  milk  with  the 
first-born  is  richer  in  water — with  a  proportionate  diminution  of  casein,  sugar, 
and  fat — than  after  several  deliveries. 

The  influence  of  menstruation  seems  to  diminish  slightly  the  milk-sugar  and 
to  increase  considerably  the  fat  and  casein  (Vernois  and  Becquerel). 

Witch's  milk  is  the  secretion  of  the  mammary  glands  of  new-born  children 
of  both  sexes  immediately  after  birth.  This  secretion  has  from  a  qualitative 
standpoint  the  same  constitution  as  milk,  but  may  show  important  differences  and 
variations  from  a  quantitative  point  of  view.  Schlossberger  and  Hauff, 
Gubler  and  Quevenne,  and  v.  Genser,4  have  made  analyses  of  this  milk  and 
give  the  following  results:  10.5-28  p.  m.  proteins,  8.2-14.6  p.  m.  fat,  and  9-60 
p.  m.  sugar. 

1  See  Hoppe-Seyler,  Physiol.  Chem.,  734. 

2  Sourdat,  Compt.  Rend.,  71;  Brunner,  Pfluger's  Arch.,  7. 

8  l'Heritier,  cited  from  Hoppe-Seyler,  Physiol.  Chem.,  738;  Vernois  and  Becquerel, 
Du  lait  chez  la  fen.n.e  dans  l'etat  de  sante,  etc.,  (Paris,  1853);  Tolmatscheff,  Hoppe- 
Seyler,  Med.-chem.  Untersuch.,  272. 

4  Schlossberger  and  Hauff,  Annal.  d.  Chem.,  u.  Pharm.,  96;  Gubler  and  Quevenne, 
cited  from  Hoppe-Seyler 's  Physiol.  Chem.,  723;  v.  Genser,  ibid. 


666  MILK. 

As  milk  is  the  only  form  of  nourishment  during  a  certain  period  of 
the  life  of  man  and  mammals,  it  must  contain  all  the  nutriment  necessary 
for  life.  This  fact  is  shown  by  the  milk  containing  representatives  of 
the  three  principal  groups  of  organic  nutritive  substances — proteins, 
carbohydrates,  and  fat,  and  the  last  two  groups  can  here  also  in  part 
mutually  substitute  each  other.  Besides  this  all  milk  seems  to  contain, 
without  doubt,  some  lecithin  and  nucleon.  The  mineral  bodies  in  milk 
must  also  occur  in  proper  proportions,  and  on  this  point  the  experiments 
of  Bunge  on  dogs  are  of  special  interest.  He  found  that  the  mineral 
bodies  of  the  milk  occur  in  about  the  same  relative  proportion  as  they 
do  in  the  body  of  the  sucking  animal.  Bunge  l  found  in  1000  parts  of 
the  ash  the  following  results  (A  represents  results  from  the  new-born 
dog,  and  B  the  milk  from  the  bitch) : 

A  B 

K,0 114.2  149.8 

Na20 106.4  88.0 

CaO 295.2  272.4 

MgO 18.2  15.4 

Fe203 7.2  1.2 

P„05 394.2  342.2 

CI 83.5  169.0 

Bunge  explains  the  fact  that  the  milk-ash  is  richer  in  potash  and 
poorer  in  soda  than  the  new-born  animal  by  saying  that  in  the  growing 
animal  the  ash  of  the  muscles  rich  in  potash  relatively  increases  and  the 
cartilage  rich  in  soda  relatively  decreases.  In  regard  to  the  amount 
of  iron  we  find  an  unexpected  condition,  the  ash  of  the  new-born  animal 
containing  six  times  as  much  as  the  milk-ash.  This  condition  Bunge 
explains  by  the  fact  founded  on  his  and  Zalesky's  experiments,  that  the 
quantity  of  iron  in  the  entire  organism  is  highest  at  birth.  The  new-born 
has  therefore  its  own  supply  of  iron  for  the  growth  of  its  organs  even  at 
birth. 

The  investigations  of  Hugounenq,  de  Lange,  Camerer  and  Soldner  2 
have  shown  that  in  man  the  conditions  are  different  from  those  in  animals, 
as  the  ash  of  the  child  has  an  entirely  different  composition  as  compared 
with  the  milk.  As  an  example  the  following  analyses  are  given  (of 
Camerer  and  Soldner).  (A,  the  ash  of  the  sucking  infant,  and  B,  the 
ash  of  the  milk.)     The  results  are  in  1000  parts  of  the  ash. 

A  B 

K20 78  314 

Na20 91  119 

CaO 361  164 

MgO 9  26 

FfcO, 8  6 

p.,Q5 389  135 

CI 77  200 

1  Zeitschr.  f.  physiol.  Chem.,  13. 

2  Hugounenq,  Compt.  Rend.,  128;  de  Lange,  Zeitschr.,  f.  Biologie,  40;  Camerer 
and  Soldner,  ibid.,  39,  40,  and  44. 


INFLUENCE  OF  THE  FOOD.  667 

We  cannot  therefore  state  as  a  definite  fact  that  the  composition  of 
the  ash  of  the  sucking  young  and  the  ash  of  the  corresponding  milk  coin- 
cide. Binge  l  nevertheless  claims  that  the  composition  of  the  ash  of 
the  sucking  young  of  various  mammals  is  nearly  the  same,  but  that  the 
ash  of  the  milk  differs  from  the  ash  of  the  young  in  bo  far  as  the  slower 
the  young  grows  the  richer  it  is  in  alkali  chlorides  and  relatively  poorer 
in  phosphates  and  lime-salts.  The  constituents  of  the  ash  have  two 
functions  to  perform,  namely,  the  building  up  of  the  tissues  and  secondly 
the  preparation  of  the  excreta,  especially  the  urine.  The  faster  the 
young  grows  the  more  is  the  first  in  evidence,  while  the  slower  it  develops, 
the  more  prominent  is  the  second. 

The  quantity  of  mineral  l;odies  in  the  milk,  and  especially  the  amount 
of  lime  and  phosphoric  acid,  as  shown  by  Bunge  and  Proscher  and 
Pages,  stands  in  close  relation  to  the  rapidity  of  growth,  because  the 
amount  of  these  mineral  constituents  in  the  milk  is  greater  in  animals 
which  grow  and  develop  quickly  than  in  those  which  grow  only  slowly. 
A  similar  relation  also  exists,  as  shown  by  the  researches  of  Proscher, 
and  especially  of  Abderhalden,2  between  the  quantity  of  protein  in 
the  mill:  and  the  rapidity  of  development  of  the  sucking  young.  The 
amount  of  protein  is  greater  in  the  milk  the  quicker  the  animal  develops. 

The  influence  of  the  food  on  the  composition  of  the  milk  is  of  interest 
from  many  points  of  view  and  has  been  the  subject  of  many  investigations. 
From  these  we  learn  that  in  human  beings  as  well  as  in  animals  an  insuffi- 
cient diet  decreases  the  quantity  of  milk  and  the  quantity  of  solids,  while 
abundant  food  increases  both.  From  the  observations  of  Decai^ne  '•' 
on  nursing  women  during  the  siege  of  Paris  in  1871,  the  amount  of  casein, 
fat,  sugar,  and  salts,  but  especially  the  fat,  was  found  to  decrease  with 
insufficient  food,  while  the  quantity  of  lactalbumin  was  found  to  be  some- 
what increased.  Food  rich  in  proteins  increases  the  quantity  of  milk, 
and  also  the  solids  contained,  especially  the  fat,  according  to  most 
reports.  The  quantity  of  sugar  in  woman's  milk  is  found  by  certain 
investigators  to  be  increased  after  food  rich  in  proteins,  while  others 
claim  it  is  diminished.  A  diet  rich  in  fat  may,  as  the  researches  of  Soxhlet 
and  many  others4  have  shown,  cause  a  marked  increase  in  the  fat  of 
the  milk  when  the  fat  partaken  is  in  a  readily  digestible  and  assimilable 
form.     The  presence  of  large  quantities  of  carbohydrates  in  the  food 


1  Bunge,  "  Die  zunehmende  (Tnfuhigkeit  der  Frauen  ihre  Kinder  zu  stillen,"  Miin- 
chen,  1900,  cited  by  Camerer,  Zeitschr.  f.  Biologie,  40. 

2  Proscher,  Zeitschr.   f.  physiol.  Chem.,  24;    Abderhalden,  il>id.,  27;  Pages.  Arch, 
de  Physiol.  (5),  7. 

3  Cited  from  Hoppe-Seyler,  1.  c,  739. 

4  See  Maly's  Jahresber.,  26.     See  also  Basch,  Ergebnisse  der  Physiologie,  2,  Abt.  1. 


668  MILK. 

seems  to  cause  do  constant,  direct  action  on  the  quantity  of  the  milk 
constituents.1  From  feeding  experiments  with  different  foods  we  come 
to  the  conclusion  that  the  character  of  the  food  is  of  comparatively  little 
influence,  while  the  race  and  other  conditions  play  an  important  role. 
Watery  food  gives  a  milk  containing  an  excess  of  water  and  having  little 
value.  In  the  milk  from  cows  which  were  fed  on  distillers'  grain  Com- 
maille2  found  906.5  p.  .m.  water,  26.4  p.  m.  casein,  4.3  p.  m.  albumin, 
18.2  p.  m.  fat,  and  33.8  p.  m.  sugar.  Such  milk  has  sometimes  a  peculiar 
sharp  after-taste,  although  not  always.  Tangl  and  Zaitschek  3  could 
not  find  any  difference  in  the  average  composition  of  the  milk  produced 
after  feeding  with  dry  and  with  moist  fodder. 

Chemistry  of  Milk-secretion.  That  the  constituents  which  occur 
actually  dissolved  in  milk  pass  into  the  secretion  and  not  alone  by  filtra- 
tion or  diffusion,  but  more  likely  are  secreted  by  a  specific  secretory 
activity  of  the  granular  elements,  is  shown  by  the  fact  that  milk-sugar, 
which  is  not  found  in  the  blood,  is  to  all  appearances  formed  in  the  glands 
themselves.  A  further  proof  lies  in  the  fact  that  the  lactalbumin  is  not 
identical  with  seralbumin;  and  lastly,  as  Bunge4  has  shown,  the  mineral 
bodies  secreted  by  the  milk  are  in  quite  different  proportions  from  those 
in  the  blood-serum. 

Little  is  known  in  regard  to  the  formation  and  secretion  of  the  specific 
constituents  of  milk.  The  older  theory,  that  the  casein  was  produced 
from  the  lactalbumin  by  the  action  of  an  enzyme,  is  incorrect,  and  prob- 
ably originated  from  mistaking  an  alkali  albuminate  for  casein.  Better 
founded  is  the  theory  that  the  casein  originates  from  the  protoplasm 
of  the  gland-cells.  According  to  Basch's  researches,  the  casein  is  formed 
in  the  mammary  gland  by  the  nucleic  acid  of  the  nucleus  being  set 
free  and  uniting  intra-alveolar  with  the  transudated  serum,  thus  form- 
ing a  nucleoalbumin,  the  casein.  The  untenableness  of  this  view  has 
been  shown  by  Lobisch,  and  the  investigations,  of  Hildebrandt  5 
upon  the  proteolytic  enzyme  of  the  mammary  gland,  and  the  autolysis 

1  In  regard  to  the  literature  on  the  action  of  various  foods  on  woman's  milk,  see 
Zalesky,  "  Ueber  die  Einwirkung  der  Nahrung  auf  die  Zusammensetzung  und  Nahr- 
haftigkeit  der  Frauenmilch,"  Berlin,  klin.  Wochenschr.,  1888,  which  also  contains  the 
literature  on  the  importance  of  diet  on  the  composition  of  other  kinds  of  milk.  In 
regard  to  the  extensive  literature  on  the  influence  of  various  foods  on  the  milk  pro- 
duction of  animals,  see  Konig,  Chem.  d.  menschl.  Nahrungs  und  Genussmittel.  3.  Aufl., 
1.  298.  See  also  Maly's  Jahresber.,  29-40,  and  Morgen,  Beger  and  Fingerling,  Landw. 
Versuchsst.,  61,  and  Raudnitz,  Monatschr.  f.  Kinderheilk. 

'Cited  from  Konig,  2,  235. 

•  Landwirt.  Vers.  .St.  1911. 

*  Lehrbuch  d.  physiol.  und  pathol.  Chem.,  3.  Aufl.,  93. 

■  Basch,  Jahrb.  f.  Kinderheilkunde,  1898;  Hildebrandt,  Hofmeister'a  Beitrage,  5; 
Lobisch.  ibid.,  8. 


CHEMISTRY  OF  MILK-SECRETION.  669 

of  the  gland  have  not  given  any  clue  as  to  the  mode  of  formation  of 
casein.  The  findings  of  Mandbl  l  that  the  hydrolytic  cleavage  products 
of  the  nucleoprotein  from  the  mammary  glands  occur  approximately 
quantitatively  in  the  same  proportions  as  in  casein,  are  important  in  this 
connection. 

That  the  milk-fat  is  produced  by  a  formation  of  fat  in  the  protoplasm, 
and  that  the  fat-globules  are  set  free  by  their  destruction,  is  a  generally 
admitted  opinion,  which,  however,  does  not  exelude  the  possibility  that 
the  fat  is  in  part  taken  up  by  the  glands  from  the  blood  and  eliminated 
with  its  secretion.  That  the  fats  of  the  food  can  pass  into  the  milk 
follows  from  the  investigations  of  Winternitz,  as  he  has  been  able  to 
detect  the  passage  of  iodized  fats  in  the  milk,  and  these  observations 
have  been  substantiated  by  the  investigations  of  Caspari  and  Parascht- 
schuk.2 The  abundant  quantities  of  iodized  fat  which  were  eliminated 
with  the  milk  in  these  cases  without  doubt  depend,  at  least  in  great  part, 
upon  the  iodized  fat  of  the  food,  hence  it  cannot  be  said  that  all  of  the 
milk-fat  containing  iodine  was  unchanged  iodized  fat  of  the  food.  The 
previously-mentioned  older  investigations  of  Lebedeff  and  Rosenfeld 
and  also  the  recent  ones  of  Spampani  and  Daddi,  Paraschtschuk,  Gogi- 
tidse  and  others  on  the  passage  of  foreign  fats  into  the  milk  also  indicate 
the  passage  of  the  fat  of  the  food  into  the  milk,  although  we  are  still  uncer- 
tain on  this  point.  According  to  Soxhlet  the  fat  of  the  food  does  not 
pass  into  the  milk  directly,  but  is  destroyed  in  place  of  the  body-fat, 
which  then  becomes  available  and  is,  as  it  were,  pushed  into  the  milk. 
Hexriques  and  Hansen  could  not  detect  any  mentionable  quantity  of 
linseed-oil  in  the  milk  after  feeding  with  this  oil;  the  milk-fat  was  not 
normal,  but  had  a  higher  iodine  equivalent  and  a  higher  melting-point, 
from  which  they  also  concluded  that  a  transformation  of  the  food-fat 
in  the  glandular  cells  is  possible.  The  results  of  the  experiments  of 
Gogitidse  3  with  soaps  also  indicate  that  the  mammary  glands  have  the 
property  of  forming  fats  by  synthesis  from  their  components.  As  a 
formation  of  fat  from  carbohydrates  in  the  animal  organism  is  at  the 
present  day  considered  as  positively  proved,  it  is  likewise  possible  that 
the  milk-glands  also  produce  fats  from  the  carbohydrates  brought  to 
them  by  the  blood.  It  is  a  well-known  fact  that  an  animal  gives  off 
for  a  long  time,  daily,  considerably  more  fat  in  the  milk  than  it  receives 

1  Bioch.  Zeitschr.,  22. 

:  Winternitz,  Zeitschr.  f.  physiol.  Chem.,  24;  Caspari,  Arch.  f.  (Anat.  u.)  Physiol., 
1899,  Supplbd.  and  Zeitschr.  f.  Biologie, -46;  with  Winternitz,  ibid.,  49;  Paraschtschuk, 
Chem.  Centralbl.,  1903,  1. 

3  Lebedeff,  Pfluger's  Arch.  31;  Rosenfeld,  Ergebn.  d.  Physio!.  1  and  2;  Spampani 
and  Daddi,  Maly's  Jahresber.,  26;  Hennques  and  Hansen,  ibid.,  29;  Gogitidse,  Zeitschr. 
f.  Biologie,  45,  46,  and  47.     See  also  Basch,  Ergebnisse  d.  Physiol.,  2,  Abt.  1. 


670  MILK. 

as  food,  and  this  proves  that  at  least  a  part  of  the  fat  secreted  by  the 
milk  is  produced  from  proteins  or  carbohydrates,  or  perhaps  from  both. 
The  question  as  to  how  far  this  fat  is  produced  directly  in  the  milk- 
glands,  or  from  other  organs  and  tissues,  and  brought  to  the  gland  by 
means  of  the  blood,  cannot  be  decided. 

The  origin  of  milk-sugar  is  not  known.  Muntz  calls  attention  to  the 
fact  that  a  number  of  very  widely  diffused  bodies  in  the  vegetable  king- 
dom— vegetable  mucilage,  gums,  pectin  bodies — yield  galactose  as  a 
product  of  decomposition,  and  he  believes,  therefore,  that  milk-sugar 
may  be  formed  in  herbivora  by  a  synthesis  from  glucose  and  galactose. 
This  origin  of  milk-sugar  does  not  apply  to  carnivora,  as  they  produce 
milk-sugar  when  fed  on  food  consisting  entirely  of  lean  meat.  The 
observations  of  Bert  and  Thierfelder  *  that  a  mother-substance  of 
the  milk-sugar,  a  saccharogen,  occurs  in  the  glands,  does  not  explain 
the  formation  of  milk-sugar,  as  the  nature  of  this  mother-substance 
is  still  unknown.  As  the  animal  body  has  undoubtedly  the  power  of 
converting  one  variety  of  sugar  into  another,  the  origin  of  the  milk- 
sugar  can  be  sought  simply  in  the  glucose  introduced  as  food  or  formed 
in  the  body.  Certain  observations  of  Porcher  indicate  such  an  origin 
as  he  found  in  sheep,  cows,  and  goats  whose  mammary  glands  "were 
extirpated,  that  glucose  appeared  in  the  urine  after  delivery.  He  also 
found  that  milk  secreting  animals  became  glycosuric  on  the  removal 
of  the  mammary  glands,  and  he  explains  this  glycosuria  by  the  fact  that 
the  lactose-forming  action  of  the  gland  was  removed  at  the  time  of  delivery, 
when  large  amounts  of  glucose  were  being  produced.  The  experiments 
of  Kaufmann  and  Magne  upon  cows  also  indicate  a  formation  of  lactose 
from  glucose.  They  found  that  during  secretion  the  glands  took  sugar 
from  the  blood,  so  that  the  venous  gland-blood  was  poorer  in  sugar  than 
otherwise.  Noel-Paton  and  Cathcart  2  have  carried  on  experiments 
on   phlorhinized   dogs   which   show   a   lactose   formation   from   glucose. 

The  passage  of  foreign  substances  into  the  milk  stands  in  close  connec- 
tion with  the  chemical  processes  of  milk  secretion. 

It  is  a  well-known  fact  that  milk  acquires  a  foreign  taste  from  the 
food  of  the  animal,  which  is  in  itself  a  proof  that  foreign  bodies  pass  into 
the  milk.  This  fact  becomes  of  special  importance  in  reference  to  such 
injurious  substances  as  may  be  introduced  into  the  organism  of  the  nurs- 
ing child  by  means  of  the  milk. 

Among  these  substances  may  be  mentioned  opium  and  morphine, 
which  after  large  doses  pass  into  the  milk  and  act  on  the  child.     Alcohol 


1  Muntz,  Compt.  Rend.,  102;  Bert  and  Thierfelder,  footnote  1,  p.  644. 

2  Porcher,  Compt.   Rend.   138  and  141  and  Bioch.  Zeitschr.,  23;  Kaufmann  and 
Magne,  Compt.  Rend.,  143;  Noel-Paton  and  Cathcart,  Journ.  of  Physiol.,  42. 


MILK  IN  DISEASES.  671 

may  also  pass  into  the  milk,  but  probably  not  in  such  quantities  as  to 
have  any  direct  action  on  the  nursing  child.1  Alcohol  is  claimed  to  have 
been  detected  in  the  milk  after  feeding  cows  with  brewer's  grains. 

Among  inorganic  bodies,  iodine,  arsenic,  bismuth,  antimony,  zinc, 
lead,  mercury,  and  iron  have  been  found  in  milk.  In  icterus  neither 
bile-acids  nor  bile-pigments  pass  into  the  milk. 

Under  diseased  conditions  no  constant  change  has  been  found  in  woman's 
milk.  In  isolated  cases  Schlossberger,  Joly  and  Filhol  -  have  indeed  observed 
a  markedly  abnormal  composition,  but  no  positive  conclusion  can  be  derived 
therefrom. 

The  changes  in  cow's  milk  in  disease  have  been  little  studied.  In  tuber- 
culosis of  the  udder,  Storch  3  found  tubercle  bacilli  in  the  milk,  and  he  also  noted 
that  the  milk  became  more  and  more  diluted,  during  the  disease,  with  a  serous 
liquid  similar  to  blood-serum,  so  that  that  the  glands  finally,  instead  of  yielding 
milk,  gave  only  blood-serum  or  a  serous  fluid.  Husson  4  found  that  milk  from 
murrain  cows  contained  more  proteins  but  considerably  less  fat  and  (in  severe 
cases)  less  sugar  than  normal  milk. 

The  milk  may  be  blue  or  red  in  color,  due  to  the  development  of  micro-organisms. 

The  formation  of  concrements  in  the  exit-passages  of  the  cow's  udder  is  often 
observed.  These  consist  chiefly  of  calcium  carbonate,  or  of  carbonate  and  phos- 
phate with  only  a  small  amount  of  organic  substances. 

xSee   Klingemann,   Virehow's  Arch.,    126,   and   Rosemann,    Pfliiger's  Arch.,  78. 
*  Schlossberger,    Annal.  d.  Chem.  u.  Pharm.,    96;    Joly  and  Filhol,  cited  from  v. 
Gorup-Besanez,  Lehrb.,  4,  Aufl.,  438. 

3  See  Bang,  Om  Tuberkulose  i  Koens  Yver  og  om  tuberkulos  Malk,  Xord.  Med. 
Arkiv,  16,  and  also  Maly's  Jahresber.,  14,  170;  Storch,  Maly's  Jahresber.,  14. 

4  Compt.  Rend.,  73. 


CHAPTER  XIV. 
URINE. 

Urine  is  the  most  important  excretion  of  the  animal  organism;  it 
is  the  means  of  eliminating  the  nitrogenous  metabolic  products,  also 
the  water  and  the  soluble  mineral  substances;  and  in  many  cases  it 
furnishes  important  data  relative  to  the  metabolism,  quantitatively 
by  its  variation,  and  qualitatively  by  the  appearance  of  foreign  bodies 
in  the  excretion.  Moreover,  in  many  cases  we  are  able,  from  the  chemical 
or  morphological  constituents  which  the  urine  abstracts  from  the  kidneys, 
ureter,  bladder,  and  urethra,  to  judge  of  the  condition  of  these  organs;  and 
lastly  urinary  analysis  affords  an  excellent  means  of  deciding  the  question 
as  to  how  certain  medicinal  agents  or  other  foreign  substances  intro- 
duced into  the  organism  are  absorbed  and  chemically  changed.  In  this 
respect,  urinary  analysis  has  furnished  very  important  particulars  especially 
in  regard  to  the  nature  of  the  chemical  processes  taking  place  within 
the  organism,  and  it  is  therefore  not  only  an  important  aid  to  the 
physician  in  diagnosis,  but  it  is  also  of  the  greatest  importance  to  the 
toxicologist  and  the  physiological  chemist. 

In  studying  the  secretions  and  excretions,  the  relation  must  be 
sought  between  the  chemical  structure  of  the  secreting  organ  and  the 
chemical  composition  of  its  secreted  products.  Investigations  with 
respect  to  the  kidneys  and  the  urine  have  led  to  very  few  results  from 
this  standpoint.  Although  the  anatomical  relation  of  the  kidneys  has 
been  carefully  studied,  their  chemical  composition  has  not  been  the  sub- 
ject of  thorough  analytical  research.  In  cases  in  which  a  chemical 
investigation  of  the  kidneys  has  been  undertaken,  it  has  been  in  general 
only  of  the  organ  as  such,  and  not  of  the  different  anatomical  parts. 
An  enumeration  of  the  chemical  constituents  of  the  kidneys  known  at 
the  present  time  can,  therefore,  only  have  a  secondary  value. 

In  the  kidneys  we  find  proteins  of  different  kinds.  According  to 
Halliburton  the  kidneys  do  not  contain  any  albumin,  but  only  a 
globulin  and  a  nucleoprotein.  The  globulin  coagulates  at  about  52°  C, 
and  the  nucleoprotein  contains  0.37  per  cent  phosphorus.  Lieber- 
mann  claims  that  the  kidneys  contain  a  lecithalbwnin,  and  he  ascribes 
to  this  body  a  special  importance  in  the  secretion  of  acid  urines.     The 

672 


THE  KIDNEYS.  673 

kidneys  also  contain,  according  to  Lonnberg,  a  mucin-like  substance. 
This  substance  yields  no  reducing  body  on  boiling  with  acids,  and  belongs 
chiefly  to  the  papillae,  and  is,  this  author  says,  a  nucleoalbumin 
(nucleoproteid?).  The  cortical  substance  is  richer  in  another  nucleoal- 
bumin (nucleoproteid)  unlike  mucin.  It  has  not  been  decided  what 
relation  this  last  substance  bears  to  Halliburton's  nucleoprotein. 
Chondroitin  sulphuric  acid  also  occurs  as  traces.  Mandel  and  Levenb 
have  also  obtained  glncothionic  acid  from  the  kidneys,  and  the  question 
as  to  the  relation  of  this  to  the  renosulphuric  acid  described  by  Mandel 
and  Neuberg  l  is  still  undecided.  This  renosulphuric  acid  to  all  appear- 
ances is  not  a  unit  substance  but  a  sulphuric  acid  ester,  and  a  com- 
ponent related  to  glucuronic  acid  which  contained  2.63  p.  c.  S.,  4.53 
p.  c,  N.,  and  1.34  p.  c.  P. 

Fat  occurs  only  in  very  small  amounts  and  this  fat,  like  the  organ 
fat  in  general,  is  relatively  rich  in  unsaturated  fatty  acids.  The  phos- 
phatides seem  to  be  of  different  kinds.  Frankel  and  Nogueira  2  found 
a  cephalin-like  substance,  a  triaminodiphosphatide  and  a  diamino- 
monophosphatide.  Dunham  and  Jacobson  3  found  in  beef-kidneys  a 
substance  which  they  called  carnaubon  which  is  soluble  in  alcohol  but 
insoluble  in  ether,  and  which  is  a  triaminomonophosphatide  with  the 
formula  C74H150N3PO13.  Carnaubon  does  not  contain  any  glycerin 
but  an  amino-sugar,  two  choline  groups  and  a  molecule  of  each  of  the 
following  acids:  stearic,  palmitic  and  carnaubic  (C24H4SO2)  acids.  Among 
the  extractive  bodies  of  the  kidneys  one  finds  purine  bases,  betaine,4  urea, 
uric  acid  (traces),  glycogen,  leucine,  inosite,  taurine,  and  cystine  (in  ox- 
kidneys).  The  quantitative  analyses  of  the  kidneys  thus  far  made 
possess  little  interest.  In  the  kidney  of  a  healthy  suicide  Magnus- 
Levy  5  found  in  1000  parts  of  the  fresh  substance  756  p.  m.  water,  244 
p.  m.  solids,  52.7  p.  m.  fat,  2.08  p.  m.  CI.,  0.192  p.  m.  Ca.,  0.207  p.  m. 
Mg  and  0.158  p.  m.  Fe. 

The  fluid  collected  under  pathological  conditions,  as  in  hydronephrosis,  is 
thin  with  a  variable  but  generally  low  specific  gravity.  Usually  it  is  straw-yellow 
or  paler  in  color,  and  sometimes  colorless.  Most  frequently  it  is  clear,  or  only 
faintly  cloudy  from  white  blood-corpuscles  and  epithelium-cells;  in  a  few  cases 
it  is  so  rich  in  form-elements  that  it  appears  like  pus.  Protein  generally  occurs 
in  small  amounts;   occasionally  it  is  entirely  absent,  but  in  a  few  rare  cases  the 

1  Halliburton,  Journ.  of  Physiol.,  13,  Suppl.,  and  18;  Liebermann,  Pfliiger's  Arch., 
50  and  54;  Lonnberg,  see  Maly's  Jahresber.,  20;  Mandel  and  Levene,  Zeithschr.  f. 
physiol.  Chem.,  47;  Mandel  and  Neuberg,  Bioch.  Zeitschr.,  13;  Morner,  Skand. 
Arch.  f.  Physiol.,  6. 

2  Bioch.  Zeitschr.,  16. 

3  Zeitschr.  f.  physiol.  Chem.,  64. 

4  Bebeschin,  Zeitschr.,  f.  physiol.  Chem.,  72. 

5  Bioch.  Zeitschr.,  24. 


674  URINE.    . 

amount  is  nearly  as  large  as  in  the  blood-serum.  Urea  occurs  sometimes  in 
considerable  amounts  when  the  parenchyma  of  the  kidneys  is  only  in  part  atro- 
phied; in  complete  atrophy  the  urea  may  be  entirely  absent. 

I.    PHYSICAL  PROPERTIES  OF  URINE. 

Consistency,  Transparency,  Odor,  and  Taste  of  Urine.  Under 
physiological  conditions  urine  is  a  thin  liquid  and  gives,  when  shaken 
with  air,  a  froth  which  quickly  subsides.  Human  urine,  or  urine  from 
carnivora,  which  is  habitually  acid,  appears  clear  and  transparent,  often 
faintly  fluorescent,  immediately  after  voiding.  When  allowed  to  stand  for 
a  little  while  human  urine  shows  a  light  cloud  (nubecula),  which  consists  of 
the  so-called  "  mucus,"  and  generally  also  contains  a  few  epithelium 
cells,  mucus-corpuscles,  and  urate-granules.  The  presence  of  a  larger 
quantity  of  urates  renders  the  urine  cloudy,  and  a  clay-yellow,  yellowish- 
brown,  rose-colored,  or  often  brick-red  precipitate  (sedimentum  lateri- 
tium)  settles  on  cooling,  because  of  the  greater  insolubility  of  the  urates 
at  the  ordinary  temperature  than  at  the  temperature  of  the  body. 
This  cloudiness  disappears  on  gently  warming.  In  new-born  infants 
the  cloudiness  of  the  urine  during  the  first  4-5  days  is  due  to  epithelium, 
mucus-corpuscles,  uric  acid,  and  urates.  The  urine  of  herbivora,  which 
is  habitually  neutral  or  alkaline  in  reaction,  is  very  cloudy  on  account 
of  the  carbonates  of  the  alkaline  earths  present.  Human  urine  may 
sometimes  be  alkaline  under  physiological  conditions.  In  this  case  it 
is  cloudy,  due  to  the  earthy  phosphates,  and  this  cloudiness  does  not 
disappear  on  warming,  differing  in  this  respect  from  the  sedimentum 
lateritium.  Urine  has  a  salty  and  faintly  bitter  taste  produced  by  sodium 
chloride  and  urea.  The  odor  of  urine  is  peculiarly  aromatic;  the  bodies 
which  produce  this  odor  are  unknown. 

The  color  of  urine  is  normally  pale  yellow  when  the  specific  gravity 
is  1.020.  The  color  otherwise  depends  on  the  concentration  of  the  urine 
and  varies  from  pale  straw-yellow,  when  the  urine  contains  small  amounts 
of  solids,  to  a  dark  reddish-yellow  or  reddish-brown  in  stronger  con- 
centration. As  a  rule  the  intensity  of  the  color  corresponds  to  the  con- 
centration, but  under  pathological  conditions,  exceptions  occur  such  as 
are  found  in  diabetic  urine,  which  contains  a  large  amount  of  solids  and 
has  a  high  specific  gravity  and  a  pale-yellow  color. 

The  reaction  of  urine  depends  essentially  upon  the  composition  of  the 
food.  The  carnivora,  as  a  rule,  void  an  acid,  the  herbivora,  a  neutral 
or  alkaline  urine.  If  a  carnivore  is  put  upon  a  vegetable  diet,  its  urine 
may  become  less  acid  or  neutral,  while  the  reverse  occurs  when  an  herbi- 
vore is  starved,  that  is,  when  it  lives  upon  its  own  tissues,  as  then  the 
urine  voided  is  acid. 

The  urine  of  a  healthy  man  on  a  mixed  diet  has  an  acid  reaction, 


PHYSICAL  PROPERTIES  OF  THE  URINE.  G75 

and  the  sum  of  the  acid  equivalents  is  greater  than  the  sum  of  the  basic 
equivalents.  This  depends  upon  the  fact  that  in  the  physiological 
combustion  of  neutral  substances  (proteins  and  others)  within  the 
organism,  acids  are  produced,  chiefly  sulphuric  acid,  hut  also  phosphoric 
and  organic  acids,  such  as  hippuric,  uric,  and  oxalic  acids,  aromatic 
oxyacids,  oxyproteic  acids  and  others.  From  this  it  follows  that  the 
acid  reaction  is  not  due  to  one  acid  alone.  The  various  acids  take  part 
in  the  acid  reaction  in  proportion  to  their  dissociation,  since,  according 
to  the  ion  theory,  the  acid  reaction  of  a  mixture  is  dependent  upon  the 
number  of  hydrogen  ions  present.  Hence  the  theory  that  the  acidity 
is  due  entirely  to  dihydrogen  phosphate  is  incorrect  although  this  salt 
takes  such  a  great  part  in  the  acid  reaction  that  its  quantity  is  often 
taken  as  a  measure  of  the  degree  of  acidity  of  the  urine.1 

The  composition  of  the  food  is  not  the  only  influence  which  affects  the  degree 
of  acidity  of  human  urine.  For  example,  after  taking  food  at  the  beginning  of 
digestion,  when  a  larger  amount  of  gastric  juice  containing  hydrochloric  acid 
is  secreted,  the  urine  may  be  neutral  or  even  alkaline.2  As  to  the  time  of  the 
appearance  of  the  maximum  and  minimum  of  acidity,  the  various  investigators 
do  not  agree,  which  may  in  part  be  explained  by  the  varying  individuality  and 
conditions  of  life  of  the  persons  investigated.  It  has  not  infrequently  been 
observed  that  perfectly  healthy  persons  in  the  morning  void  a  neutral  or  alkaline 
urine  which  is  cloudy  from  earthy  phosphates.  The  effect  of  muscular  activity 
on  the  acidity  of  urine  has  not  been  positively  determined.  According  to  Hoff- 
mann, Ringstedt,  Oddi,  and  Tarulli  and  Vozarik  muscular  work  raises  the 
degree  of  acidity,  but  Aducco  3  claims  that  it  decreases  it.  Abundant  perspira- 
tion reduces  the  acidity  (Hoffmann). 

In  man  and  especially  in  carnivora  it  seems  that  the  degree  of  acidity 
of  the  urine  cannot  be  increased  above  a  certain  point,  even  though 
mineral  acids  or  organic  acids  which  are  burned  up  with  difficulty  are 
ingested  in  large  quantities.  Under  such  conditions  a  different  behavior 
has  been  repeatedly  observed  between  carnivora  and  herbivora.  In  the 
first  (and  also  in  man)  it  has  been  found  that  the  acids  are  in  part  neu- 
tralized by  the  alkalies  and  alkaline  earths  of  the  body,  but  that  the 
excess  of  acid  is  combined  with  ammonia,  split  off  from  the  proteins  or 
their  cleavage  products,  and  eliminated  in  the  urine  as  ammonium  salt. 
In  herbivora  such  a  combination  of  the  excess  of  acid  with  ammonia 


1  In  regard  to  the  acidity  of  the  urine  see  the  recent  works  of  Ringer,  Zeitschr.  f. 
physiol.  Chem.  60;  Henderson,  Bioch.  Zeitschr.  24,  with  Spiro,  ibid.,  15;  De  Jager, 
Maly's  Jahresb.  39  and  Bioch.  Zeitschr.  38;  v.  Skramlik,  Zeitschr.  f.  physiol.  Chem. 
71;  Klein  and  Moritz,  Deutsch.  Arch.  f.  klin.  Med.  99;  Quagliariello,  Chem.  Cen- 
tralbl.  1912,  1,  506. 

2  Contradictory  statements  are  found  in  Linossier,  Maly's  Jahresber.,  27. 

3  Hoffmann,  see  Maly's  Jahresber.,  14;  Ringstedt,  ibid.,  20;  Oddi  and  Tarulli, 
ibid.,  24;  Aducco,  ibid.,  17;  Vozarik,  Pfliiger's  Arch.,  111. 


676  URINE. 

seems  not  to  take  place,  or  not  to  the  same  extent,1  and  this  is  given  as 
a  reason  why  herbivora  soon  die  when  acids  are  given.  This  is  true  at 
least  for  rabbits,  while  according  to  Baer  this  power  of  increasing  the 
elimination  of  ammonia  exists  also  in  the  goat,  monkey,  and  pig,  hence 
no  definite  difference  in  this  regard  exists  between  herbivora  and  carnivora. 
The  differences  which  have  been  observed  are,  according  to  Eppinger, 
not  of  a  special  kind,  and  they  may  be  caused,  he  says,  from  a  different 
amount  of  protein  in  the  food  which  yields  ammonia.  Thus  dogs  with 
food  poor  in  protein  behave  like  rabbits  while,  according  to  Eppinger, 
in  herbivora  (rabbits)  a  de-toxification  of  the  acid  can  be  brought  about 
by  the  abundant  supply  of  proteins  or  their  cleavage  products.  The 
correctness  of  this  statement  is  still  disputed  (Pohl)  or  has  only  been 
partly  confirmed  (Bostock).  The  point  is  disputed  and  it  must  not  be 
forgotten  that,  as  A.  Loewy2  found,  the  sensitiveness  toward  the  action 
of  acids  varies  very  much  in  different  individuals. 

Although  one  cannot  raise  the  degree  of  acidity  of  the  urine  above  a 
certain  limit  by  the  introduction  of  acid,  still  it  may  be  easily  diminished, 
so  that  the  reaction  becomes  neutral  or  alkaline.  This  occurs  after  the 
taking  of  carbonates  of  the  fixed  alkalies  or  of  such  alkali  salts  of  vege- 
table acids — citric  acid,  and  malic  acid — as  are  easily  burned  into  car- 
bonates in  the  organism.  Under  pathological  conditions,  as  in  the 
absorption  of  alkaline  transudates,  or  the  alkaline  fermentation  within 
the  bladder,  the  urine  may  become  alkaline. 

A  urine  with  an  alkaline  reaction  caused  by  fixed  alkalies  has  a  very 
different  diagnostic  value  from  one  whose  alkaline  reaction  is  caused  by 
the  presence  of  ammonium  carbonate.  In  the  latter  case  we  have  to 
deal  with  a  decomposition  of  the  urea  of  the  urine  by  the  action  of  micro- 
organisms. 

If  one  wishes  to  determine  whether  the  alkaline  reaction  of  the  urine 
is  due  to  ammonia  or  to  fixed  alkalies,  a  piece  of  red  litmus  paper  is  dipped 
into  the  urine  and  allowed  to  dry  exposed  to  the  air  or  to  a  gentle  heat. 
If  the  alkaline  reaction  is  due  to  ammonia,  the  paper  becomes  red  again; 
but  if  it  is  caused  by  fixed  alkalies,  it  remains  blue. 

Determination  of  the  Acidity.  As  the  quantity  of  phosphoric  acid 
present  as  dihydrogen  salt,  as  above  stated,  cannot  be  used  as  a  measure 
of  the  acidity,  none  of  the  older  methods  suggested  for  the  estimation 
of  this  portion  of  the  phosphoric  acid  is  suited  for  acidity  determinations. 

1  See  Winterberg,  Zeitschr.  f.  phyaiol.  Chem.,  25,  and  J.  Baer,  Arch.  f.  exp.  Path.  u. 
Pharm.,  54. 

2  Eppinger,  Zeitschr.  f.  exp.  Path.  u.  Therap.,  3;  with  Tedesko,  Bioch.  Zeitschr., 
16;  Pohl.  ibid.,  18;  Staal,  Zeitschr.  f.  physiol.  Chem.,  58;  Bostock,  ibid.,  84;  A. 
Loewy,  Centralbl.  f.  Physiol.,  20;  337. 


ACIDITY  OF  THE   URINE.  677 

We  now  determine  the  acidity  simply  by  acidimetric  methods,  titrat- 
ing with  N/10  caustic  alkali,  using  phenolphthalein  as  an  indicator 
(Naegeli,  Hober,  Folin).  On  account  of  the  color  of  the  urine  and  the 
presence  of  ammonium  salts  and  alkaline  earths,  this  method  cannot 
yield  entirely  exact  results.  The  greatest  error  is  due  to  the  alkaline 
earths,  which,  on  titration  with  caustic  alkali,  precipitate  as  earthy 
phosphates  in  variable  amounts  and  of  variable  composition.  This 
error  can  be  prevented,  according  to  Folin,  by  the  addition  of  neutral 
potassium  oxalate,  which  precipitates  the  lime,  and  in  this  way  the  dis- 
turbing action  of  the  ammonium  salts  is  also  inhibited.  Perfectly 
accurate  results  are  not  obtained  by  this  method,  but  it  is  the  best  of 
those  which  have  been  suggested. 

It  is  performed  as  follows:  25  cc.  of  urine  are  placed  in  an  Erlenmeyer 
flask  (about  200  cc.  capacity),  treated  with  1-2  drops  of  |-per  cent 
phenolphthalein  solution,  and  shaken  with  15-20  grams  of  powdered 
potassium  oxalate  and  immediately  titrated  with  N/10  caustic  soda 
with  constant  shaking  until  a  pronounced  pale-rose  color  appears. 
Vozarik  1  titrates  the  diluted  urine  without  the  addition  of  oxalate 
and  uses  phenolphthalein  as  indicator. 

The  acidity,  as  determined  by  titration,  varies  considerably  under 
physiological  conditions,  but  calculated  as  hydrochloric  acid  it  amounts 
in  man  to  about  1.5-2.3  grams  in  the  twenty-four  hours. 

By  titration  we  learn  the  amount  of  hydrogen  present  which  can 
be  substituted  by  a  metal,  i.e.,  the  acidity  in  the  ordinary  older  sense, 
but  not  the  true  acidity,  the  ion  acidity,  which  is  given  by  the  concentra- 
tion of  the  hydrogen  ions  of  the  urine.  For  similar  reasons,  as  previously 
indicated  in  treating  of  the  alkalinity  of  the  blood-serum  (page  272), 
the  ion  acidity  cannot  be  determined  by  titration,  while  it  can  be  deter- 
mined according  to  the  principle  of  the  electrometric  gas-chain  method 
as  there  given.  Such  estimations  have  been  made  by  v.  Rhorer  and  by 
Hober.  For  normal  urine  v.  Rhorer  found  as  a  minimum  4X10-7, 
as  a  maximum  76X10-7,  and  as  an  average  30X10-7.  Hober  found 
4.7X10"7,  100X10"7,  and  49X10"7,  respectively.  On  an  average  the 
urine  therefore  contains  30-50  grams  of  hydrogen  ions  in  10  million 
liters.  Henderson2  has  obtained  much  lower  values,  namely  10.10 ~7 
as  the  average  of  50  investigations,  and  has  rather  great  differences 
for  different  persons.     From  the  comparative  estimation  of  the  titration 

1  In  regard  to  the  degree  of  acidity  and  its  estimation  see  Naegeli,  Zeitschr.  f . 
physiol.  Chem.,  30;  Hober,  Hofmeister's  Beitrage,  3;  Folin,  Amer.  Journ.  of  Physiol., 
9;  Vozarik,  1.  c;  de  Jager,  Zeitschr.  f.  physiol.  Chem.,  55;  and  Ringer,  ibid.,  60;  Grim- 
bert  and  Morel,  Compt.  Rend.,  154. 

2  v.  Rhorer,  Pfliiger's  Arch.,  86;  Hober,  1.  c.  See  also  Jolles,  Bioch.  Zeitschr., 
13;  Henderson,  Bioch.  Zeitschr.,  24. 


678  UKINE. 

acidity  and  the  ion  acidity  it  follows  that  no  direct  relation  exists  between 
these  and  that  the  extent  of  these  two  acidities  may  be  independent  of 
each  other. 

The  osmotic  pressure  of  the  urine  varies  considerably  even  under 
physiological  conditions.  The  limit  for  the  freezing-point  depression 
has  been  found  by  a  number  of  investigators  to  be  A  1.3°  to  2.3°  C.  After 
partaking  of  considerable  water  it  may  be  markedly  lower,  and  on 
diminished  supply  of  water  it  may  be  considerably  higher. 

In  regard  to  the  further  physical-chemical  imvestigations  of  the  urine 
and  as  to  the  conclusions  drawn  from  a  combination  of  the  chemical 
and  the  physico-chemical  investigations  of  the  urine,  we  must  refer  to 
the  extensive  work  of  Carl  Neuberg.1 

The  specific  gravity  of  urine,  which  is  dependent  upon  the  relation 
existing  between  the  quantity  of  water  secreted  and  the  solid  urinary 
constituents,  especially  the  urea  and  sodium  chloride,  may  vary  con- 
siderably, but  is  generally  1.017-1.020.  After  drinking  large  quantities 
of  water  it  may  fall  to  1.002,  while  after  profuse  perspiration  or  after 
drinking  very  little  water  it  may  rise  to  1.035-1.040.  In  new-born 
infants  the  specific  gravity  is  low,  1.007-1.005.  The  determination 
of  the  specific  gravity  is  an  important  means  of  learning  the  average 
amount  of  solids  eliminated  from  the  organism  in  the  urine,  and  on  this 
account  the  determination  becomes  of  true  value  only  when  at  the  same 
time  the  quantity  of  urine  voided  in  a  given  time  is  determined.  The 
different  portions  of  urine  voided  in  the  course  of  the  twenty-four  hours 
are  collected,  mixed  together,  the  total  quantity  measured,  and  then  the 
specific  gravity  taken. 

The  determination  of  the  specific  gravity  is  most  accurately  obtained 
with  the  pycnometer.  For  ordinary  cases  the  specific  gravity  may  be 
determined  with  sufficient  accuracy  by  means  of  areometers.  The 
areometers  found  in  the  trade,  or  urinometers,  are  graduated  from  1.000 
to  1.040;  for  exact  observations  it  is  better  to  use  two  urinometers,  one 
graduated  from  1.000  to  1.020,  and  the  other  from  1.020  to  1.040. 

To  determine  the  specific  gravity  of  urine,  if  necessary  filter  the 
urine,  or  if  it  contains  a  urate  sediment,  first  dissolve  it  by  gentle  heat, 
then  pour  the  clear  urine  into  a  dry  cylinder,  avoiding  the  formation  of 
froth.  Air  bubbles  or  froth,  when  present,  must  be  removed  with  a  glass 
rod  or  filter-paper.  The  cylinder,  which  should  be  about  four-fifths  full, 
must  be  wide  enough  to  allow  the  urinometer  to  swim  freely  in  the  liquid 
without  touching  the  sides.  The  cylinder  and  urinometer  should  both 
be  dry  or  previously  washed  with  the  urine.  On  reading,  the  eye  is 
brought  on  a  level  with  the  lower  meniscus — which  occurs  when  the  sur- 
face of  the  liquid  and  the  lower  limb  of  the  meniscus  coincide;   the  read- 

1  Der  Ham  sowie  die  iibrigen  Ausscheidungen  und  Korperfliissigkeiten  von  Mensch 
und  Tier.  Teil.  2,  Berlin,  1911. 


ORGANIC  PHYSIOLOGICAL  CONSTITUENTS.  679 

ing  is  then  made  from  the  point  where  this  curved  line  coincides  with 
the  scale  of  the  urinometer.  If  the  eye  is  not  in  the  same  horizontal 
plane  with  the  convex  line  of  the  meniscus,  hut  is  too  high  or  too  low, 
the  surface  of  the  liquid  assumes  the  shape  of  an  ellipse,  and  the  reading 
in  this  position  is  incorrect.  Before  reading,  press  the  urinometer  gently 
down  into  the  liquid  and  then  allow  it  to  rise,  and  wait  until  it  is  at  rest. 

Each  urinometer  is  graduated  for  a  certain  temperature,  which, 
at  least  in  the  case  of  the  better  ones,  is  marked  on  the  instrument. 
If  the  urine  is  not  at  the  proper  temperature,  the  following  corrections 
must  be  made:  For  every  three  degrees  above  the  normal  temperature 
one  unit  of  the  last  order  is  added  to  the  reading,  and  for  every  three 
degrees  below  the  normal  temperature  one  unit  (as  above)  is  subtracted 
from  the  specific  gravity  observed.  For  example,  when  a  urinometer 
graduated  for  15°  C.  shows  a  specific  gravity  of  1.017  at  24°  C,  then  the 
specific  gravity  at  15°  C.  =  1.017+0.003  =  1.020. 

When  great  exactitude  is  required,  as,  for  instance,  a  determina- 
tion to  the  fourth  decimal  point,  we  make  use  of  a  urinometer  constructed 
by  Lohnstein.1  Jolles  2  has  also  devised  a  small  urinometer  for  the 
determination  of  the  specific  gravity  of  small  amounts  of  urine,  20-25 
cc.  The  specific  gravity  may  also  be  determined  by  the  Westphal 
hydrostatic  balance. 

II.     ORGANIC   PHYSIOLOGICAL   CONSTITUENTS   OF  URINE. 

+  /NH2 

Urea,  Ur,  CON2H4  =  CO^  ,  has  been  synthetically  prepared  in  sev- 

NNH2 
eral  ways,  especially,  as  Wohler  showed  in  1828,  by  the  metameric 
transformation  of  ammonium  isocyanate:  CO.N.NH4  =  CO(NH2)2-  It 
is  also  produced  by  the  decomposition  or  oxidation  of  certain  bodies 
found  in  the  animal  organism,  such  as  purine  bodies,  creatine,  arginine, 
other  amino-acids,  and  other  substances. 

Urea  is  found  most  abundantly  in  the  urine  of  carnivora  and  man,, 
but  in  smaller  quantities  in  that  of  herbivora.  In  carnivora  (dog)  the 
urea  nitrogen  by  abundant  protein  feeding  may  amount  to  97-98  per  cent 
of  the  total  nitrogen  of  the  urine  (Schondorff  3) .  The  quantity  in  human 
urine  is  ordinarily  20-30  p.  m.  It  has  also  been  found  in  small  quantities 
in  the  urine  of  amphibians,  fishes,  and  certain  birds.  Urea  occurs  in 
the  perspiration  in  small  quantities,  and  as  traces  in  the  blood  and  in 
most  of  the  animal  fluids.  It  also  occurs  in  rather  large  quantities  in  the 
blood,  liver,  muscle,4  and  bile  5  of  sharks,  even  in  rather  large  quantities. 
Urea  is  also  found  in  certain  tissues  and  organs  of  mammals,  especially 

1  Pfliiger's  Arch.,  59;  Chem.  Centralbl.,  1895,  1,  and  1896,  2. 

!  Wien.  med.  Presse,  1897,  No.  8 

s  Pfluger's  Arch.,  117. 

4  v.  Schroeder,  Zeitschr.  f.  physiol.  Chem.,  14. 

6  Hammarsten,  ibid.,  24. 


€80  URINE. 

in  the  liver,  spleen,  muscles  and  others,  although  only  in  small  amounts. 
Under  pathological  conditions,  as  in  obstructed  excretion,  urea  may 
appear  to  a  considerable  extent  in  the  animal  fluids  and  tissues. 

The  quantity  of  urea  which  is  voided  in  twenty-four  hours  on  a  mixed 
diet  is  in  a  grown  man  about  30  grams,  in  women  somewhat  less.  While 
children  void  less,  the  excretion  relative  to  their  body  weight  is  greater 
than  in  grown  persons.  The  physiological  significance  of  urea  lies  in 
the  fact  that  this  body  forms  in  man  and  carnivora,  from  a  quantitative 
standpoint,  the  most  important  nitrogenous  end-product  of  the  metabolism 
of  protein  bodies.  On  this  account  the  elimination  of  urea  varies  to  a 
great  extent  with  the  catabolism  of  the  protein,  and  above  all  with  the 
quantity  of  absorbable  proteins  in  the  food  ingested.  The  elimination 
of  urea  is  greatest  after  an  exclusive  meat  diet,  and  lowest,  indeed  less 
than  during  starvation,  after  the  consumption  of  non-nitrogenous  sub- 
stances, since  these  diminish  the  metabolism  of  the  proteins  of  the  body. 

If  the  consumption  of  the  proteins  of  the  body  is  increased,  then 
the  elimination  of  nitrogen  is  correspondingly  increased.  This  is  found  to 
be  the  case  in  fevers,  after  poisoning  with  arsenic,  antimony,  phosphorus, 
and  other  protoplasmic  poisons,  and  when  there  is  a  diminished  supply 
of  oxygen — as  in  severe  and  continuous  dyspncea,  poisoning  with  carbon 
monoxide,  hemorrhage,  etc.  In  these  cases  it  used  to  be  considered  that 
the  rise  in  the  excretion  of  nitrogen  was  due  to  an  increased  elimination 
of  urea,  because  no  exact  difference  was  made  between  the  quantity 
of  urea  and  of  total  nitrogen  in  the  urine.  Recent  researches  have  con- 
clusively demonstrated  Ihe  untrustworthiness  of  these  observations. 
Since  Pfluger  and  Bohland  have  shown  that  16  per  cent  of  the  total 
nitrogen  of  the  urine  exists  under  physiological  conditions  in  other  com- 
pounds, not  urea,  attention  has-been  called  to  the  relation  of  the  dif- 
ferent nitrogenous  constituents  of  the  urine  to  each  other,  and  it  has 
been  found,  under  pathological  conditions,  that  this  relation  may  vary 
considerably,  especially  in  regard  to  the  urea.  We  have  numerous 
determinations  by  different  investigator's,1  on  the  relation  of  the  different 
nitrogenous  constituents  to  each  other  in  the  normal  urine  of  adults. 


1  Pfluger  and  Bohland,  Pfluger' s  Arch.,  38  and  43;  Bohland,  ibid.,  43;  Schultze, 
ibid.,  45;  Camerer,  Zeitschr.  f.  Biologie,  24,  27,  and  28;  Voges,  Ueber  die  Mischung 
der  stickstoffhaltigen  Bestandtheile  im  Ham.  etc.  (Inaug.-Diss.  Berlin.  1892),  cited 
from  Maly's  Jahresber.,  22;  K.  Morner  and  Sjoqvist,  Skand.  Arch.  f.  Physiol.,  2. 
See  also  Sjoqvist,  Nord.  Med.  Arkiv.,  1892,  No.  36,  and  1894,  No.  10;  Gumlich,  Zeitschr. 
f.  physiol.  Chem.,  17;  Bodtker,  see  Maly's  Jahresber.,  26;  Folin,  Amer.  Journ.  of 
Physiol.,  13;  Osterberg  and  Wolff,  Journ.  of  biol.  Chem.,  3;  Haskins,  ibid.,  2;  Donze 
et  Lambling,  Journ.  de  Physiol,  et  de  Path.,  5;  Bouchet,  ibid.,  14;  Lambling  et 
Bouchet,    Compt.    rend.    soc.  biol.,   71;    Long    and    Gephart,  Journ.  Amer.    Chem. 


UREA.  681 

Thus  Long  and  Gephart  found  in  the  urine  of  six  healthy  men  to  whom 
the  same  qualitative  diet  was  fed  for  a  long  time,  the  following  division 
of  the  nitrogen  in  percentage  of  the  total  nitrogen:  urea  79.87-84. ill, 
creatinine  5.21-6.87,  ammonia  3.6-4.74,  uric  acid  1.57-1.99,  purine 
0.33-0.96  and  rest  nitrogen  4.23-6.01  per  cent.  Sjoqvist  has  made 
similar  determinations  on  new-born  babes  from  1  to  7  days  old.  From 
all  these  analyses  we  obtain  the  following  figures  (A  for  adults  and  B  for 
new-born  babes).     Of  the  total  nitrogen  there  exists: 

A.  B. 

Per  Cent.  Per  Cent. 

Urea 84-91  73-76 

Ammonia 2-5  7.8-9.6 

Uric  acid 1-3  3.0-8.5 

Remaining  nitrogenous  substances 7-12  7.3-14.7 

The  variable  relation  between  uric  acid,  ammonia,  and  urea  nitro- 
gen in  children  and  adults  is  remarkable,  since  the  urine  of  children 
is  considerably  richer  in  uric  acid  and  ammonia,  and  considerably  poorer 
in  urea,  than  the  urine  of  adults.  A  much  larger  number  of  analyses 
of  children's  urine  is  necessary  to  explain  the  division  of  the  nitrogen 
.therein.  The  absolute  quantity  of  urea  nitrogen  in  adults  amounts  to 
about  10-16  grams  per  day.  In  disease  the  proportion  of  the  nitroge- 
nous substances  may  be  markedly  changed,  and  a  decrease  in  the  quan- 
tity of  urea  and  an  increase  in  the  quantity  of  ammonia  have  been  observed 
in  certain  diseases  of  the  liver.  This  will  be  considered  in  detail  in 
connection  with  the  formation  of  urea  in  the  liver.  It  is  natural  that 
there  should  be  a  diminished  formation  of  urea  after  a  decrease  in  the 
ingestion  of  proteins  or  in  a  lowered  catabolism.  In  diseases  of  the 
kidneys  which  disturb  or  destroy  the  integrity  of  the  epithelium  of 
the  convoluted  urinary  tubules,  the  elimination  of  urea  is  considerabl}'' 
diminished. 

Recently  by  means  of  Pfaundler's  l  method,  by  precipitating  the  urine  with 
phosphotungstic  acid  and  closely  studying  the  precipitate  as  well  as  the  filtrate, 
it  has  been  possible  to  learn  further  about  the  division  of  the  nitrogen  of  the  urine. 
We  determine  a,  the  total  nitrogen;  b,  the  nitrogen  of  the  phosphotungstate  pre- 
cipitate; and  c,  the  nitrogen  in  the  filtrate  from  the  phosphotungstate  pre- 
cipitate. This  last  contains  the  urea,  hippuric  acid,  o'xyproteie  acids,  and  other 
bodies  whose  nitrogen  is  ordinarily  designated  as  monamino-acid  nitrogen.  The 
urea  nitrogen  is  especially  determined.  The  bodies  precipitated  by  phospho- 
tungstic acid  are  not  all  known;  but  uric  acid  and  purine  basest  ammonia, 
creatinine,  pigments,  diamino-acids,  diamines  and  ptomaines  (if  they  occur),  sul- 
phocyanides,  carbamic  acid,  urine  mucoid,  and  proteid  belong  to  this  group. 
Special  methods  have  been  suggested  for  the  determination  of  several  of  these 
substances  (see  below). 

The  urea  nitrogen  is  always  the  greatest  part  of  the  total  nitrogen, 
but  otherwise  the  division  of  the  nitrogen  undergoes  considerable  varia- 

1  Zeitschr.  f.  physiol.  Chem.,  30. 


682  URINE. 

tion  and  very  great  variations  seem  to  occur  not  only  in  the  healthy 
individual,  but  also  and  to  a  greater  degree  in  diseased  conditions.1 

Formation  of  Urea  in  the  Organism.  The  older  statements  of  Bechamp 
that  urea  is  directly  formed  from  proteins  by  oxidation  has  been  denied 
by  several  investigators  but  according  to  recent  statements  of  Fosse  2 
this  is  correct.  On  the  hydrolysis  of  proteins  arginine  is  found  among 
other  products,  and  as  it  is  also  produced  in  tryptic  digestion,  it  is  possible 
that  a  small  portion  of  the  urea  is  produced  in  this  manner,  varying 
according  to  the  kind  of  protein.  Drechsel  claims  that  about  10  per 
cent  of  the  urea  can  be  accounted  for  in  this  way. 

The  possibility  of  a  formation  of  urea  from  arginine  has  gained  in 
interest  since  Kossel  and  Dakin  have  discovered  the  presence  of  an 
enzyme,  arginase,  in  the  liver  and  other  organs,  which  has  the  power 
of  splitting  arginine  with  the  formation  of  urea.  Thompson  3  has  given 
a  direct  proof  for  the  formation  of  urea  from  arginine.  The  introduc- 
tion of  arginine  into  the  body  of  a  dog  either  per  os  or  subcutaneously 
has  in  his  experiments  led  t©  an  elimination  of  urea.  While  outside  of 
the  body  only  one-half  of  the  nitrogen  of  arginine  is  split  off  as  urea 
and  the  other  half  as  ornithine,  in  the  above  experiments  the  increase 
in  urea  in  several  instances  corresponded  to  the  greater  part  if  not  the 
whole  of  the  nitrogen  of  the  arginine  introduced.  This  increased  forma- 
tion of  urea  makes  it  probable  that  also  ornithine  is  deamidized  and  the 
urea  is  formed  from  the  ammonia  split  off. 

By  the  action  of  alkalies,  as  above  mentioned  (Chapter  X),  urea  may 
be  formed  from  creatinine ;  still  such  an  origin  of  urea  in  the  animal  body 
has  not  thus  far  been  proved. 

The  amino-acids  are  considered  as  special  mother-substances  of  urea. 
By  numerous,  generally  older  experiments  with  these  acids,  it  has  been 
proved  that  the  amino-acids  of  the  animal  body  are  transformed  in  part 
into  urea.  The  investigations  by  Salaskin  with  the  three  amino-acids, 
glycocoll,  leucine,  and  aspartic  acid,  have  unmistakably  shown  that  the 
surviving  dog-liver,  supplied  with  arterial  blood,  has  the  property  of 
transforming  the  above  amino-acids  into  urea  or  a  closely  allied  sub- 
stance.4    Like  the  amino-acids  the  polypeptides  are  also  transformed  into 

1  See  Satta,  Hofmeister's  Beitrage,  6,  which  also  gives  the  literature,  and  Erben, 
Zeitschr.  f.  Heilkunde,  25. 

2  Compt.  Rend.,  154. 

3  Kossel  and  Dakin,  Zeitschr.  f.  physiol.  Chem.,  41;  Thompson,  Journ.  of  Physiol., 
32  and  33. 

4  Schultzen  and  Nencki,  Zeitschr.  f.  Biologie,  8;  v.  Knieriem,  ibid.,  10;  Salkowski, 
Zeitschr.  f.  physiol.  Chem.,  4;  Salaskin,  ibid.,  25;  Stolte,  Hofmeister's  Beitrage,  5; 
Levene  and  Meyer,  Arner.  Journ.  of  Physiol.,  25;  see  also  Loewi,  Zeitschr.  f.  physiol. 
Chem.,  25;  Richet,  Compt.  Rend.,  118,  and  Compt.  rend.  Soc.  biol.,  49;  Ascoli, 
Pfliiger's  Arch.,  72. 


FORMATION  OF  UREA.  683 

urea  in  the  animal  body,  as  shown  by  the  investigations  of  Abderhalden 

and  his  collaborators.1 

There  is  no  doubt  that  the  ammonia  formation  is  of  great  importance 
in  the  production  of  urea  in  the  animal  body. 

A  great  number  of  older  investigations2  on  the  behavior  of  ammo- 
nium salts  in  the  animal  body  have  shown  that  not  only  ammonium  car- 
bonate, but  also  those  ammonium  salts  which  are  burned  into  carbonate 
in  the  organism,  are  transformed  into  urea  by  carnivora  as  well  as  her- 
bivora.  v.  Schroeder,3  by  irrigating  the  surviving  dog's  liver  with 
blood  treated  with  ammonium  carbonate  or  ammonium  formate,  has 
shown  that  the  formation  of  urea  takes  place,  at  least  in  part,  in  this  organ. 
Nencki,  Pawlow,  Zaleski  and  Salaskin4  have  also  found  that,  in  dogs, 
the  quantity  of  ammonia  in  the  blood  from  the  portal  vein  is  considerably 
greater  than  that  from  the  hepatic  vein,  and  they  claim  that  the  liver 
retains  in  great  part  the  ammonia  thus  supplied.  The  formation  of  urea 
from  ammonia  in  the  liver  is  a  positively  proved  fact. 

The  assumption  of  a  splitting  off  of  ammonia  from  amino-acids 
stands  in  agreement  with  the  experience  that  a  deamidation  of  the  amino- 
acids  takes  place  in  the  animal  body.  The  ammonia  split  off  finds,  in 
the  blood  and  tissues,  the  carbon  dioxide  necessary  for  the  formation 
of  carbonate,  and  the  investigations  of  Nolf,  as  well  as  those  of  Macleod 
and  Haskins,5  on  the  equilibrium  of  carbonate  and  carbamate  solutions 
and  the  conditions  for  the  formation  of  both  salts,  must  also  be  abundant 
evidence  of  a  carbamate  formation. 

Important  observations  have  been  made  which  give  support  to  the 
views  of  Schultzen  and  Nencki,6  namely,  that  the  amino-acids  are 
transformed  into  urea  with  ammonium  carbamate,  H4N.O.CO.NH2, 
as  an  intermediate  step.  Drechsel  has  shown  that  the  amino-acids 
yield  carbamic  acid  by  oxidation  in  alkaline  fluid  outside  of  the  organism, 
and  he  obtained  urea  from  ammonium  carbamate  by  alternate  oxidation 
and  reduction.  Carbamate  has  also  been  found  in  the  blood  (Drechsel) 
as  well  as  in  the  urine  (Drechsel,  Abel  and  Muirhead)  7  and  Nexckt 

1  Abderhalden  with  Teruuchi  and  with  Babkin,  Zeitschr.  f.  physiol.  Chem.,  47, 
with  Schittenhelm,  ibid.,  51. 

2v.  Knieriem,  Zeitschr.  f.  Biologie,  10;  Feder,  ibid.,  13;  Salkowski,  Zeitschr.  f. 
Biologie,  1;  Munk,  ibid.,  2;  Coranda,  Arch.  f.  exp.  Path.  u.  Pharm.,  12;  Schmiede- 
berg  and  Walter,  ibid.,  7;  Hallervorden,  ibid.,  10;  Pohl  and  Miinzer,  Arch.,  f.  exp. 
Path.  u.  Pharm.,  43. 

3  Arch.  f.  exp.  Path.  u.  Pharm.,  15.     See  also  Salomon,  Virchow's  Arch.,  97. 

4  Arch,  des  sciences  biol.  de  St.  Petersbourg,  4;  see  also  Chapter  V,  p.  336. 

6  Nolf,  Zeitschr.  f .  physiol.  Chem.,  23;  Macleod  and  Haskins,  Journ.  of  biol.  Chem.,  1. 

6  Zeitschr.  f.  Biologie,  8. 

7  Drechsel,  Ber.  d.  sachs.  Gesellsch.  d.  Wissensch.,  1875.  See  also  Journ.  f.  prakt. 
Chem.  (N.  F.),  12,  16,  and  22;  Abel,  Arch.  f.  (Anat.  u.)  Physiol.,  1891;  Abel  and 
Muirhead,  Arch.  f.  exp.  Path.  u.  Pharm.,  31. 


684  URINE. 

and  Hahn  have  made  further  observations  on  dogs  with  Eck's  fistular 
which  substantiate  this  view.  In  such  fistula  dogs,  they  observed  that 
when  meat  was  fed,  violent  poisonous  symptoms  developed  which  were 
almost  identical  with  those  produced  when  carbamate  was  introduced 
into  the  blood.  The  same  symptoms  also  appeared  on  the  introduction 
of  carbamate  into  the  stomach  of  the  fistula  animal,  while  the  intro- 
duction of  carbamate  into  the  stomach  of  a  normal  dog  had  no  action.1 
As  these  observers  also  found  that  the  urine  of  the  dog  on  which  the 
operation  was  made  was  richer  in  carbamate  than  that  of  the  normal 
dog,  they  concluded  that  the  symptoms  were  due  to  the  non-transforma- 
tion of  the  ammonium  carbamate  into  urea  in  the  liver,  and  they  consider 
the  ammonium  carbamate  as  the  substance  from  which  the  urea  is  derived 
in  the  mammalian  liver. 

Besides  the  above  view  of  the  formation  of  urea  from  ammonium 
carbonate  and  carbamate,  which  has  been  called  the  anhydride  theory, 
we  also  have  the  oxidation  theory  of  Hofmeister. 

F.  Hofmeister2  found  in  the  oxidation  of  different  members  of 
the  fatty  series,  as  well  as  in  amino-acids  and  proteins,  that  urea  was 
formed  in  the  presence  of  ammonia,  and  he  therefore  suggests  the  pos- 
sibility that  urea  may  be  formed  by  an  oxidation-synthesis.  Accord- 
ing to  him,  in  the  oxidation  of  nitrogenous  substances  a  radical  CONH2, 
containing  the  amide  group,  unites  at  the  moment  of  formation  with  the 
radical   NH2   remaining   on  the   oxidation   of   ammonia,   forming   urea. 

Besides  the  above-mentioned  theories  as  to  the  formation  of  urea, 
there  are  others  which  will  not  be  given,  because  the  only  theory  which 
has  thus  far  been  positively  demonstrated  is  the  formation  of  urea  in 
the  liver  from  ammonium  compounds  and  amino-acids. 

The  liver  is  the  only  organ  in  which,  up  to  the  present  time,  a  forma- 
tion of  urea  has  been  directly  detected;3  and  the  question  arises,  what 
importance  has  this  urea  formation  which  takes  place  in  the  liver?  Is 
the  urea  wholly  or  chiefly  formed  in  the  liver? 

If  the  liver  is  the  only  organ  capable  of  forming  urea,  it  is  to  be 
expected,  on  the  extirpation  or  atrophy  of  that  organ,  that  a  reduced 


1  Hahn,  Massen,  Nencki  et  Pawlow,  La  fistule  d'Eck  de  la  veine  cave  inferieure  et 
de  la  veine  porte,  etc.  Arch,  des  sciences  biol.  de  St.  P<5tersbourg,  1,  No.  4,  1892. 
In  regard  to  certain  differences  between  the  symptoms  with  carbamate  poisoning  and 
after  meat  feeding  with  Eck  fistula  dogs,  see  Rothberger  and  Winterberg,  Zeitschr. 
f .  exp.  Path.  u.  Therap.,  1 ;  Hawk,  Amer.  Journ.  of  Physiol.,  21. 

2  Arch.  f.  exp.  Path.  u.  Pharm.,  37. 

3  In  regard  to  the  investigations  of  Prevost  and  Dumas,  Meissner,  Voit,  Grehant, 
Gscheidlen  and  Salkowski,  and  others,  on  the  r61e  of  the  kidneys  in  the  formation  of 
urea,  see  v.  Schroeder,  Arch.  f.  exp.  Path.  u.  Pharm.,  15  and  19,  and  Voit,  Zeitschr. 
f.  Biologie,  4. 


FORMATION  OF  UREA.  685 

or,  in  short  experiments,  at  least  a  strongly  diminished  elimination  of 
urea  should  occur.  As  at  hast  a  part  of  the  urea  is  formed  in  the  liver 
from  ammonium  compounds,  a  simultaneous  increase  in  the  elimination 
of  ammonia  is  to  be  expected. 

The  extirpation  and  atrophy  experiments  made  on  animals  by  dif- 
ferent methods  l  have  shown  that  sometimes  a  rather  marked  increase 
of  ammonia  and  a  diminished  elimination  of  urea  takes  place  after  the 
operation,  but  that  there  are  also  cases  in  which,  irrespective  of  the  pro- 
nounced atrophy,  an  abundant  formation  of  urea  occurs,  and  no  appre- 
ciable. ;f  any,  change  in  the  proportion  of  ammonia  to  the  total  nitrogen 
and  urea  is  observed.  After  shutting  out  from  the  circulation  the  organs 
of  the  posterior  part  of  the  body,  especially  the  liver  and  kidneys,  Kauf- 
maxx  2  also  found  an  important  increase  in  the  urea  of  the  blood,  and  these 
different  observations  show  that  the  liver  is  not  the  only  organ,  in  the 
various  animals  experimented  upon,  in  which  urea  is  formed. 

The  observations  made  by  numerous  investigators  3  on  human  beings 
with  cirrhosis  of  the  liver,  acute  yellow  atrophy  of  the  liver,  and  phos- 
phorus poisoning  have  led  to  the  same  result.  These  investigations 
teach  that  in  certain  cases  the  proportion  of  the  nitrogenous  substances 
may  be  so  changed  that  urea  is  only  50-60  per  cent  of  the  total  nitrogen, 
while  in  other  cases,  on  the  contrary,  even  in  very  extensive  atrophy 
of  the  liver-cells,  the  formation  of  urea  is  not  diminished,  neither  is  the 
proportion  between  the  total  nitrogen,  urea,  and  ammonia  essentially 
changed.  Even  in  the  cases  in  which  the  formation  of  urea  was  relatively 
diminished  and  the  elimination  of  ammonia  considerably  increased,  fur- 
ther investigation  must  be  instituted  before  it  will  be  possible  to  assume 
a  reduced  ability  of  the  organism  to  produce  urea.  An  increased  elimi- 
nation of  ammonia  may,  as  shown  by  Munzer  in  the  case  of  acute 
phosphorus  poisoning,  be  dependent  upon  the  formation  of  abnormally 
large  quantities  of  acids,  caused  by  abnormal  metabolism,  and  these  acids 
require  a  greater  quantity  of  ammonia  for  their  neutralization  according 
to  the  law  of  elimination  of  ammonia.     That  an  abnormal  formation 


1  Xencki  and  Hahn,  1.  c;  Slosse,  Arch.  f.  (Anat.  u.)  Physiol.,  1890;  Lieblein,  Arch, 
f.  exp.  Path.  u.  Pharm.,  33;  Nencki  and  Pawlow,  Arch,  des  science,  biol.  de  St.  Peters- 
bourp,  5.  See  also  v.  Meister,  Maly's  Jahresber.,  25;  Salaskin  and  Zaleski,  Zeitschr.  f . 
physiol.  Chem.,  29;   Fischler  and  Bardach,  ibid.,  78. 

2Compt.  rend.  soc.  biol.,  46,   and  Arch,  de  Physiol.  (5),  6. 

3  See  Hallervorden,  Arch.  f.  exp.  Path.  u.  Pharm.,  12;  Weintraud,  ibid.,  31;  Miinzer 
and  Winterberg,  ibid.,  33;  Stadelmann,  Deutsch.  Arch.  f.  klin.  Med.,  33;  Fawitzki, 
ibid.,  45:  Miinzer,  ibid.,  52;  Frankel,  Berlin,  klin.  Wochenschr.,  1878;  Richter,  ibid., 
1896;  Morner  and  Sjoqvist,  Skand.  Arch.  f.  Physiol.,  2,  and  Sjoqvist,  Nord.  Med. 
Arkiv.  1892;  Guinlich.  Zeitschr.  f.  physiol.  Chem.,  17;  v.  Xoorden,  Lehrb.  d.  Pathol, 
des  Stoffwechsels,  2.  Aufl.,  Bd.  1,  104. 


686  URINE. 

of  acid  occurs  after  the  cutting  out  of  the  liver  has  been  especially  shown 
by  Salaskin  and  Zaleski.1 

For  the  present  we  are  not  justified  in  the  statement  that  the  liver 
is  the  only  organ  in  which  urea  is  formed,  and  only  continued  investiga- 
tion can  yield  further  information  as  to  the  extent  and  importance  of  the 
formation  of  urea,  from  ammonium  compounds,  in  the  liver. 

Properties  and  Reactions  of  Urea.  Urea  crystallizes  in  needles  or  in 
long,  colorless,  four-sided,  often  hollow,  anhydrous  rhombic  prisms. 
It  has  a  neutral  reaction,  and  produces  a  cooling  sensation  on  the  tongue 
like  saltpeter.  It  melts  at  132°  C.  At  ordinary  temperatures  it  dis- 
solves in  an  equal  weight  of  water  and  in  five  parts  alcohol;  it  requires 
one  part  boiling  alcohol  for  solution:  it  is  insoluble  in  alcohol-free  anhy- 
drous ether,  and  also  in  chloroform.  If  urea  in  substance  is  heated  in  a 
test-tube,  it  melts,  decomposes,  gives  off  ammonia,  and  finally  leaves  a 
non-transparent  white  residue  which,  among  other  substances,  contains 
cyanuric  acid  and  biuret,  which  latter  dissolves  in  water,  giving  a  beautiful 
reddish-violet  liquid  with  copper  sulphate  and  alkali  (biuret  reaction). 
On  heating  with  baryta-water  or  caustic  alkali,  also  in  the  so-called 
alkaline  fermentation  of  urine  caused  by  micro-organisms,  urea  splits 
into  carbon  dioxide  and  ammonia  with  the  addition  of  water.  The 
same  decomposition  products  are  produced  when  urea  is  heated  with 
concentrated  sulphuric  acid.  An  alkaline  solution  of  sodium  hypo- 
bromite  decomposes  urea  into  nitrogen,  carbon  dioxide,  and  water  accord- 
ing to  the  equation 

CON2H4+3NaOBr  =  3NaBr-r-C02+2H20+N2. 

With  a  concentrated  solution  of  furfuroi  and  hydrochloric  acid,  urea 
in  substance  gives  a  coloration  passing  from  yellow,  green,  blue,  to  violet, 
and  then  after  a  few  minutes  beautiful  purple-violet  (Schiff's  reaction). 
According  to  Huppert2  the  test  is  best  performed  by  taking  2  cc.  of  a 
concentrated  furfuroi  solution,  4-6  drops  of  concentrated  hydrochloric 
acid,  and  adding  to  this  mixture,  which  must  not  be  red,  a  small  crystal 
of  urea.     A  deep  violet  coloration  appears  in  a  few  minutes. 

Urea  forms  crystalline  compounds  with  many  acids.  Among  these 
the  one  with  nitric  acid  and  the  one  with  oxalic  acid  are  the  most 
important. 

Urea  Nitrate,  CO(NH2)2-HN03.  On  crystallizing  quickly  this 
compound  forms  thin  rhombic  or  six-sided  overlapping  tiles,  or  colorless 


1  Zeitschr.  f.  physiol.  Chem.,  29. 

1  Huppert-Neubauer,  Analyse  des  Harns,  10.  Aufl.,  296. 


PROPERTIES  AND   REACTIONS   OF  UREA.  C87 

plates,  with  an  angle  of  82°.  When  crystallizing  slowly,  larger  and 
thicker  rhombic  pillars  or  plates  are  obtained.  This  compound  is  rather 
easily  soluble  in  pure  water,  but  is  considerably  less  soluble  in  water 
containing  nitric  acid;  it  may  be  obtained  by  treating  a  concentrated 
solution  of  urea  with  an  excess  of  strong  nitric  acid  free  from  nitroufl 
acid.     On  heating  this  compound  it  volatilizes  without  leaving  a  residue. 

This  compound  may  bo  employed  with  advantage  in  detecting  small  amounts 
of  urea.  A  drop  of  the  concentrated  solution  is  placed  on  a  microscope  slide  and 
the  cover-glass  placed  upon  it;  a  drop  of  nitric  acid  is  then  placed  on  the  side 
of  the  cover-glass  and  allowed  to  flow  under.  The  formation  of  crystals  begins 
where  the  solution  and  the  nitric  acid  meet.  Alkali  nitrates  may  crystallize 
very  similarly  to  urea  nitrate  when  they  are  contaminated  with  other  bodies; 
therefore,  in  testing  for  urea,  the  crystals  must  be  identified  as  urea  nitrate  by 
heating  and  by  other  means. 

Urea  Oxalate,  2.CO(NH2)2-H2C204.  This  compound  is  nu  re 
sparingly  soluble  in  water  than  the  nitric-acid  compound.  It  is  obtained 
in  rhombic  or  six-sided  prisms  or  plates  on  adding  a  saturated  oxalic- 
acid  solution  to  a  concentrated  solution  of  urea. 

Urea  also  forms  combinations  with  mercuric  nitrate  in  variable 
proportions.  If  a  very  faintly  acid  mercuric-nitrate  solution  is  added 
to  a  2  per  cent  solution  of  urea  and  the  mixture  carefully  neutralized, 
a  compound  is  obtained  of  a  constant  composition  which  contains  for 
every  10  parts  of  urea  72  parts  of  mercuric  oxide.  This  compound  serves 
as  the  basis  of  Liebig's  titration  method.  Urea  also  combines  with 
salts,  forming  mostly  crystallizable  combinations,  as,  for  instance,  with 
sodium  chloride,  with  the  chlorides  of  the  heavy  metals,  etc.  An  alka- 
line but  not  a  neutral  solution  of  urea  is  precipitated  by  mercuric  chloride. 

If  urea  is  dissolved  in  dilute  hydrochloric  acid  and  then  an  excess  of  formal- 
dehyde is  added,  a  thick,  white,  granular  precipitate  is  obtained  which  is  dif- 
ficultly soluble  and  whose  composition  is  somewhat  disputed.1  With  phenyl- 
hydrazine,  urea  in  strong  acetic  acid  gives  a  colorless  crystalline  compound  of 
phenylsemicarbazid,  C\Jd6NH.NH:CONH2,  which  is  soluble  with  difficulty  in  cold 
water  and  melts  at  172°  C.  (Jaffe  2). 

The  method  of  preparing  urea  from  urine  is  in  the  main  as  follows: 
Concentrate  the  urine,  which  has  been  faintly  acidified  with  sulphuric 
acid,  at  a  low  temperature,  add  an  excess  of  nitric  acid,  at  the  same  time 
keeping  the  mixture  cool,  press  the  precipitate  well,  decompose  it  in 
water  with  freshly  precipitated  barium  carbonate,  dry  on  the  water- 
bath,  extract  the  residue  with  strong  alcohol,  decolorize  when  necessary 
with  animal  charcoal,  and  filter  while  warm.     The  urea  which  crystallizes 


1  See  Tollens  and  his  pupils,  Ber.  d.  deutsch.  chem.  Gesellsch.,  29,  2751;  Gold- 
schmidt,  ibid.,  29;  and  Chem.  Centralbl.,  1897,  1,  33;  Thorns,  ibid.,  2,  144  and  737. 

2  Zeitschr.  f.  physiol.  Chem.,  22. 


<C8S  URINE. 

on  cooling  is  purified  by  recrystallization  from  warm  alcohol.  A  fur- 
ther quantity  of  urea  may  be  obtained  from  the  mother-liquor  by  con- 
centration. The  urea  is  purified  from  contaminating  mineral  bodies 
by  redissolving  in  alcohol-ether.  If  it  is  only  necessary  to  detect  the 
presence  of  urea  in  urine,  it  is  sufficient  to  concentrate  a  little  of  the 
urine  on  a  watch-glass  and,  after  cooling,  treat  it  with  an  excess  of  nitric 
acid.     In  this  way  we  obtain  crystals  of  urea  nitrate. 

Quantitative  Estimation  of  the  Total  Nitrogen  and  Urea  in  Urine. 
Among  the  various  methods  proposed  for  the  estimation  of  the  total 
nitrogen,  that  suggested  by  Kjeldahl  is  to  be  recommended.  Liebig's 
method  for  the  estimation  of  urea  is  really  a  method  for  determining 
the  total  nitrogen,  but  as  it  is  very  seldom  used  now,  we  can  refer  to 
larger  works  in  regard  to  details. 

Kjeldahl 's  method  consists  in  transforming  all  the  nitrogen  of  the 
organic  substances  into  ammonia  by  heating  with  a  sufficiently  con- 
centrated sulphuric  acid.  The  ammonia  is  distilled  off,  after  super- 
saturating with  alkali,  and  collected  in  standard  sulphuric  acid.  The 
following  reagents  are  necessary: 

1.  Sulphuric  Acid.  Either  a  mixture  of  equal  volumes  of  pure  con- 
centrated and  fuming  sulphuric  acid,  or  else  a  solution  of  200  grams 
phosphoric  anhydride  in  1  liter  of  pure  concentrated  sulphuric  acid. 
2.  Caustic  soda  free  from  nitrates,  30-40  per  cent  solution.  The  quantity 
of  this  caustic-soda  sohition  necessary  to  neutralize  10  cc.  of  the  acid 
mixture  must  be  determined.  3.  Metallic  mercury  or  pure  yellow  mercuric 
oxide.  (The  addition  of  this  facilitates  the  destruction  of  the  organic 
substances.)  4.  A  potassium-sulphide  solution  of  4  per  cent,  whose 
object  is  to  decompose  any  mercuric  amide  combination  which  might 
not  evolve  its  ammonia  completely  during  the  distillation  with  caustic 
:soda.     5.  1/5  normal  sulphuric  acid  and  1/5  normal  caustic  soda  solution. 

In  performing  the  determination  5  cc.  of  the  carefully  measured  and 
filtered  urine  are  placed  in  a  long-necked  Kjeldahl  flask,  a  drop  of  mercury 
or  about  0.3  gram  of  mercuric  oxide  added,  and  then  treated  with  10-15 
cc.  of  the  strong  sulphuric  acid.  The  contents  are  heated  very  care- 
fully, placing  the  flask  at  an  angle,  until  they  just  begin  to  boil  gently; 
this  is  continued  for  about  half  an  hour  after  the  mixture  becomes  color- 
less. On  cooling,  the  contents  are  transferred  to  a  voluminous  distilling- 
flask,  carefully  washing  the  Kjeldahl  flask  with  water  and  the  greater 
part  of  the  acid  is  neutralized  by  caustic  soda.  A  few  zinc  shavings  are 
added  to  prevent  too  rapid  ebullition  on  distillation,  and  then  an  excess 
of  caustic-soda  solution  which  has  previously  been  treated  with  30-40 
cc.  of  the  potassium-sulphide  solution.  The  flask  is  quickly  connected 
with  the  condenser-tube  and  all  the  ammonia  distilled  off.  In  order 
to  prevent  loss  of  ammonia  it  is  best  to  lower  the  end  of  the  exit-tube 
below  the  surface  of  the  acid,  the  regurgitation  of  the  acid  being  prevented 
by  having  a  bulb  blown  on  the  exit-tube.  Not  less  than  25-30  cc.  of 
the  standard  acid  is  used  for  every  5  cc.  of  urine,  and  on  completion 
[of  the  distillation  the  acid  is  retitrated  with  1/5  normal  caustic  soda 
u.-ing  rosolic  acid,  tincture  of  cochineal,  or  lacmoid  as  indicator.  Each 
cubic  centimeter  of  the  acid  corresponds  to  2.8  milligrams  nitrogen.  As  a 
control  and  in  order  to  test  the  purity  of  the  reagents,  or  to  eliminate 
any  error  caused  by  an  accidental  quantity  of  ammonia  in  the  air,  we 
always  make  a  blank  determination  with  the  reagents. 


METHODS  FOR  THE   DETERMINATION  OF   UREA.  089 

Recently  Folin  and  Farmer  1  have  suggested  a  method  for  the  estima- 
tion of  the  total  nitrogen  in  very  small  quantities  of  urine,  1  cc.  dilute 
urine.  After  hydrolysis  with  acid  the  ammonia  formed  is  colorimetrically 
determined  by  means  of  Nessler's  reagent. 

Among  the  methods  suggested  f<<r  the  special  estimation  of  urea, 
that  of  Morner-Sjo ovist,  in  combination  with  Folin's  method,  is  the 
one  that  is  generally  used. 

Principle  of  M&rner-Sjoqvist's  Method.2  According  to  this  method 
the  nitrogenous  constituents  of  the  urine,  with  the  exception  of  urea, 
ammonia,  hippuric  acid,  creatinine,  and  traces  of  allantoin,3  are  pre- 
cipitated by  a  mixture  of  alcohol  and  ether  after  the  addition  of  a  solu- 
tion of  barium  chloride  and  barium  hydroxide,  or  in  the  presence  of  sugar 
with  solid  barium  hydroxide.  The  urea  is  determined  in  the  concentrated 
filtrate,  after  driving  off  the  ammonia,  by  Kjeldahl's  nitrogen  estima- 
tion. The  slight  error  due  to  the  presence  of  hippuric  acid  and  creatinine 
can  be  prevented  according  to  Morner  by  a  combination  of  his  method 
with  Folin's  method. 

Principle  of  Folin's  Method.*  On  heating  urea  with  hydrochloric 
acid  and  crystalline  magnesium  chloride,  which  melts  in  its  water  of 
crystallization  at  112-115°  C.  and  then  boils  at  about  150-155°  C,  the 
urea  is  completely  decomposed,  while  no  appreciable  decomposition 
of  the  hippuric  acid  and  creatinine  takes  place.  The  ammonia  produced 
from  the  urea  is  distilled  off  and  determined  by  titration.  The  amount 
of  ammonia  previously  existing  in  the  urine  must  be  specially  determined. 

Determination  of  Urea  by  the  Mdrner-Sjoqvist  and  Folin  Method.5 
Five  cc.  of  the  urine  are  treated  with  1.5  grams  of  powdered  barium 
hydroxide,  and  when  as  much  of  this  is  dissolved  as  possible  by  gently 
mixing,  it  is  precipitated  by  100  cc.  of  the  alcohol  and  ether  mixture 
(I  vol.  ether).  On  the  following  day  it  is  filtered  and  the  precipitate 
washed  with  the  alcohol  and  ether  mixture.  The  alcohol  and  ether 
are  distilled  off  from  the  filtrate  at  about  55°  C.  (not  above  60°  C).  The 
remaining  liquid  is  treated  with  2  cc.  of  hydrochloric  acid  of  sp.gr. 
1.124  (for  5  cc.  urine),  and  carefully  transferred  to  a  flask  of  200  cc. 
capacity,  and  evaporated  to  dryness  on  the  water-bath.  Then  add  20 
grams  of  crytalline  magnesium  chloride  to  the  contents  of  the  flask 
and  2  cc.  of  concentrated  hydrochloric  acid,  and  boil  on  a  wire  gauze 
over  a  small  flame  for  two  hours,  making  use  of  a  proper  return  cooler. 


1  Journ.  of  biol.  Chem.,  11. 

2  Skand.  Arch.  f.  Physiol.,  2,  and  Morner,  ibid.,  14,  where  the   recent  literature 
may  also  be  found. 

3  According  to  Wiechowski,  Hofmeister's  Beitriige,  11,  the  quantity  of    allantoin 
is  so  great  in  urine  that  it  must  be  considered  in  this  method. 

4  Zeitschr.  f.  physiol.  Chem.,  32,  3<>  and  37. 
8  See  Morner,  Skand.  Arch.  f.  Physiol.,  14. 


690  URINE. 

After  cooling  it  is  diluted  to  about  f  to  1  liter  with  water,  the  ammonia 
completely  distilled  off,  after  making  it  alkaline  with  caustic  soda,  and 
the  ammonia  collected  in  standard  acid.  After  boiling  in  order  to  drive 
off  the  CO2  and  cooling,  the  acid  is  retitrated. 

In  recent  years  objections  of  various  kinds  have  been  made  against 
these  methods,  which  are  directed  towards  their  exactness  and  which  have 
led  to  changes  in  several  directions  (Benedict  and  Gephart,  Levene 
and  Meyer,  Gill,  Allison  and  Grindley).  These  changes  are: 
precipitation  of  the  other  nitrogenous  substances  (nearly  all  the  ammonia) 
with  phosphotungstic  acid,  decomposition  of  the  urea  in  the  nitrate  by 
heating  with  acid  in  an  autoclave  to  150,°  and  distilling  off  the  ammonia 
from  the  solution,  made  alkaline,  not  by  boiling  with  alkali,  but  by  the 
aid  of  a  vacuum  or  by  means  of  a  current  of  air.  These  changes  have 
been  carefully  studied  by  Henriques  and  Gammeltoft  *  and  they  have 
suggested  the  following  method: 

Henriques  and  Gammeltoft  Method.  First  determine  in  5  cc.  urine 
how  much  of  a  10  per  cent  phosphotungstic  acid  solution  (in  N/2  H2SO4) 
is  necessary  to  exactly  cause  a  complete  precipitation.  Then  place  10  cc. 
of  the  urine  in  a  100  cc.  flask,  add  the  determined  quantity  of  phospho- 
tungstic acid  solution  and  fill  the  flask  up  to  the  100  cc.  mark  with  N/2 
H2SO4.  The  liquid  is  allowed  to  stand  after  mixing  until  it  has  settled 
and  it  is  then  filtered.  Two  portions  of  10  cc.  each  are  placed  in  test- 
tubes  of  Jena  glass,  covered  with  tin-foil  and  placed  in  the  autoclave  at 
150°  C.  for  1^  hours.  The  contents  of  the  test-tubes  are  now  placed  in  a 
flask,  and  the  ammonia  determined  either  by  passing  a  current  of  air 
through  it  (after  the  addition  of  sodium  carbonate)  or  by  distillation  in  a 
vacuum  (after  the  addition  of  barium  hydrate  dissolved  in  methyl  alcohol). 
Folin  and  Pettibone2  have  suggested  a  method,  according  to  which  the 
ammonia  is  determined  colorimetrically  with  Nessler  reagent. 

Kxop-Hupner's  method  3  is  based  on  the  fact  that  urea,  by  the  action  of 
sodium  hypobromite,  splits  into  water,  carbon  dioxide  (which  dissolves  in  the 
alkali),  and  nitrogen,  whose  volume  is  measured  (see  page  686).  This  method 
is  less  accurate  than  the  preceding  ones,  and  therefore  in  scientific  work  it  is  dis- 
carded. It  is  of  value  to  the  physician  and  for  practical  purposes,  because  of 
the  ease  and  rapidity  with  which  it  may  be  performed,  even  though  it  may  not 
give  very  accurate  results.  For  practical  purposes  a  number  of  different  appa- 
ratus have  been  constructed  to  facilitate  the  use  of  this  method. 


1  Benedict  and  Gephart,  Journ.  of  Amer.  Chern.  Soc,  30;  Levene  and  Meyer, 
ibid.,  81;  Gill,  Allison  and  Grindley,  ibid.,  31;  Henriques  and  Gammeltoft,  Skand. 
Arch.  f.  Physiol.,  25. 

:in  and  Pettibone,  Journ.  of  Biol.  Chem.,  11. 

'  Knop,  Zeitechr.  f.  analyt.  Chern.,  9;  Hiifner,  Journ.  f.  prakt.  Chem.  (N.  F.),  3. 
In  regard  to  the  extensive  literature,  see  Huppert-Neubauer,  10.  Aufl.,  304,  and  follow- 
ing.    See  also  Keogh,  Zeitechr.  f.  physiol.  Chern.,  84. 


CARBAMIC  ACID.  G91 

In  regard  to  other  met  lux  Is  such  as  Bunben's  method  with  its  man. 
modifications  as  suggested  by  Pfluger,  Bohland  and  Bleibtreu, 
we  refer  to  more  complete  handbooks. 

For  the  quantitative  estimation  of  urea  in  blood  or  other  animal 
fluids,  as  well  as  in  the  tissues.  ScHONDOBFF  has  proposed  a  method 
where  the  proteins  and  extractives  are  first  precipitated  by  a  mixture 
of  phosphotungstic  acid  and  hydrochloric  acid,  and  then  the  filtrate 
made  alkaline  with  lime.  The  quantity  of  ammonia  formed  on  heating 
a  part  of  this  filtrate  to  150°  C.  with  phosphoric  acid  and  the  amount 
of  carbon  dioxide  produced  by  heating  the  other  part  to  150°  C.  are 
determined.  In  regard  to  the  principles  of  this  method,  as  well  a-  to 
the  details,  we  refer  to  the  original  article  (Pfluger's  Arch.,  (>2).  Sal- 
cowski  '  has  recently  suggested  a  method  for  estimating  the  urea  in 
tissues. 

Urein  is  the  name  given  by  Ovid  Moor  to  a  product  which  he  obtained  by 
extracting  urine,  which  had  been  evaporated  to  a  syrup,  with  absolute  alcohol 
and  precipitating  the  urea  with  alcohol  containing  oxalic  acid,  or  by  cooling  and 
treatment  with  alcohol.  Urein  is  a  golden-yellow  oil  which  is  poisonous;  it 
reduces  permanganate  in  the  cold,  and  it  forms  the  chief  portion  of  the  nitro- 
genous extractives  of  urine.  There  is  no  doubt  that  urein  is  a  mixture  of  several 
substances.  According  to  Moor,2  the  amount  of  urea  in  the  urine  is  only  about 
one-half  that  ordinarily  given,  and  he  has  suggested  a  new  method  for  the  deter- 
mination of  the  true  quantity  of  urea.  The  possibility  that  in  the  urine  we  have 
other  bodies  besides  urea  which  have  been  determined  with  the  urea  cannot  be 
denied  a  priori.  From  the  investigations  published  so  far  it  must  be  said  that 
Moor's  assertions  are  not  sufficientlv  grounded.3 

/XH2 

Carbamic  Acid,  CH3XOj=CO<  .     This  acid  is  not  known  in  the  free 

xOH 
state,  but  only  as  salts.  Ammonium  carbamate  is  produced  by  the  action  of 
dry  ammonia  on  dry  carbon  dioxide,  but  also  after  the  addition  of  Xa.-C'O,  to  a 
solution  which  contains  an  ammonium  salt  (Macleod  and  Haskixs).  Carbamic 
acid  is  also  produced  by  the  action  of  potassium  permanganate  on  protein  and 
several  other  nitrogenous  organic  bodies. 

The  occurrence  of  carbamic  acid  in  human  and  animal  urines  has  already 
been  considered  in  connection  with  the  formation  of  urea.  The  calcium  salt 
which  is  soluble  in  water  and  ammonia  but  insoluble  in  alcohol,  is  the  most  impor- 
tant in  the  detection  of  this  acid.  The  solution  of  the  calcium  salt  in  water 
becomes  cloudy  on  standing,  but  much  more  quickly  on  boiling,  and  calcium  car- 
bonate separates.  Xolf,  Macleod  and  Haskixs  have  made  experiments  as  to 
the  method  of  formation  of  carbamic  acid.  The  latter  have  indicated  a  new 
method  for  the  quantitative  estimation  of  carbamates.4 

1  Arbeiten  aus  deni  pathol.  Institute,  Berlin,  1906. 

20.  Moor,  BuM.  Acad,  de  St.  Petersbourg,  14  (also  Maly's  Jahresber.,  31,  415), 
and  Zeitschr.  f.  Biologie,  44  and  45,  and  Zeitschr.  f.  physiol.  Cheai.,  41)  and  48. 

3  See  Kuliabko.  Maly's  Jahresber.,  31,  415;  Erben,  Zeitschr.  f.  physiol.  Chem., 
38;  Folin,  ibirl.,  37;  Gies,  Journ.  Amer.  Chem.  Soc,  25;  Haskins,  Anier.  Journ.  of 
Physiol.,  12;  Lippich,  Zeitschr.  f.  physiol.  Chem.,  48  and  52. 

4  Xolf,  Zeitschr.  f.  physiol.  Chem.,  23;  Macleod  and  Haskins,  Amer.  Journ.  of 
Physiol.,  12,  and  Journ.  of  biol.  Chem.,  1. 


692  URINE. 

Carbamic-acid  ethylester  (urethane),  as  shown  by  Jaffe,1  may  pass,  by  the 
mutual  action  of  alcohol  and  urea,  into  the  alcoholic  extract  of  urine  when  one 
is  working  with  large  quantities. 

Folin  2  claims  that  all  human  urine  contains  a  body  which  is  probably  methyl- 
urea. 

7NH CO 

Creatinine,  C4H7N3O,  or  NH :  C\  ,   is  the  anhydride  of 

\N(CH3).CH2 

/NH2 
Creatine,  NH:C\  ,    which    occurs  in   the    muscles, 

xN(CH3).CH2.COOH 
bird  urine  and  sometimes  also  in  human  urine. 

Creatinine  occurs  in  human  urine  and  in  that  of  certain  mammalia. 
It  has  also  been  found  in  ox-blood,  milk,  though  in  very  small  amounts, 
in  meat  extracts,  and  in  the  flesh  of  certain  fishes. 

The  quantity  of  creatinine  in  human  urine  is,  in  a  grown  man  voiding 
a  normal  quantity  of  urine  in  the  course  of  a  day,  0.6-1.3  grams  (Netj- 
bauer),  or  on  an  average  i  gram.  Johnson3  found  1.7-2.1  grams  per 
day,  and  similar  results  have  been  obtained  by  v.  Hoogenhuyze  and 
Verploegh.4  The  quantity  of  creatinine  with  a  diet  free  from  meat 
is,  Folin  5  says,  variable  for  different  individuals,  but  is  constant  for  the 
same  person.  He  never  found  the  quantity  below  1  gram  and  often 
between  1.3  and  1.7  grams.  Nurslings  also  eliminate  creatinine,  although 
the  quantity  is  small  (v.  Hoogenhuyze  and  Verploegh).  The  quantity 
of  creatinine  nitrogen  in  per  cent  of  the  total  nitrogen  varies  under 
different  conditions,  but  is  on  an  average  about  4.5-6.9  per  cent,  as 
determined  by  several  experimenters. 

Creatine  occurs  especially  in  the  urine  of  birds  and  also  in  the  urine 
of  nurslings,  but  also  in  older  children  (Rose,  Folin  and  Denis).  It 
has  also  been  found  in  the  urine  of  pregnant  women  (Kratjse  and  Cramer) 
but  otherwise  only  in  starvation,  in  diabetes,  diseases  of  the  liver,  fevers 
and  diseases  accompanied  by  a  destruction  of  the  body  proteins,  espe- 
cially muscle-proteins.  Between  creatine  and  creatinine  elimination  a 
relation  exists  it  seems,  at  least  for  certain  cases,  namely  with  a  decrease 
in  the  quantity  of  creatinine  eliminated  the  quantity  of  creatine  increases 
(Levene  and  Kristellerj.6 


1  Zeitschr.  f.  physiol.  Chem.,  14. 

7  Journ.  of  biol.  Chem.,  3. 

J  Huppert-Neubauer,  Harnanalyse,  10.  Aufl.,  387. 

*  Zeitschr.  f .  physiol.  Chem.,  4f>. 

*  Amer.  Journ.  of  Physiol.  13;  af.  Klercker,  Hofmeister's  Beitrage,  8. 

6  Rose.  Journ.  of  biol.  Chem.,  10;  Folin  and  Denis,  ibid.,  11;  Krauee  and  Cramer, 
Journ.  of  Physiol.,  40  (Proc.  physiol.  Soc,  July,  1910,  LXI);  Schaffer,  Amer.  Journ. 
of  Physiol.,  23;  Levene  and  Kristeller,  ibid.,  24. 


CREATININE.  693 

As  the  two  bodies,  creatine  and  creatinine,  can  easily  be  transformed 
into  each  other,  it  has  been  considered  for  a  long  time  that  the  urinary 
creatinine  is  formed  from  the  creatine  of  the  muscles  and  other  organs. 
Unfortunately  the  authorities  disagree  on  this  question.  Folin  in  his 
investigations  found  that  about  80  per  cent  of  the  creatinine  intro- 
duced was  again  eliminated,  while  the  creatine  taken  did  not  appear  in 
the  urine  as  creatinine,  but  was  partly  retained  by  the  body  and  in 
part  eliminated,  as  such.  An  intravital  transformation  of  creatine  into 
creatinine  is  disputed  by  v.  Klercker,  Mellanby  and  Lefmann,1  while 
it  is  accepted  by  Gottlieb,  Stangassinger,  S.  Weber,  v.  Hoogen- 
huyze and  Verploegh  and  Rothmann.  The  observations  of  Myers 
and  Fine  indicate  a  production  of  urinary  creatinine  from  creatine,  that  is 
they  found  that  the  creatinine  elimination  by  the  urine  in  rabbits  was 
greater  according  to  the  total  creatine  content  of  the  respective  animal. 
The  investigations  of  Pekelharing  and  v.  Hoogenhuyze  on  the  behavior 
of  parenterally  introduced  creatine  in  rabbits  and  dogs,  show  without 
any  doubt  that  a  part  of  the  creatine  is  actually  transformed  into  creatinine. 
Towles  and  Voegtlin  2  have  also  observed  that  the  subcutaneously 
injected  creatine  increases  somewhat  the  creatinine  elimination,  while  this 
is  not  the  case  with  creatine  taken  per  os.  The  condition  of  the  digestive 
apparatus  also  seems  to  be  of  importance  here.  Pekelharing  and  v. 
Hoogenhuyze  found  that  in  dogs  of  the  parenterally  introduced  creatine 
always  a  smaller  part  (as  creatine  and  creatinine)  passed  into  the  urine 
during  the  digestion  than  during  rest  of  the  digestive  organs.  They 
explain  this  by  the  accepted  ability  of  the  liver  to  partly  destroy  the 
creatine  and  partly  by  an  anhydride  formation  of  transforming  the  creatine 
into  creatinine. 

As  mentioned  in  Chapter  X  the  proteins  and  the  guanidine  groups 
therein  are  considered  as  the  mother-substance  of  these  two  bodies. 
If  the  creatinine  (creatine)  originates  from  the  protein  it  is  evident  that 
we  must  differentiate  between  food-protein  and  body-protein.  The  quan- 
tity of  creatinine  is,  inasmuch  as  it  is  increased  by  meat  diet,  dependent 
upon  the  food;  but  otherwise,  as  found  by  Folin  and  in  chief  substantiated 
by  others,  is  rather  independent  of  the  food.  Its  elimination  does  not 
run  parallel  with  the  urea  and  the  total  nitrogen,  and  consequently  is 
not  in  general  greater  with  food  rich  in  protein  than  with  food  poor  therein. 
On  the  contrary,  its  extent,  as  shown  by  other  conditions,  is  dependent 
upon  the  intensity  of  the  metabolism  in  the  cells,  especially  the  muscle 

1  Folin,  Hammarsten's  Festschrift,  1906;  v.  Klercker,  Bioch.  Zeitschr.,  3;  Mellanby, 
Journ.  of  Physiol.,  36;  Lefmann,  Zeitschr.  f.  physiol.  Chem.,  57. 

2  See  footnote  1,  page  574,  and  v.  Hoogenhuyze  and  Verploegh,  Zeitschr.  f.  physiol. 
Chem.,  59;  Pekelharing  and  v.  Hoogenhuyze,  ibid.,  69;  Towles  and  Voegtlin,  Journ. 
of  biol.  Chem.,  10;    Myers  and  Fine,  ibid.,  14. 


694  URINE. 

tissue,  and  the  creatinine,  according  to  Folin,  is  a  product  of  the  endo- 
genous protein  metabolism. 

Reports  as  to  the  behavior  of  the  creatinine  elimination  with  work 
are  conflicting,  v.  Hoogenhuyze  and  Verploegh,  who  made  use  of 
a  much  more  trustworthy  method  of  quantitative  estimation  than  their 
predecessors,  find  that  muscular  activity  as  a  rule  does  not  cause  any 
rise  in  the  creatinine  elimination,  and  that  in  man  such  a  rise  with  work 
occurs  only  when  the  body  is  obliged  to  live  upon  its  own  tissues.  S. 
Weber  l  also  finds  an  absolute  increase  in  the  elimination  of  creatinine 
onty  in  starving  dogs.  Other  investigators  could  not  find  any  increase 
in  the  elimination  of  creatinine  by  work,  although  such  a  rise  was  found 
as  shown  by  Pekelharing  and  Harkink,2  by  the  muscle  tonus. 

In  starvation,  a  decrease  in  the  creatinine  but  a  simultaneous  increase 
in  the  elimination  of  creatine  has  been  found  in  man  (v.  Hoogenhuyze 
and  Verploegh,  Cathcart,  Benedict  and  Myers3).  Such  an  increase 
in  the  creatinine  elimination  only  occurs  in  those  conditions  which  are 
accompanied  by  acidosis,  and  correspondingly  it  can  be  prevented  by 
the  introduction  of  carbohydrates  (Cathcart,  Mendel  and  Rose) 
The  creatinine  elimination  in  certain  cases  has  therefore  been  explained 
by  a  disturbed  carbohydrate  metabolism.  This  is  neverthless  on  the 
other  hand  disputed  by  Wolf  and  Oesterberg  4  who  find  that  the  crea- 
tinine elimination  in  starvation  can  be  arrested  by  the  introduction  of 
proteins  alone. 

Little  is  known  about  the  behavior  of  creatinine  in  disease,  nor  are 
the  observations  in  accord.  In  anaemia  and  cachexia  the  elimination 
of  creatinine  is  diminished,  and  when  the  metabolism  is  increased  the 
elimination  is  also  increased.  That  this  is  the  case,  at  least  in  fevers, 
seems  to  be  borne  out  by  several  concurrent  observations.5  In  diseases 
of  the  liver  a  diminished  elimination  of  creatinine  may  occur,  and  in  cases 
of  carcinoma  of  the  liver  considerable  creatine  has  been  found  in  the  urine 
(v.  Hoogenhuyze  and  Verploegh,  Mellanby).  The  role  of  the  liver 
in  the  creatine-creatinine  metabolism,  is,  as  has  already  been  mentioned 
in  Chapter  X,  not  clear.  The  exclusion  of  the  liver  from  the  metabolism 
of  a  dog  with  Eck  fistula  had  no  result  in  the  experiments  of  Towles 


1  Arch.  f.  exp.  Path.  u.  Pharm.,  58.  Further  literature  may  be  found    in  v.  Hoog- 
enhuyze and  Verploegh,  Zeitschr.  f.  physiol.  Chem.,  46. 

2  Maillard  and  Clausmann,  Journ.  de  Physiol,  et  de  Path.,  12;    Prayon,  Maly's 
Jahresb.,  40;  Pekelharing  and  Harkink,  Zeitschr.  f.  physiol.  Chem.,  75. 

3  v.  Hoogenhuyze  and  Verploegh,  Zeitschr.  f.  physiol.  Chem.,  57;  Cathcart,  Bioch. 
Zeitschr.,  6;  Benedict  and  Myers,  Amer.  Journ.  of  Physiol.,  18;  Jaffe,  1.  c. 

4  Cathcart,  Jour,  of  Physiol.,   39:  Mendel  and  Rose,  Journ.  of  biol.  Chem.,  10; 
Wolf  and  Osterberg,  Bioch.  Zeitschr.,  35;  Wolf,  Journ.  of  biol.  Chem.,  10. 

6  See  0.  af  Klercker,  Zeitschr.  f.  klin.  Med.,  68. 


PROPERTIES   OF  CREATININE.  895 

and  Voegtlin.     The  dogB,   after  feeding  with  creatine  and  creatinine, 

I  ehaved  like  normal  d<  gs,  and  the  observations  of  other  investigators 
such  as  London  and  Boljabski,  Foster  and  Fisher1  upon  dogs  with 
Eck  fistula  have  not  had  any  unanimous  results  or  they  are  hard  to  explain. 
Properties  of  Creatinine.  Creatinine  crystallizes  in  colorless,  Bhinin; 
monoclinic  prisms  which  differ  from  creatine  crystals  in  not  becoming 
white  with  loss  of  water  when  heated  to  100°  C.  It  dissolves  in  11  parts 
cjld  water,  but  more  easily  in  warm  water.  It  is  difficultly  soluble  in 
cold  alcohol,  but  the  reports  in  regard  to  its  solubility  differ  widely.2 
It  is  more  soluble  in  warm  alcohol  and  nearly  insoluble  in  ether.  In 
alkaline  solution  creatinine  is  very  easily  converted  into  creatine  on 
warming. 

Creatinine  gives  an  easily  soluble  crystalline  compound  with  hydro- 
chloric acid.  A  solution  of  creatinine  acidified  with  mineral  acids  gives 
crystalline  precipitates  with  phosphotungstic  and  phosphomolybdic 
acids  even  in  very  dilute  solutions  (1:10000),  (Kerner,  Hofmeister3). 
It  is  precipitated,  like  urea,  by  mercuric-nitrate  solution  and  also  by 
mercuric  chloride.  On  treating  a  dilute  creatinine  solution  with  sodium 
acetate  and  then  with  mercuric  chloride  a  precipitate  of  glassy  globules 
having  the  composition  4(C4H7N30.HCl.HgO)3HgCl2  separates  on 
standing  some  time  (Johnson).  Among  the  compounds  of  creatinine, 
that  with  zinc  chloride,  creatinine-zinc  chloride,  (C^H^NsO^ZnClo.  is 
of  special  interest.  This  combination  is  obtained  when  a  sufficiently 
concentrated  solution  of  creatinine  in  alcohol  is  treated  writh  a  concentrated, 
faintly  acid  solution  of  zinc  chloride.  Free  mineral  acids  dissolve  the  com- 
pound, hence  they  must  not  be  present;  this,  however,  may  be  prevented 
by  an  addition  of  sodium  acetate.  In  the  impure  state,  as  from  urine, 
creatinine-zinc  chloride  forms  a  sandy,  yellowish  powder  which  under 
the  microscope  appears  as  fine  needles,  forming  concentric  groups, 
mostly  complete  rosettes  or  yellow  balls  or  tufts,  or  grouped  as  brushes. 
On  slowly  crystallizing  or  when  very  pure,  more  sharply  defined  prismatic 
crystals  are  obtained.     The  compound  is  slightly  soluble  in  water. 

Creatinine  acts  as  a  reducing  agent.  Mercuric  oxide  is  reduced  to 
metallic  mercury,  and  oxalic  acid  and  methylguanidine  (methyluramine) 
are  formed.  Creatinine  also  reduces  cupric  hydroxide  in  alkaline  solution, 
forming  a  colorless  soluble  compound,  and  only  after  continued  boiling 
with  an  excess  of  copper  salt  is  free  suboxide  of  copper  formed.     Creat- 


1  Towles  and  Voegtlin,   1.  c;  London  and  Boljarski,   Zeitschr.  f.  physiol.  Chem., 
62;  Foster  and  Fischer,  Journ.  of  biol.  Chem.,  9. 

2  See    Huppert-Xeubauer,   10.  Aufl.,  and    Hoppe-Sevler-Thierfelder's    Handbuch. 
8.  Aufl. 

3  Kerner,  Pfliiger's  Arch.,  8;  Hofmeister,  Zeitschr.  f.  physiol.  Chem.,  o. 


696         .  URINE. 

inine  interferes  with  Trommer's  test  for  sugar,  partly  because  it  has  a 
reducing  action,  and  partly  by  retaining  the  copper  suboxide  in  solution. 
The  compound  with  copper  suboxide  is  not  soluble  in  a  saturated  soda 
solution,  and  if  a  little  creatinine  is  dissolved  in  a  cold  saturated  soda 
solution  and  then  a  few  drops  of  Fehling's  reagent  added,  a  white  flocculent 
compound  separates  after  heating  to  50-60°  C.  and  then  cooling  (v. 
Maschke's1  reaction).  An  alkaline  bismuth  solution  (see  Sugar  Tests) 
is  not  reduced  by  creatinine. 

An  aqueous  solution  of  creatinine  is  precipitated  by  picric  acid. 
The  precipitate  consists  on  recrystallization  from  hot  water,  of  thin, 
silky,  pale  yellow  needles  (Jaffe).  If  the  urine  is  treated  with  picric 
acid  (20  cc.  of  a  5  per  cent  solution  in  alcohol  for  each  100  cc.  urine), 
then  a  double  picrate  of  creatinine  and  potassium  is  precipitated  (Jaffe). 
If  a  solution  of  creatinine  in  water  (or  urine)  is  treated  with  a  watery 
solution  of  picric  acid  and  a  few  drops  of  a  dilute  caustic-soda  solution, 
a  red  coloration,  lasting  several  hours,  immediately  occurs  at  the  ordinary 
temperature,  which  turns  yellow  on  the  addition  of  acid  (Jaffa's  2  reac- 
tion). Acetone  gives  a  more  reddish-yellow  color.  Glucose  gives 
with  this  reagent  a  red  coloration  only  after  heating.  If  we  add  a  few 
drops  of  a  freshly  prepared  very  dilute  sodium-nitrcprusside  solution 
(sp.gr.  1.003)  to  a  dilute  creatinine  solution  (or  to  the  urine)  and  then  a 
few  drops  of  caustic  soda,  a  ruby-red  liquid  is  obtained  which  quickly 
turns  yellow  again  (Weyl's3  reaction).  If  the  cold  yellow  solution  is 
neutralized  and  treated  with  an  excess  of  acetic  acid,  a  crystalline  pre- 
cipitate of  a  nitroso-compound  (C4H6N4O2)  of  creatinine  separates  on 
stirring  (Kramm)  or  creatininoxim  (Schmidt4).  If,  on  the  contrary, 
the  yellow  solution  is  treated  with  an  excess  of  acetic  acid  and  heated, 
the  solution  becomes  first  green  and  then  blue  (Salkowski  5) ;  finally 
a  precipitate  of  Prussian  blue  is  obtained. 

A  reaction  which  in  description  is  similar  and  which,  although  not  solely 
(Arnold)  but  at  least  partially  (Holobut),  appears  after  partaking  of  protein 
food  or  meat  soup  is  Arnold's  reaction.6  This  reaction  is  due  to  an  unknown 
endogenous  metabolism  product.  If  10-20  cc.  urine  are  treated  with  a  few  drops 
of  a  4  per  cent  sodium  nitroprusside  solution  and  then  with  5-10  cc.  of  a  5  per 
cent  sodium  or  potassium  hydroxide  solution,  at  first  a  strong  and  pure  violet 
color  is  obtained  with  an  absorption  band  between  D  and  E,  then  it  becomes 
purple-red  and  then  brown-red  and  finally  yellow.      On  the  addition  of  acetic 


1  Zeitschr.  f.  analyt.  Chem.,  17. 

2  Zeitschr.  f.  physiol.  Chem.,  10. 

3  Ber.  d.  deutsch.  chem.  Gesellsch.,  11. 

'Kramm,  Centralbl.  f.  d.  med.  Wissensch.,   1897;    Schmidt,  cited  from  Chem. 
Centralbl.,  1012,  2. 

5  Zeitschr.  f.  physiol.  Chem.,  4. 

8  Arnold,  Zeitschr.  f.  physiol.  Chem.,  49  and  83;  Holobut,  ibid.,  56. 


ESTIMATION   OF  CREATININE.  697 

acid  the  violet  or  purple-red  color  passes  into'blue,  which  soon  becomes  pale 
and  finally  a  pale  yellow  color.  It  differs  from  the  creatinine  in  color  and 
the  absorption  band  as  well  as  in  that  the  creatinine  reaction  requires  more 
sodium  oitroprusside. 

The  best  method  for  preparing  creatinine  is  the  following,  suggested 
by  Folin.1  The  creatinine  is  first  precipitated  as  the  double  picrate 
of  creatinine  and  potassium  by  means  of  picric  acid  according  to  Jaffa's 
method,  and  then  this  precipitate,  while  still  moist,  is  decomposed  by 
KHCO3  and  water.  The  solution,  which  contains  the  creatinine  besides 
potassium  carbonate  and  small  amounts  of  impurities,  is  neutralized 
with  sulphuric  acid  and  the  sulphate  precipitated  by  alcohol.  The 
creatinine  is  now  converted  into  the  double  zinc-chloride  salt  and  this 
last  treated  with  moist  lead  hydroxide.  After  the  removal  of  the  lead, 
the  solution  contains  a  mixture  of  creatinine  and  creatine,  which  last  is 
completely  transformed  into  creatinine  by  heating  for  forty-eight  hours 
with  normal  sulphuric  acid.  After  exact  neutralization  with  barium- 
hydroxide   solution   it   is   concentrated   to  the  point   of   crystallization. 

According  to  recent  work  of  Folin  and  Blanck  the  creatinine-zinc  chloride 
can  be  dissolved  in  warm  10  per  cent  sulphuric  acid  when  creatinine-zinc  alum 
(C\H7NsO)2SOiZnSOi,  8H20  is  obtained  and  from  this  the  creatinine  can  be 
obtained  by  decomposing  with  barium  acetate  and  removing  the  zinc  by  H2S. 
Creatine  can,  according  to  Folin  and  Denis,2  be  transformed  into  creatinine  by 
heating  in  an  autoclave  for  3  hours  under  a  pressure  of  4-5  kg.  per  qcm. 

The  quantitative  estimation  of  creatinine  used  to  be  performed  accord- 
ing to  Neubauer's  method  for  the  preparation  of  creatinine,  or  more 
simply  by  Salkowski's3  modification  of  this  method.  As  this  method 
is  now  seldom  used  we  refer  the  reader  to  other  hand-books. 

Folin  4  has  suggested  a  colorimetric  method  for  determining  creatinine 
which  is  based  upon  Jaffe's  picric-acid  reaction  and  is  as  follows:  10 
cc.  of  the  urine  are  treated  in  a  graduated  flask  of  500  cc.  capacity 
with  15  cc.  of  a  1.2  per  cent  solution  of  picric  acid  and  5  cc.  of  a  10 
per  cent  NaOH  solution.  After  shaking  and  allowing  to  stand  for  five 
minutes  it  is  diluted  with  water  to  500  cc.  and  mixed.  This  solution 
is  now  compared  in  a  Duboscq  colorimeter  "with  a  1/2  normal  potassium- 
dicromate  solution.  The  latter  solution  has  in  a  layer  8  mm.  thick 
exactly  the  same  intensity  of  color  as  a  layer  8.1  mm.  thick  of  a  solution 
of  10  milligrams  creatinine  after  the  addition  of  15  cc.  picric-acid  solu- 
tion and  5  cc.  NaOH  solution  and  dilution  to  500  cc.  The  calculations 
are  simple.  For  example,  in  case  the  urine  tested  in  a  layer  7.2  mm. 
thick  has  the  same  color  as  the  dichromate  solution  in  a  layer  8  mm. 
thick,  then  the  quantity  of  creatinine  in   10  cc.  of  the  urine  will  be 

8  1 
=  ^2X10,  or  11.25  milligrams.     This  method  has  been  tried  by  many 

authorities  and  found  to  be  trustworthy. 


1  Zeitschr.  f.  physiol.  Chem.,  41. 

2  Folin  and  Blanck,  Journ.  of  biol.  Chem.,  8,  with  Denis,  ibid.,  8. 

3  Zeitschr.  f.  physiol.  Chem.,  10  and  14. 
*  Ibid.,  41. 


698  URINE. 

The  same  method  is  used  in  the  determination  of  creatine,  which 
for  this  purpose  is  first  converted  into  creatinine  by  warming  with  dilute 
mineral  acid.  The  quantity  of  creatine  is  the  difference  obtained  between 
the  values  for  creatinine  before  and  after  treatment  with  acid.  More 
detailed  directions  can  be  found  in  the  cited  works  of  Folin,  v.  Hoogen- 
htjyze  and  Verploegh,  Gottlieb  and  Stangassinger. 

In  regard  to  other  methods,  see  the  works  of  Kolisch  and  Gregor.1 

Xanthocreatinine,  C5Hi0N4O.  This  body,  which  was  first  prepared  from 
meat  extract  by  Gautier,  has  been  found,  by  Monari,  in  dog's  urine  after  the 
injection  of  creatinine  into  the  abdominal  cavity,  and  in  human  urine  after  several 
hours  of  exhaustive  inarching.  According  to  Colasanti  it  occurs  to  a  relatively 
greater  extent  in  lion's  urine.  Stadthagen  2  considers  the  xanthocreatinine 
isolated  from  human  urine  after  strenuous  muscular  activity  as  impure  creatinine. 

Xanthocreatinine  forms  thin  sulphur-yellow  plates,  similar  to  cholesterin, 
which  have  a  bitter  taste.  It  dissolves  in  cold  water  and  in  alcohol,  and  gives 
a  crystalline  compound  with  hydrochloric  acid  and  a  double  compound  with 
gold  and  platinum  chloride.  It  gives  a  compound  with  zinc  chloride,  which 
crystallizes  in  fine  needles.     Xanthocreatinine  has  a  poisonous  action. 

Methylguanidine  occurs,  according  to  Achelis,  Kutscher  and  Lohmann, 
to  a  slight  extent  as  a  regular  constituent  of  the  urine  of  man,  horse  and  dog. 
It  has  been  found  in  urines  associated  with  dimethylguanidine  by  Engeland.3 

HN— CO 

I       I 
Uric  Acid,  Ur,  C5H4N4O3;  2,  6,  8-trioxypurine,  OC     C— NHV        ,  has 

I      II  >CO 

HN— C— NH' 
been  prepared  synthetically  by  Horbaczewski  by  fusing  urea  anu 
glycocoll,  or  by  heating  trichlorlactic-acid  amide  with  an  excess  of  urea. 
Behrend  and  Roosen  prepared  it  from  isodialuric  acid  and  urea;  it 
is  also  readily  produced  from  isouric  acid  on  boiling  with  hydrochloric 
acid  (E.  Fischer  and  Tullner)  and  finally  E.  Fischer  and  Ach  4  have 
prepared  uric  acid  from  pseudouric  acid  by  heating  with  oxalic  acid  to 
145°  C. 

On  strongly  heating  uric  acid  it  decomposes  with  the  formation  of 
urea,  hydrocyanic  acid,  cyanuric  acid,  and  ammonia.  On  heating  with 
concentrated  hydrochloric  acid  in  sealed  tubes  to  170°  C.  it  splits  into 
glycocoll,  carbon  dioxide,  and  ammonia.  By  the  action  of  oxidizing 
agents  splitting  and  oxidation  take  place,  and  either  monoureides  or 
diureides  are  produced.     By  oxidation  with  lead  peroxide,  carbon  dioxide, 

1  Kolisch,  Centralbl.  f.  innere  Med.,  1805;  Gregor,  Zeitschr.  f.  physiol.  Chem.,  31. 

1  Gautier,  Bull,  de  l'acad.  demed.  (2),  15,  and  Bull,  de  la  soc.  chim.  (2),  48;  Monari, 
Maly's  Jahresber.,  17;  Colasanti,  Arch.  ital.  d.  Biologie,  15,  Fasc.  3;  Stadthagen, 
Zeitschr.  f.  klin.  Med.,  15. 

1  Achelis,  Centralbl.  f.  Physiol.,  20,  455,  and  Zeitschr.  f.  physiol.  Chem.,  50;  Kut- 
Bcher  and  Lohmann,  ibid.,  49;  Engeland,  ibid.,  57. 

4  Horbaczewski,  Monatshefte  f.  Chem.,  6  and  8;  Behrend  and  Roosen,  Ber.  d.  d. 
chem.  Gesellsch.,  21 ;  Fischer  and  Tullner,  ibid.,  35;  Fischer  and  Ach,  ibid.,  28. 


URIC  ACID.  G99 

oxalic  acid,  urea,  and  allantoin,  which  last  is  glyoxyldiureide,  arc  pro- 
duced (see  below).  By  oxidation  with  nitric  acid  in  the  cold,  urea  and 
a  monoureide,  the  mesoxalyl  urea,  or  alloxan,  are  obtained,  ('.-,114X403+ 
0  +  H2O  =  C'4H2N204+(NH2)2C0.  On  warming  with  nitric  acid,  alloxan 
yields  carbon  dioxide  and  oxalyl  urea,  or  parabanic  acid,  C3H2N2O3. 
By  the  addition  of  water  the  parabanic  acid  passes  into  oxaluric  acid, 
C3H4N2O4,  traces  of  which  are  found  in  the  urine  and  which  easily  splits 
into  oxalic  acid  and  urea.  In  alkaline  solution  uric  acid  may,  by  taking 
up  water  and  oxygen,  be  transformed  into  a  new  acid,  uroxanic  acid, 
CsEfeNiOe,  which  may  then  be  changed  into  oxonic  acid,  C4H5N3O4.1 
On  the  oxidation  of  uric  acid  by  hydrogen  peroxide  in  alkaline  solution 
Sc  iiittenhelm  and  Wiener2  have  obtained  urea  with  carbonyl  diurea 
as  intermediary  product.  Uric  acid  may,  as  F.  and  L.  Sestini  as  well 
as  Gerard  have  shown,  undergo  bacterial  fermentation  with  the  forma- 
tion of  urea.  According  to  Ulpiani  and  Cingolani,3  uric  acid  is  quan- 
titatively split  into  urea  and  carbon  dioxide,  according  to  the  equation 

C5H^V>:,+2H20  +  30  =  3C02+2CO(NH2)2. 

Uric  acid  occurs  most  abundantly  in  the  urine  of  birds  and  of  scaly 
amphibians,  in  which  animals  the  greater  part  of  the  nitrogen  of  the  urine 
appears  in  this  form.  Uric  acid  frequently  occurs  in  the  urine  of  carniv- 
orous mammalia,  but  is  sometimes  absent;  in  urine  of  herbivora  it  is 
habitually  present,  though  only  as  traces;  in  human  urine  it  occurs  in 
greater  but  still  small  and  variable  amounts.  Traces  of  uric  acid  are 
also  found  in  several  organs  and  tissues,  as  in  the  spleen,  lungs,  heart, 
pancreas,  liver  (especially  in  birds),  and  in  the  brain.  It  always  occurs 
in  the  blood  of  birds.  Traces  have  been  found  in  human  blood  under 
normal  conditions.  Under  pathological  conditions  it  occurs  to  an 
increased  extent  in  the  blood,  as  in  pneumonia  and  nephritis,  but  espe- 
cially in  leucaemia  and  sometimes  also  in  arthritis.  Uric  acid  also  occurs 
in  large  quantities  in  "  chalk-stones,"  certain  urinary  calculi,  and  in 
guano.  It  has  also  been  detected  in  the  urine  of  insects  and  certain 
snails,  as  also  in  the  wings  (which  it  colors  white)  of  certain  butterflies 
(Hopkins4). 

The  amount  of  uric  acid  eliminated  with  human  urine  is  subject  to 
considerable   individual   variation,   but   amounts  on  an  average   to  0.7 

1  See  Sundwik,  Zeitschr.  f.  physiol.  Chem.,  20  and  41;  also  Behrend,  Annal.  d. 
Chem.  u.  Pharm.,  333. 

2  Zeitschr.  f .  physiol.  Chem.,  62. 

3  See  Chem.  Centralbl.,  1903,  where  the  other  investigators  are  cited,  and  Centralbl. 
f.  Physiol.,  19. 

4  Philos.  Trans.  Roy.  Soc,  186,  B,  661. 


700  URINE. 

gram  per  day  on  a  mixed  diet.  The  ratio  of  uric  acid  to  urea  varies  con- 
siderably with  a  mixed  diet,  but  is  on  an  average  1 :  50-1 :  70.  In  new- 
born infants  and  in  the  first  days  of  life  the  elimination  of  uric  acid  is 
relatively  increased,  and  the  relation  between  uric  acid  and  urea  has  been 
found  to  be  1:6.42-17.1. 

We  used  to  ascribe  an  increasing  action  upon  the  elimination  of  uric 
acid  to  protein  food,  but  the  investigations  of  Hirschfeld,  Rosen- 
feld  and  Orgler,  Siven,  Burian,  and  Shur,1  and  many  others  have 
positively  proven  that  a  diet  rich  in  protein  does  not  itself  increase  the 
elimination  of  uric  acid,  but  only  according  to  the  amount  of  nucleins 
or  purine  bodies  contained  therein.  The  common  assumption  that  the 
elimination  of  uric  acid  is  smaller  with  a  vegetable  diet  than  with  an  ani- 
mal diet,  when  the  quantity  may  be  2  grams  or  more  per  twenty-four 
hours,  is  explained  by  this.2 

Still  a  purine-free  diet  is  not  without  some  influence  upon  the  elimina- 
tion of  uric  acid,  as  the  quantity  of  uric  acid  eliminated  with  a  purine- 
free  diet  is  considerably  greater  than  in  starvation  and  can  be  increased 
by  protein  feeding.  The  action  of  the  food-protein  is  here  probably  an 
indirect  one,  consisting  in  that  the  proteins  raise  the  work  of  the  digestive 
glands  and  the  metabolism  of  their  cells  and  thereby  also  raise  the  endo- 
genous uric  acid  formation  (see  below)  somewhat.3  Work  and  rest  do 
not  seem  to  have  any  special  influence  upon  the  uric  acid  elimination, 
although  according  to  the  confirmed  statement  of  Siven  and  Leathes4 
the  elimination  in  the  night  is  less  than  in  the  morning  hours. 

The  reports  in  regard  to  the  influence  of  other  circumstances,  as  well 
as  of  different  substances,  on  the  elimination  of  uric  acid  are  diverse. 
This  is  in  part  due  to  the  fact  that  the  earlier  investigators  used  an 
inaccurate  method  (Heintz),  and  also  that  the  extent  of  uric-acid  elimina- 
tion is  dependent  in  the  first  place  upon  the  individuality.  Thus  the 
investigators  are   not   in  accord   in   regard   to   the   action  of   drinking- 


1  See  the  extensive  review  of  the  literature  in  Wiener,  "  Die  Harnsaure,"  in  Ergeb- 
nisse  der  Physiologie,  1,  Abt.  1,  1902. 

2  J.  Ranke,  Beobachtungen  und  Versuche  uber  die  Ausscheidung  der  Harnsaure, 
etc.  (Miinchen,  1858);  Mares,  Centralbl.  f.  d.  med.  Wissensch.,  1888;  Horbaczewski, 

Wien.  Sitzungsber.,  100,  Abt.  3,  1891.  In  regard  to  the  action  of  various  diets  the 
reader  is  referred  to  the  above-cited  authors,  and  especially  to  A.  Hermann,  Arch.  f. 
klin.  Med.,  43,  and  Camerer,  Zeitschr.  f.  Biologie,  33,  and  Folin,  Amer.  Journ.  of  Physiol., 
13. 

3  See  Hirschstein  Arch.  f.  exp.  Path.  u.  Pharm.,  57;  Smetanka,  Pfliiger's  Arch., 
]'.'>*>  und  140  ;  Mares,  ibid.,  134  and  140.  Contrary  views,  Brugsch  and  Schitten- 
helrn,  Zeitschr.  f.  exp.  Path.  u.  Therp.,  4,  and  Siven,  Pfliiger's  Arch.,  146. 

4  Siven,  Skand.  Arch.  f.  Physiol.,  11;  Leathes,  Journ.  of  Physiol,  35;  see  also  Ken- 
naway,  Journ.  of  Physiol.,  38. 


FORMATION  OF  URIC  ACID.  701 

water  l  and  of  alkalies.2  Certain  medicines,  such  as  quinine  and  atro- 
pine, diminish,  while  others,  such  as  pilocarpine  and,  as  it  seems,  salicylic 
acid,3  increase  the  elimination  of  uric  acid. 

There  .is  much  diversity  of  opinion  regarding  the  elimination  of  uric 
acid  in  disease,4  although  it  is  known  that  it  is  increased  after  an  abun- 
dant destruction  of  nucleated  cells  as  in  pneumonia,  after  the  crisis, 
and  in  leucaemia.  In  the  latter  in  most  cases  not  only  is  the  elimina- 
tion to  the  urea  increased  absolutely,  but  also  relatively ;  and  the  relation 
between  uric  acid  and  urea  (total  nitrogen  calculated  as  urea)  may  in 
lineal  leucaemia  even  be  1 : 9,  while  under  normal  conditions,  accord- 
ing to  different  investigators,  it  is  1:50  to  70  to  100.  As  to  the  behavior 
of  uric  acid  in  gout,  authorities  are  by  no  means  agreed.  That  the 
blood  contains  uric  acid  in  gout  has  been  repeatedly  shown,  and 
it  is  also  found  in  this  disease  with  a  purine-free  diet  (Brugsch  and 
Schittenhelm).  According  to  these  investigators  a  diminished  enzy- 
motic  decomposition  of  uric  acid  occurs  in  the  body  in  gout  and  this 
causes  the  occurrence  of  uric  acid  in  the  blood  and  its  accumulation  in 
certain  tissues.  Strong  arguments  against  this  view  have  been  presented 
by  others  such  as  Wells  and  Corper,  Miller  and  Jones.5 

Formation  of  Uric  Acid  in  the  Organism.  Since  Horbaczewski 
first  showed  that  uric  acid  could  be  produced  by  oxidation  from  the 
nuclein-rich  spleen-pulp  or  nucleins  outside  of  the  body,  he  also  showed 
that  nucleins  when  introduced  into  the  animal  body  caused  an  increase 
in  the  elimination  of  uric  acid.  These  observations  have  been  confirmed, 
and  at  the  same  time  developed  by  the  work  of  a  great  number  of  investi- 
gators, and  we  are  sure  that  uric  acid  can  be  produced  from  purine 
bases  either  outside  or  inside  the  animal  body,  and  also  that  food  rich 
in  nucleins  (especially  the  thymus  gland)  increases  the  elimination  of 
uric  acid.  It  is  nevertheless  true  that  a  few  investigators  after  intro- 
ducing pure  purine  bases  into  the  organism  could  not  observe  any  essential 
rise  in  the  uric  acid  or  its  transformation  products;  still  we  have  a  large 
number  of  recent  investigations  which  positively  show  that  nucleic  acids, 
as  well  as  purine  bases,  when  introduced  into  the  animal  body  are  trans- 
formed in  abundant  quantities  into  uric  acid  in  tho  body.6     At  present 

1  See  Schondorff.  Pfliiger's  Arch.,  46,  which  contains  the  pertinent  literature. 

2  See  Clar,  Centralbl.  f.  d.  ined.  Wissensch.,  1888;  Haig,  Journ.  of  Physiol.,  8;  and 
A.  Hermann,  Arch.  f.  klin.  Med.,  43. 

3  See  Bohland,  cited  from  Maly's  Jahresber.,  26;  Schreiber  and  Zaudy,  ibid.,  30. 
*  In  regard  to  the  extensive  literature  on  the  elimination  of  uric  acid  in  disease 

we  must  refer  to  special  works  on  internal  diseases. 

6  Brutisch  and  Schittenhelm,  Zeitschr.  f.  exp.  Path.  u.  Therp.,  4;  Wells  and  Corper, 
Journ.  of  biol.  Chem.,  6;  Miller  and  Jones,  Zeitschr.  f.  physiol.  Chem.,  61. 

6  As  it  is  not  within  the  scope  of  this  book  to  enter  into  a  discussion  of  the  numer- 
ous researches  on  this  subject,  we  will  refer  to  Wiener,  "  Die  Harnsiiure,"  Ergebnisse 


702  URINE. 

we  consider  the  formation  of  uric  acid  from  the  purine  bases  of  the  nuclein 
substances  as  a  positively  proven  fact. 

According  to  the  original  view  of  Horbaczewski  the  nucleins  do 
not  directly  (by  their  purine  bases)  cause  an  increased  elimination  of  uric 
acid,  but  indirectly  by  causing  a  leucocytosis  with  a  consequent  destruc- 
tion of  leucocytes.  This  view  has  been  justly  discarded  on  account, 
of  the  above-mentioned  conditions;  still  on  the  other  hand  it  cannot 
be  denied  that  the  formation  of  uric  acid  is  alsto  in  certain  regards  related 
to  the  formation  or  the  destruction  of  leucocytes  and  to  the  metabolism 
in  the  cells  as  a  whole.1 

The  uric  acid,  in  so  far  as  it  is  produced  from  nuclein  bases,  is  in  part 
derived  from  the  nucleins  of  the  destroyed  cells  of  the  body  and  in  part 
from  the  nucleins  or  free  purine  bases  introduced  with  the  food.  It 
is  therefore  possible  to  admit,  with  Burian  and  Schur,2  of  a  double  origin 
for  the  uric  acid  as  well  as  the  urinary  purines  (all  purine  bodies  of  the 
urine,  including  the  uric  acid),  namely,  an  endogenous  and  an  exogenous 
origin.  Burtan  and  Schur  attempted  to  determine  the  quantity  of 
endogenous  urinary  purines  by  feeding  with  sufficient  food,  but  as  free 
as  possible  from  purine  bodies,  and  they  found  that  this  quantity  was 
constant  for  every  individual,  while  it  was  variable  for  different  persons. 
The  observations  of  many  other  investigators  have  led  to  similar  con- 
clusions, and  we  are  now  unanimous  in  our  opinion  that  the  uric  acid 
originating  from  the  nucleins  is  partly  endogenous  and  partly  exogenous, 
and  that  the  amount  of  endogenous  uric  acid  is  only  very  slightly  dependent 
upon  the  protein  content  of  the  food. 

The  formation  of  uric  acid  from  the  nucleins  or  the  purine  bases  seems 
at  least  in  great  part  to  be  of  an  enzymotic  kind.  After  it  was  shown  that 
certain  organs,  such  as  the  liver  and  spleen,  had  the  power  of  converting 
oxypurines  into  uric  acid  in  the  presence  of  oxygen  (Horbaczewski, 
Spitzer  and  Wiener3),  recently  Schittenhelm,  Burian,  Jones  and 
co-workers4,  by  more  careful  investigations  have  shown  that  enzymes 

der  Physiol.,  1,  Abt.  1,  1002.  See  also  Schittenhelm,  Zeitschr.  f.  physiol.  Chem.,  62, 
with  Frank,  ibid.,  03,  with  Seisser,  Zeitschr.  f.  exp.  Path.  u.  Ther.,  7;  Abderhaklen, 
London  and  Schittenhelm,  Zeitschr.  f.  physiol.  Chem.,  61;  Mendel  and  Lyman.,  Journ. 
of  bioL  Chern.,  8. 

1  See  Plimmer,  Dick  and  Lieb,  Journ.  of  Physiol.,  39;  Mares  Pfliiger's  Arch.,  134, 
and  Smotanka,  ibid.,  138. 

2  Pfliiger's  Arch.,  80,  87,  and  94. 
1  See  footnote,  6,  page  701. 

•Schittenhelm,  Zeitschr.  f.  physiol.  Chem.,  42,  43,  45,  46,  57,  63,  66,  with  Schmid, 
ibid.,  50,  and  Zeitschr.  f.  exp.  Path.  u.  Therap.,  4;  Burian,  Zeitschr.  f.  physiol. 
Chem.,  43;  Jones  and  Partridge,  ibid.,  42;  Jones  with  Winternitz,  ibid.,  44  and  60; 
Jones,  ibid.,  45,  05,  with  Austrian,  ibid.,  48,  with  Miller,  ibid.,  61;  Jones,  Journ.  of 
biol.  Chem.,  '.»;  Wells,  ibid.,  7;  Mendel  and  Mitchell,  Amer.  Journ.  of  Physiol.,  20. 


FORMATION  OF  URIC  ACID.  703 

of  different  kinds  act  together.  By  means  of  the  two  deamidizing  enzymes 
adenase  and  gttanase,  the  adenine  and  guanine  are  transformed  into 
hypoxanthine  and  xanthine  respectively,  and  from  the  latter  by  means 
of  an  oxidizing  enzyme,  called  xanthine  oxidase  by  Burian,  the  uric 
acid  is  formed.  In  the  formation  of  uric  acid  from  the  nucleoproteins 
we  must  admit  of  a  gradual  decomposition  of  these  by  the  aid  of  different 
enzymes,  proteases,  nucleases  and  deamidases.  The  deamidases  seem 
to  be  present  in  most  organs,  and  we  have  numerous  investigations  upon 
their  distribution,  especially  those  of  Jones  and  Schittenhelm  and 
his  collaborators.1  The  distribution  is  not  the  same  in  all  animals  and 
the  reports  regarding  it  are  unfortunately  conflicting  (Schittenhelm, 
Jones  and  Miller).  We  must  exercise  the  greatest  caution  in  drawing 
conclusions  as  to  the  occurrence  of  these  enzymes,  and  from  experiments 
made  with  the  extracts  of  organs,  because  it  seems  as  if  also  other 
unknown  factors  must  be  considered  in  the  formation  of  uric  acid. 
Thus  Jones  has  with  Rohde  2  shown  that  in  rats  the  organs  do  not  con- 
tain any  xanthine  oxidase,  and  that  nevertheless  the  urine  of  this  animal 
contains  uric  acid.  On  the  other  hand  deamidases  occur  in  the  organs 
of  monkeys  (and  xanthine  oxidase  in  the  liver)  but  the  urine  does  not 
contain  any  uric  acid  and  only  traces  of  allantoin  (Wells).3  The  pos- 
sibility of  a  uric  acid  formation  in  man  and  mammalia  in  another  way 
from  the  enzymotic  destruction  of  the  purines  cannot,  for  several  reasons, 
be  denied. 

In  birds  the  conditions  are  different,  v.  Mach  4  has  shown  that  in 
the  bird  family  a  part  of  the  uric  acid  may  be  formed  from  the  purine 
bodies.  The  chief  quantity  of  uric  acid,  however,  is  undoubtedly  formed 
in  birds  by  synthesis. 

The  formation  of  uric  acid  in  birds  is  increased  by  the  administra- 
tion of  ammonium  salts  (v.  Schroder),  and  urea  acts  in  a  similar 
manner  (Meyer  and  Jaff£).  Minkowski  observed,  in  geese  with 
extirpated  livers,  a  very  significant  decrease  in  the  elimination  of  uric 
acid,  while  the  elimination  of  ammonia  was  increased  to  a  corresponding 
degree.  This  indicates  a  participation  of  ammonia  in  the  formation 
of  uric  acid  in  the  organism  of  birds;  and  as  Minkowski  has  also  found, 
after  the  extirpation  of  the  liver,  that  considerable  amounts  of  lactic 
acid  occur  in  the  urine,  it  is  probable  that  the  uric  acid  in  birds  is  pro- 
duced in  the  liver  by  synthesis,  perhaps  from  lactic  acid  and  ammonia ; 


1  See  footnote  4,  page  703. 

1  Jones,  Zeitschr.  f.  physiol.  Chem.,  65,  with  Alice  Rohde,  Journ.  of  biol.  Chem.,  7; 
see  also  Voegtlin  and  Jones,  Zeitschr.  f.  physiol.  Chem.,  66. 
3  Journ.  of  biol.  Chem.,  7. 
*  Arch.  f.  exp.  Path.  u.  Pharm.,  24. 


704  URINE. 

although,  as  Salaskin  and  Zaleski  and  Lang  have  shown,  after  the 
extirpation  of  the  liver,  and  increase  in  the  formation  of  lactic  acid  pri- 
marily occurs,  and  this  causes  an  increase  in  the  elimination  of  ammonia 
(neutralization  ammonia).  The  direct  proof  for  the  uric-acid  formation 
from  ammonia  and  lactic  acid  in  the  liver  of  birds  has  been  given  by 
Kowalewsky  and  Salaskin1  by  means  of  blood-transfusion  experiments 
on  geese  with  extirpated  livers.  They  observed  a  relatively  abundant 
formation  of  uric  acid  after  the  addition  of  ammonium  lactate  and  a 
still  greater  formation  after  arginine.  They  not  only  consider  ammonium 
lactate  but  also  amino-acids  as  substances  from  which  the  uric  acid  can 
be  produced  in  the  liver  by  synthesis.  That  these,  for  example,  leucine, 
glycocoll,  and  aspartic  acid,  increase  the  elimination  of  uric  acid  in 
birds  was  first  shown  by  v.  Knieriem.2 

The  possibility  of  a  formation  of  uric  acid  from  lactic  acid  has  been 
shown  in  another  manner  by  Wiener,3  namely,  by  feeding  birds  with 
urea  and  lactic  acid  and  different  non-nitrogenous  substances,  oxy-, 
keto-,  and  dibasic  acids  of  the  aliphatic  series.  The  dibasic  acids,  with  a 
chain  of  3  carbon  atoms  or  their  ureides,  showed  themselves  most  active 
as  uric-acid  formers,  and  Wiener  is  therefore  of  the  opinion  that  the 
active  substances  must  first  be  converted  into  dibasic  acids.  By  the 
attachment  of  a  urea  residue  the  corresponding  ureide  is  produced, 
according  to  Wiener,  and  from  this  the  uric  acid  is  derived  by  the  attach- 
ment of  a  second  urea  residue. 

Among  the  substances  tested,  only  tartronic  acid  and  its  ureide,  dialuric  acid, 
have  shown  themselves  active  in  the  experiments  with  the  isolated  organs,  and 
Wiener  therefore  also  considers  that  the  other  acids  must  be  first  converted  into 
tartronic  acid  by  oxidation  or  reduction.  From  lactic  acid,  CH3.CH(OH).COOH, 
we  first  obtain  tartronic  acid,  COOH.CH(OH).COOH,  which  by  the  attachment 

/NH— CO\ 
of  a  urea  residue  forms  dialuric  acid,  CO\  /CHOH,  and  from  this,  by 

\NH— CCK 
the  attachment  of  a  second  urea  residue,  uric  acid  is  formed. 

Recently  Izar  4  has  shown  on  perfusing  blood  containing  urea  and 
dialuric  acid  through  the  liver  of  a  dog  and  at  the  same  time  saturating 
the  blood  with  carbon  dioxide,  that  an  abundant  formation  of  uric  acid 
occurred,  and  that  a  combined    action    between    an   enzyme  occurring 

1  v.  Schroder,  Zeitschr.  f.  physiol.  Chem.,  2;  Meyer  and  Jaffa,  Ber.  d.  f.  Chem. 
Gesellsch.,  10;  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  21  and  31;  Salaskin  and 
Zaleski,  Zeitschr.  f.  physiol.  Chem.,  29;  Lang,  ibid.,  32;  Kowalewsky  and  Salaskin, 
ibid.,  33. 

2  Zeitschr.  f.  Biologic,  13. 

'  Hofmeistcr's  Beitrage,  2.  See  also  Arch.  f.  exp.  Path.  u.  Pharm.,  42,  and  Ergeb- 
nissc  fi.  Physiol.,  1,  Abt.  1,  1902. 

4  Zeitschr.  f.  physiol.  Chem.,  73,  see  also  ibid.,  65. 


FORMATION  OF  URIC  ACID.  705 

in  the  blood  and  an  alcohol-soluble  co-enzyme  occurring  in  the  liver 
and  spleen  took  place.  He  has  besides  this  also  given  further  proof  of 
the  formation  of  uric  acid  in  the  bird-liver  from  urea  and  ammonium 
carbonate. 

We  cannot  give  any  positive  answer  as  to  the  question  whether  uric 
acid  is  formed  by  synthesis  in  man  and  other  mammalia.  Wiener 
has  reported  experiments  which  seem  to  indicate  a  synthetic  uric-acid 
formation  in  the  isolated  mammalian  liver,  and  he  has  also  obtained 
an  increase  in  the  uric-acid  elimination,  although  only  a  slight  one,  after 
feeding  lactic  acid  and  dialuric  acid  to  man.  In  opposition  to  these 
experiments  Pfeiffer  l  could  find  no  increase  in  the  elimination  of 
uric  acid  after  feeding  malonamide  and  tartronamide  to  monkeys  as 
well  as  tartronic  acid  and  pseudouric  acid  to  monkeys  or  human  beings, 
and  he  finds  that  a  synthesis  of  uric  acid  in  mammalia  and  man  is  very 
doubtful.  According  to  Burian  2  we  have  for  the  present  no  proof 
of  a  synthetical  formation  of  uric  acid  in  the  mammalian  liver;  in 
view  of  the  above-mentioned  experiments  of  Izar,  we  cannot  deny  the 
possibility  of  a  synthetical  formation  of  uric  acid  also  in  mammalia  and 
man  even  if  we  do  not  know  to  what  extent  this  occurs. 

The  liver  seems  to  be  the  organ  in  birds  where  the  synthetical  forma- 
tion of  uric  acid  occurs,  and  the  fact  that  it  was  possible  for  Minkowski  3 
to  arrest  the  uric-acid  formation  by  the  extirpation  of  the  liver,  apparently 
shows  that  the  liver  is  the  only  organ  taking  part  in  this  synthesis.  If  a 
synthesis  of  uric  acid  also  occurs  in  man  and  other  mammalia,  we  must 
consider  the  liver  as  at  least  one  of  the  organs  taking  part  in  the  work, 
as  shown  by  Wiener's  and  Izar's  investigations.  The  liver  is  considered 
as  the  most  important  organ  in  the  oxidative  formation  of  uric  acid  from 
nucleins  and  purine  bases.  That  this  organ,  at  least  in  the  dog,  is  not 
the  only  or  at  least  not  the  most  important  follows  from  the  investiga- 
tions of  Abderhalden,  London  and  Schittenhelm  4  on  dogs  with  Eck 
fistula.  They  found  that,  on  excluding  the  liver  in  this  manner,  that  the 
transformation  of  the  nucleic  acid  fed,  the  deamidation  of  the  purine 
bases  and  the  oxidation  of  these  into  uric  acid  and  allantoin  was 
undisturbed.  In  the  dog  also  other  organs  must  be  considered  in 
this  connection.  It  is  not  known  how  other  animals  behave  in  this 
regard. 

Uric  acid  when  introduced  into  the  mammalian  organism  is,  as  first 
shown  by  Wohler  and  Frerichs,  in  the  dog,  and  later  substantiated 


1  Hofmeister's  Beit  rage,  10. 

2  Zeitschr.  f.  physiol.  Chem.,  43. 
»1.  c. 

4  Zeitschr.  f.  physiol.  Chem.,  61. 


706  URINE. 

by  several  experimenters,1  in  great  part  destroyed  and  more  or  less  com- 
pletely changed  into  urea.  As  shown  by  Wohler  and  Frerichs  for  the 
dog  and  by  later  investigators  2  also  for  cats,  rabbits  and  other  animals, 
that  allantoin  is  the  most  essential  or  indeed  the  chief  decomposition 
product  is  now  considered  as  positively  proven.  In  man,  on  the  con- 
trary, the  conditions  are  different.  According  to  Wiechowski3  probably 
also  a  formation  of  allantoin  from  uric  acid  takes  place  in  man,  but  it 
is  only  of  such  an  extent  as  to  be  without  consideration,  while  in  the  dog 
for  example  about  96  per  cent  of  the  purine  base  nitrogen  may  appear 
as  allantoin  in  the  urine.  According  to  the  investigations  of  Frank  and 
Schittenhelm  4  the  uric  acid  in  man  is  in  part  transformed  into  urea. 
This  different  behavior  of  uric  acid  in  the  metabolism  of  man  and 
animals  depends,  as  numerous  investigations 5  have  shown,  upon  the 
occurrence  of  a  urocolytic  enzyme  in  the  liver  and  also  other  organs  of 
animals,  which  transforms  the  uric  acid  into  allantoin  with  the  taking  up 
oxygen  and  splitting  off  of  carbon  dioxide.  This  enzyme,  which  has  been 
called  uricolase  and  also  uricase  and  whose  occurrence  in  the  organs  of 
different  animals  varies,  is  absent  in  the  organs  of  man.  The  results 
obtained  in  regard  to  the  enzymotic  transformation  of  uric  acid  by 
experiments  with  organ  extracts  must  be  judged  with  the  greatest  care. 
Thus  according  to  the  statements  of  Wiechowski,  Battelli  and  Stern 
and  Schittenhelm,6  in  dogs,  the  liver  is  the  only  organ  which  in  a  test- 
tube  shows  a  positive  uricolysis;  still  in  dogs,  with  excluded  livers 
(Eck  fistula)  such  an  abundant  formation  of  allantoin  from  uric  acid 
occurs  so  that  only  10-20  per  cent  of  the  uric  acid  escapes  this  trans- 
formation.7 

1  Wohler  and  Frerichs,  Annal.  d.  Chem.  u.  Pharm.,  65.  See  also  Wiener,  Ergeb- 
nisse  der  Physiologie,  1,  Abt.  1. 

2  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  35,  and  Ber.  d.  d.  Chem.  Gesellsch.,  9; 
Mendel  and  Brown,  Amer.  Jour,  of  Physiol.,  3;  Mendel  and  White,  ibid.,  12;  Wie- 
chowski, Arch.  f.  exp.  Path.  u.  Pharm.,  60,  and  Bioch.  Zeitschr.,  19  and  25,  with  Wiener, 
Hofmeister's  Beitrage,  9;  Schittenhelm,  Zeitschr.  f.  physiol.  Chem.,  62,  with  Seisser, 
Zeitschr.  f.  exp.  Path.  v.  Ther.,  7;  Abderhalden,  London  and  Schittenhelm,  Zeitschr. 
f.  physiol.  Chem.,  61. 

3  Bioch.  Zeitschr.,  25. 

4  Zeitschr.  f.  physiol.  Chem.,  63. 

5  Chassevant  and  Richet,  Comp.  rend.  soc.  biolog.,  49;  Ascoli,  Pfliiger's  Arch.,  72; 
Jacoby,  Virchow's  Arch.,  157;  Wiener,  Arch.  f.  exp.  Path.  u.  Pharm.,  42,  and  Centralbl. 
f.  Physiol.,  18;  Schittenhelm,  Zeitschr.  f.  physiol.  Chem.,  43,  45,  and  63;  Burian, 
ibid.,  43;  Almagia,  Hofmeister's  Beitrage,  7;  Pfeiffer,  ibid.,  7;  Wiechowski  and  Wiener, 
ibid.,  9;  Galeotti,  Bioch.  Zeitschr.,  20;  Battelli  and  Stern,  ibid.,  19;  Scaffidi,  ibid., 
18;  Miller  and  Jones,  Zeitschr.  f.  physiol.  Chem.,  61;  Wells,  Journ.  of  biol.  Chem.,  7, 
with  Corper,  ibid.,  6. 

6  See  Schittenhelm,  Zeitschr.  f.  physiol.  Chem.,  63,  256. 

7  Abderhalden,  London  and  Schittenhelm,  1.  c. 


PROPERTIES   AND   REACTIONS   OF  URIC  ACID.  707 

Ascoli,  Izar,  Bezzola  and  Preti  *  have  studied  the  remarkable  ability  of 
the  liver  of  destroying  uric  acid  in  the  blood  by  transfusing  the  arterial  blood 
through  this  organ  and  qd  transfusing  the  blood,  saturated  with  C02,  they  have 
regenerated  the  uric  arid.  It  is  not  known  what  becomes  of  the  uric  acid  in 
these  cases  and  from  what  substance  the  regeneration  occurs.  Preti  has  shown 
that  in  the  regeneration  a  combined  action  of  an  enzyme  in  the  blood  with  a 
co-enzyme  of  the  liver,  takes  place. 

From  this  power  of  the  various  organs  of  destroying  uric  acid  it 
follows  that  the  quantity  of  uric  acid  eliminated  is  not  a  sure  indication 
of  the  amount  of  the  acid  formed.  We  must,  therefore,  admit  that  a 
part  of  the  uric  acid  formed  in  the  body  is  destroyed  in  a  manner  similar 
to  that  introduced  from  without.  Burian  and  Schur  2  have  indeed 
suggested  a  factor,  the  so-called  "  integral  factor,"  with  which  the  quan- 
tity of  uric  acid  eliminated  in  the  twenty-four  hours  must  be  multiplied 
in  order  to  find  the  quantity  of  uric  acid  formed  during  this  time.  Such 
calculations  are  necessarily  very  uncertain  and  are  for  the  present  not 
admissible. 

Properties  and  Reactions  of  Uric  Acid.  Pure  uric  acid  is  a  white, 
odorless,  and  tasteless  powder  consisting  of  very  small  rhombic  prisms 
or  plates.  Impure  uric  acid  is  easilv  obtained  as  somewhat  larger, 
colored  crystals. 

In  rapid  crystallization,  small,  thin,  four-sided,  apparently  colorless, 
rhombic  prisms  are  formed,  which  can  be  seen  only  by  the  aid  of  the 
microscope,  and  these  sometimes  appear  as  spools  because  of  the  round- 
ing of  their  obtuse  angles.  The  plates  are  sometimes  six-sided,  irregularly 
developed;  in  other  cases  they  are  rectangular  with  partly  straight  and 
partly  jagged  sides;  and  in  other  cases  they  show  still  more  irregular 
forms,  the  so-called  dumb-Veils,  etc.  In  slow  crystallization,  as  when 
the  urine  deposits  a  sediment  or  when  treated  with  acid,  large,  invariably 
colored  crystals  separate.  Examined  with  the  microscope  these  crystals 
always  appear  yellow  or  yellowish  brown  in  color.  The  most  common 
type  is  the  whetstone  shape,  formed  by  the  rounding  off  of  the  obtuse 
angles  of  the  rhombic  plate.  The  whetstones  are  generally  connected, 
two  or  more  crossing  each  other.  Besides  these  forms,  rosettes  of  pris- 
matic crystals,  irregular  crosses,  brown-colored  rough  masses  of  broken- 
up  crystals  and  prisms  occur,  as  well  as  other  forms. 

Uric  acid  is  insoluble  in  alcohol  and  ether;  it  is  rather  easily  soluble 
in  boiling  glycerin,  but  very  insoluble  in  cold  water,  in  39480  parts  at 
18°  C.  (His  and  Paul),  and  in  15505  parts  at  37°  (Gudzent).  At 
this  temperature,  according  to  His  and  Paul,  P. 5  per  cent  of  the  uric 
acid  is  dissociated  in  the  saturated  solution.     Because  of  the  reduction 


1  See  Zeitschr.  f .  physiol.  Chem.,  58,  62,  64  and  63. 

2  Pfliiger's  Arch.,  87. 


708  URINE. 

in  the  dissociation  on  the  addition  of  strong  acids,  uric  acid  is  soluble 
with  difficulty  in  the  presence  of  mineral  acids.  It  is  soluble  in  a  warm 
solution  of  sodium  diphosphate,  and  in  the  presence  of  an  excess  of  uric 
acid,  monophosphate  and  acid  urate  are  produced.  It  is  ordinarily- 
assumed  that  sodium  diphosphate  forms  a  solvent  for  the  uric  acid  in 
the  urine,  while  according  to  Gudzent  this  is  not  dissolved  by  the  mono- 
phosphate. Rudel  l  believes  that  urea  is  an  important  solvent,  but 
this  view  has  not  been  confirmed  by  the  observations  of  His  and  Paul. 
Uric  acid  is  not  only  dissolved  by  alkalies  and  alkali  carbonates,  but  also 
by  several  organic  bases,  such  as  ethylamine  and  propylamine,  piperidine 
and  piperazine.  Uric  acid  can  form  supersaturated  solutions  with  alkalies 
and  these,  according  to  Schade  and  Boden  2  contain  colloidal  uric  acid 
and  they  may  gelatinize  on  cooling  as  well  as  under  other  conditions. 
Uric  acid  dissolves,  without  decomposing,  in  concentrated  sulphuric 
acid.  It  is  completely  precipitated  from  the  urine  by  picric  acid  (Jaffe3). 
Uric  acid  gives  a  chocolate-brown  precipitate  with  phosphotungstic 
acid  in  the  presence  of  hydrochloric  acid.4 

Uric  acid  is  dibasic  and  consequently  forms  two  series  of  salts,  neu- 
tral and  acid.  Of  the  alkali  urates  the  lithium  salts  are  the  most  soluble 
and  the  acid  ammonium  salt  is  the  most  insoluble.  The  acid  alkali 
urates  are  very  insoluble  and  separate  as  a  sediment  {sedimentum  later- 
itium)  from  concentrated  urine  on  cooling.  According  to  Gudzent 
1  liter  of  water  at  18°  C.  dissolves  (as  primary  salts)  1.5313  grams 
potassium,  0.8328  gram  sodium,  and  0.4141  gram  ammonium  urate, 
and  at  37°  C.  2.7002,  1.5043  and  0.7413  grams  of  the  respective  urates.5 
The  salts  of  the  alkaline  earths  are  soluble  with  great  difficulty.  The 
above  solubilities  apply  only,  in  Gudzent's  6  experience,  to  the  freshly 
prepared  solution,  as  the  solubility  to  a  certain  limit  gradually  dimin- 
ishes, due  to  intramolecular  transposition  (change  of  the  uric  acid  from 
the  lactam-form  into  the  lactim-form) . 

Besides  the  mono-  and  diurates  also  "  quadriurates  "  have  been  described 
and   these  occur  in  the  excrement  of   snakes  and  birds  and  in  the    sedimentum 


1  His,  Jr.,  and  Paul,  Zeitschr.  f.  physiol.  Chem.,  31;  Smale,  Centralbl.  f.  physiol., 
D;  Rudel,  Arch.  f.  exp.  Path.  u.  Pharra.,  30;  Gudzent,  Zeitschr.  f.  physiol.  Chem., 
60  and  63. 

2  Zeitschr.  f.  physiol.  Chem.,  83. 

3  IUd.,  10. 

4  In  regard  to  the  combinations  of  formaldehyde  and  uric  acid,  see  Nicolaier, 
Deutsch.  Arch.  f.  klin.  Med.,  89  (1906). 

6  Determinations  of  the  solubility  of  the  monourates  in  serum  have  been  made  by 
Gudzent,  Zeitschr.  f.  physiol.  Chem.,  63.  See  also  Bechhold  and  Ziegler,  Bioch.  Zeitschr., 
20. 

8  Zeitschr.  f.  physiol.  Chem.,  56  and  60. 


PROPERTIES  OF  URIC  ACID.  709 

lateritium.  Whether  these  quadriurates,  which  have  recently  been  studied 
by  Ringer,  Kohler  and  Schmutzer,1  are  chemical  combinations  of  2  molecules 
uric  acid  and  1  atom  of  K  or  Xa  or  are  mixtures,  so-called  solid  solutions  of  uric 
acid  in  monourates,  is  still  a  disputed  question. 

If  a  little  uric  acid  in  substance  is  treated  on  a  porcelain  dish  with 
a  few  drops  of  nitric  acid,  the  uric  acid  dissolves  on  wanning,  with  a 
strong  development  of  gas,  and  after  thoroughly  drying  on  the  water- 
bath  a  beautiful  red  residue  is  obtained,  which  turns  a  purple-red  (ammo- 
nium purpurate  or  murexide)  on  the  addition  of  a  little  ammonia.  If 
instead  of  the  ammonia  we  add  a  little  caustic  soda  (after  cooling),  the 
color  becomes  deeper  blue  or  bluish  violet.  This  color  disappears  quickly 
on  warming,  differing  from  certain  purine  bodies.  This  reaction  is  called 
the  murexide  test. 

A  solution  of  phosphotungstic  acid,  prepared  according  to  certain 
directions,  gives  with  a  solution  of  uric  acid,  when  treated  with  an  excess 
of  sodium  carbonate,  a  beautiful  blue  solution.  This  extremely  delicate 
reaction  (1:500,000)  was  suggested  by  Folin  and  Denis.2 

Uric  acid  does  not  reduce  an  alkaline  solution  of  bismuth,  while,  on 
the  contrary,  it  reduces  an  alkaline  cupric-hydroxide  solution.  In  the 
presence  of  only  a  little  copper  salt  we  obtain  a  white  precipitate  consist- 
ing of  cuprous  urate.  In  the  presence  of  more  copper  salt  red  cuprous 
oxide  separates.  The  compound  of  uric  acid  with  cuprous  oxide  is  formed 
wrhen  copper  salts  are  reduced  by  glucose  or  a  bisulphite  in  alkaline 
solution  in  the  presence  of  a  sufficient  amount  of  urate. 

If  a  solution  of  uric  acid  in  water  containing  alkali  carbonate  is  treated 
with  magnesium  mixture  and  then  a  silver-nitrate  solution  added,  a 
gelatinous  precipitate  of  silver-magnesium  urate  is  formed.  If  a  drop 
of  uric  acid  dissolved  in  sodium  carbonate  is  placed  on  a  piece  of  filter- 
paper  which  has  been  previously  treated  with  silver-nitrate  solution, 
a  reduction  of  silver  oxide  occurs,  producing  a  brownish-black  or,  in  the 
presence  of  only  0.002  milligram  of  uric  acid,  a  yellow  spot  (Schiff's 
test) . 

If  a  weak  alkaline  solution  of  uric  acid  in  water  is  treated  with  a  soluble  zinc 
salt,  a  white  precipitate  is  produced,  which  on  the  filter  in  the  presence  of  alkali 
is  oxidized  by  the  air,  and  becomes  sky-blue  in  color,  especially  in  sunlight. 
Potassium  persulphate  causes  a  blue  coloration  immediately'  (Ganassini's 
reaction  3). 

The  precipitation  of  free  uric  acid  from  its  alkali  salts  by  means  of  acids  can 
be  prevented  to  some  extent  by  the  presence  of  thymic  acid  or  nucleic  acid  (Goto). 
According  to  Seo  we  are  here  dealing  with  combinations  of  1  molecule  nucleic 

1  Ringer,  ibid.,  67  (literature)  and  75;  Kohler,  ibid.,  70  and  72;  Ringer  and  Schmut- 
zer, ibid.,  82. 

s  Journ.  of  biol.  Chem.,  12. 

*  Cited  f.  Bioch.  Centralbl.,  8,  250. 


710  URINE. 

acid  and  2  molecules  uric  acid,  which  protects  the  uric  acid  within  the  body  against 
destruction  or  transformation  into  allantoin.  This  view  is  incorrect,  accord- 
ing to  Schittenhelm  and  Seisser.1  According  to  them  no  constant  combina- 
tion between  nucleic  acid  and  uric  acid  exists,  and  in  rabbits  the  nucleic  acid  does 
not  protect  the  uric  acid  from  transformation  to  allantoin. 

Preparation  of  Uric  Acid  from  Urine.  Filtered  normal  urine  is  treated 
with  20-30  cc.  of  25-per  cent  hydrochloric  acid  for  each  liter  of  urine. 
After  forty-eight  hours  collect  the  crystals  and  purify  them  by  redis- 
Bolving  in  dilute  alkali,  decolorizing  with  animal  charcoal  and  repre- 
cipitating  with  hydrochloric  acid.  Large  quantities  of  uric  acid  are 
easily  obtained  from  the  excrement  of  serpents  by  boiling  it  with  dilute 
caustic  potash  (5-per  cent)  until  no  more  ammonia  is  developed.  A 
current  of  carbon  dioxide  is  passed  through  the  filtrate  until  it  barely 
has  an  alkaline  reaction;  dissolve  the  separated  and  washed  acid  potas- 
sium urate  in  caustic  potash,  and  precipitate  the  uric  acid  in  the  filtrate 
by  addition  of  an  excess  of  hydrochloric  acid. 

Quantitative  Estimation  of  Uric  Acid  in  the  Urine.  As  the  older 
method  suggested  by  Heintz,  even  after  recent  modifications,  gives 
inaccurate  results,  it  will  not  be  considered  here. 

Salkowski  and  Ludwig's2  method  consists  in  precipitating  the  uric 
acid,  by  silver  nitrate,  from  the  urine  previously  treated  with  magnesium 
mixture,  and  weighing  the  uric  acid  obtained  from  the  silver  precip- 
itate. Uric  acid  determinations  by  this  method  are  often  performed 
according  to  the  suggestion  of  E.  Ludwig,  which  requires  the  follow- 
ing solutions: 

1.  An  ammoniacal  silver-nitrate  solution,  which  contains  in  1  liter  26 
grams  of  silver  nitrate  and  a  quantity  of  ammonia  sufficient  to  redissolve  com- 
pletely the  precipitate  produced  by  the  first  addition  of  ammonia.  2.  Magne- 
sia mixture.  Dissolve  100  grams  of  crystallized  magnesium  chloride  in  water, 
add  ammonia  until  the  liquid  smells  strongly  of  it,  and  enough  ammonium 
chloride  to  dissolve  the  precipitate;  then  dilute  the  solution  to  1  liter.  3.  Sodium 
sulphide  solution.  Dissolve  10  grams  of  caustic  soda  which  is  free  from  nitric 
acid  and  nitrous  acid  in  1  liter  of  water.  One  half  of  this  solution  is  completely 
saturated  with  sulphuretted  hydrogen  and  then  mixed  with  the  other  half. 

The  concentration  of  the  three  solutions  is  so  arranged  that  10  cc. 
of  each  is  sufficient  for  100  cc.  of  the  urine. 

100-200  cc,  according  to  concentration,  of  the  filtered  urine,  freed 
from  protein  (by  boiling  after  the  addition  of  a  few  drops  of  acetic  acid), 
are  poured  into  a  beaker.  In  another  vessel  mix  10-20  cc.  of  the  silver 
solution  with  10-20  cc.  of  the  magnesia  mixture  and  add  ammonia, 
and  when  necessary  also  some  ammonium  chloride,  until  the  mixture 
is  clear.  This  solution  is  added  to  the  urine  while  stirring,  and  the  mix- 
ture allowed  to  stand  quietly  for  half  an  hour.  The  precipitate  isi  .col- 
lected on  a  filter,  washed  with  ammoniacal  water,  and  then  returned  to 

1  Goto,  Zeitschr.  f.  physiol.  Chem.,  30;  Seo,  Arch.  f.  exp.  Path.  u.  Pharm.,  58; 
Schittenhelm  and  Seisser,  Zeitschr.  f.  exp.  Path.  u.  Ther.,  7. 

2  Salkowski,  Virchow's  Arch.,  52;  Pfliiger's  Arch.,  5;  Salkowski,  Laboratory 
Manual  of  Physiol,  and  Path.  Chem.,  translated  by  Orndorff,  1904;  Ludwig,  Wien.. 
med.  Jahrbuch,  1884,  and  Zeitschr.  f.  anal  Chem.,  24. 


ESTIMATION  OF  UKIC  ACID.  711 

i;  e  same  beaker  by  the  aid  of  a  glass  rod  and  a  wash-bottle,  withoul 
destroying  the  filter.    Now  heat  i<>  boiling  10-20  ce.  of  the  alkali-sulphide 

solution,  which  has  previously  been  diluted  with  an  equal  volume  of 
water,  and  allow  this  solution  to  flow  through  the  above  filter  into  the 
beaker  containing  the  silver  precipitate;    wash  with  boiling  water,  and 

warm   the   contents  of  the   beaker   on   a   water-bath   for   a   time,   stirring 

constantly.  After  cooling,  filter  into  a  porcelain  dish,  wash  the  filter 
with  boiling;  water,  auidify  the  filtrate  with  hydrochloric  acid,  evaporate 
it  to  about  15  cc,  add  a  few  drops  more  of  hydrochloric  acid,  and  allow 
it  to  stand  for  twenty-four  hours.  The  uric  acid  which  has  crystallized 
is  collected  on  a  small  weighed  filter,  washed  with  water,  alcohol,  ether, 
and  carbon  disulphide,  dried  at  100-110°  C,  and  weighed.  For  each 
10  cc.  of  aqueous  filtrate  we  must  add  0.00048  gram  uric  acid  to  the 
quantity  found  directly.  Instead  of  the  weighed  filter-paper  a  glass 
tube  filled  with  glass  wool  as  described  in  other  handbooks  may  be  sub- 
stituted (Ludwig).  Too  intense  or  too  long  continued  heating  with 
the  alkali  sulphide  must  be  prevented,  otherwise  a  part  of  the  uric  acid 
may  be  decomposed. 

Salkowski  deviates  from  this  procedure  by  first  precipitating  the 
urine  with  a  magnesium  mixture  (50  cc.  to  200  cc.  urine),  filling  up  to 
300  cc,  and  filtering.  Of  the  filtrate,  200  cc.  are  precipitated  by  10-15 
cc.  of  a  3-per  cent  silver-nitrate  solution.  The  silver  precipitate  is  shaken 
with  200-300  cc.  of  water  acidified  with  a  few  drops  of  hydrochloric 
acid,  decomposed  by  sulphuretted  hydrogen,  heated  to  boiling,  the 
silver-sulphide  precipitate  boiled  with  fresh  water,  filtered,  the  filtrate 
concentrated  to  a  few  cubic  centimeters,  treated  with  5-8  drops  of  hydro- 
chloric acid,  and  allowed  to  stand  until  the  next  day.  According  to 
Salkowski  and  Kashiwabara  1  the  precipitation  with  zinc  salts  can  also 
be  used  in  the  estimation  of  uric  acid. 

Hopkins7  methcd  is  based  on  the  fact  that  the  uric  acid  is  com- 
pletely precipitated  from  the  urine  as  ammonium  urate  on  saturating 
with  ammonium  chloride.  The  uric  acid  can  either  be  weighed  after 
being  set  free  by  hydrochloric  acid  or  it  can  be  determined  in  several 
ways — by  titration  with  potassium  permanganate  or  by  the  Kjeldahl 
method.  Several  modifications  of  this  method  have  been  worked  out 
by  Folin,  Folin  and  Schaffer,  Worner,  and  Jolles.2  Of  these  methods 
we  shall  describe  only  that  suggested  by  Folin-Schaffer. 

Folin-Schaffer  Method.  Treat  300  cc.  urine  with  75  cc.  of  a  solu- 
tion containing  500  grams  of  ammonium  sulphate,  5  grams  of  uranium 
acetate,  and  60  cc.  of  10  per  cent  acetic  acid  in  a  liter,  and  filter  after 
five  minutes.  This  removes  an  unknown  constituent  of  the  urine  (a 
protein  substance)  which  would  otherwise  contaminate  the  uric  acid. 
Take  125  cc.  of  the  filtrate  (corresponding  to  100  cc.  of  the  urine)  and 
add  5  cc.  of  concentrated  ammonia.  After  twenty-four  hours  the  pre- 
cipitate is  filtered  off  and  washed  free  from  chlorine  on  the  filter  by  means 
of  an  ammonium-sulphate  solution.     The  precipitate  is  washed  off  the 


1  Zeitschr.  f.  physiol.  Cheni.,  4. 

2  Hopkins,  Journ.  of  Path,  and  Bact.,  1893,  and  Proceed.  Roy.  Soc,  52;  Folin, 
Zeitschr.  f.  physiol.  Chem.,  24;  Folin  and  Schaffer,  ibid.,  32;  Worner,  ibid.,  29;  Jolles, 
ibid.,  29;  and  Wien.  med.  Wochenschr.,  1903. 


712  URINE. 

filter  by  water  (total  100  cc.)  into  a  flask,  treated  with  15  cc.  of  con- 
centrated sulphuric  acid,  and  titrated  at  60-63°  C.  with  N/20  potassium- 
permanganate  solution.  Each  cubic  centimeter  of  this  solution  cor- 
responds to  3.75  milligrams  uric  acid.  Because  of  the  solubility  of  the 
ammonium  urate  a  correction  of  3  milligrams  must  be  added  for  every 
100  cc.  of  the  urine. 

In  regard  to  the  numerous  other  methods  for  estimating  uric  acid, 
we  must  refer  to  special  works  on  the  subject,  and  especially  to  Huppert- 
Xeubauer.  Folin  with  Macullum  Jr.  and  with  Denis  l  have  sug- 
gested a  colorimetric  method  for  estimating  uric  acid,  making  use  of 
phosphotungstic  acid. 

Purine  Bases  (Alloxuric  Bases)  .  The  purine  bases  found  in  human 
urine  are  xanthine,  (guanine),  hypoxanthine,  adenine,  paraxanthine, 
heteroxanthine,  episarkine,  epiguanine,  1-methylxanthinc.  The  occur- 
rence of  guanine  and  carnine  (Pouchet)  is,  according  to  Kruger  and  Salo- 
mon,2 not  positively  shown.  The  quantity  of  these  bodies  in  the  urine 
is  extremely  small  and  varies  in  different  individuals.  Flatow  and  Reit- 
zenstein3  found  15.6-45.1  milligrams  in  the  urine  voided  during  twenty- 
four  hours.  The  quantity  of  alloxuric  bases  in  the  urine  is  regularly 
increased  after  feeding  with  nucleins  or  food  rich  in  nucleins,  and  after  an 
abundant  destruction  of  leucocytes.  The  quantity  is  especially  increased 
in  leucaemia.  We  have  a  number  of  observations  on  the  elimination  of 
these  bodies  in  different  diseases,  but  they  are  hardly  trustworthy  on 
account  of  the  inaccuracy  of  the  methods  used  in  the  determinations. 
It  must  also  be  remarked  that  the  three  purine  bases,  heteroxanthine, 
paraxanthine,  and  1-methylxanthine,  which  form  the  chief  mass  of  the 
purine  bases  of  the  urine,  are  derived,  according  to  numerous  investiga- 
tions4 from  the  theobromine,  caffeine,  and  theophylline  which  occur 
in  the  food.  With  the  purine  bases  we  must  also  differentiate  between 
those  of  endogenous  and  those  of  exogenous  origin,5  and  the  same  factors 
apply  as  for  the  uric  acid,  viz.,  the  endogenous  purine  formation  represents 
a  value  which  is  somewhat  variable  for  different  individuals  and  relatively 


1  Journ.  of  biol.  Chem.,  13  and  14. 

2  Zeitschr.  f.  physiol.  Chem.,  24;  Pouchet,  "Contributions  a  la  connaissance  des 
matieres  extractives  de  l'urine."  Th6se,  Paris,  1880.  Cited  from  Huppert-Neubauer, 
333  and  335. 

3  Deutsch.  med.  Wochenschr.,  1897. 

«Albaneee,  Ber.  d.  d.  chem.  Gesellsch.,  32;  Arch.  f.  exp.  Path.  u.  Pharm.,  35; 
Bondzynski  and  Gottlieb,  ibid.,  36,  and  Ber.  d.  deutsch.  chem.  Gesellsch.,  28;  E. 
Fischer,  ibid.,  30,  2405;  Kruger  and  Salomon,  Zeitschr.  f.  physiol.  Chem.,  26;  Kruger 
and  Schmidt,  Ber.  d.  d.  chem.  Gesellsch.,  32,  and  Arch.  f.  exp.  Path.  u.  Pharm.,  45; 
Kotake,  Zeitschr.  f.  physiol.  Chem..  57. 

4  See  Burian  and  Schur,  footnote  2,  page  702,  and  Kaufmann  and  Mohr,  Deutsch. 
Arch.  f.  klin.  Med.,  74. 


PI  KINK   BASES.  713 

constanl  for  the  same  individual.    According  to  Siven,1  with  purine-free 

diet  the  elimination  of  purines  is  lowest  at  night  and  highest  in  the  morn- 
ing hours.  Rest  and  work  do  not  show  any  positive  difference.  As 
the  four  true  nuclein  bases  have  been  treated  in  Chapter  II,  it  only 
remains  to  describe  the  special  urinary  purine  bodies. 

HX— CO 

I       I 
Heteroxanthine,  CeHjNjOi,  7-monomethylxanthine,  OC    C.N.CHj,  was  first 

I     I'    \ch 

detected  in  the  urine  by  Salomon.  It  is  identical  with  the  monomethylxan- 
thine  which  passes  into  the  urine  after  feeding  with  theobromine  or  caffeine. 
Salomon  and  Xeuburg  2  found  heteroxanthine  in  the  urine  of  a  dog  fed  entirely 
upon  meat,  and  this  was  probably  formed  by  a  methylation  in  the  body. 

Heteroxanthine  crystallizes  in  shining  needles  and  dissolves  with  difficulty 
in  cold  water  (1592  parts  at  18°  C).  It  is  readily  soluble  in  ammonia  and  alkalies. 
The  crystalline  sodium  salt  is  insoluble  in  strong  caustic  alkali -(33-per  cent)  and 
dissolves  with  difficulty  in  water.  The  chloride  crystallizes  beautifully,  is  rela- 
tively insoluble,  and  is  readily  decomposed  into  the  free  base  and  hydrochloric 
acid  by  water.  Heteroxanthine  is  precipitated  by  copper  sulphate  and  bisul- 
phite, mercuric  chloride,  basic  lead  acetate  and  ammonia,  and  by  silver  nitrate. 
The  silver  compound  dissolves  rather  easily  in  dilute,  warm  nitric  acid;  it  crystal- 
lizes in  small  rhombic  plates  or  prisms,  often  grown  together,  forming  charac- 
teristic crosses.  Heteroxanthine  does  not  give  the  xanthine  reaction,  but  does 
give  Wiedel's  reaction,  according  to  Fischer  (see  Chapter  II). 

CH3.N— CO 

I      I 
1-Methylxanthine,  CoHsNA,     OC     C.XH       ,   was  first  isolated  from  the 

I  II  \rH 
HN— C.N/' 
urine  and  studied  by  Kruger,  and  then  by  Kruger  and  Salomon.3  It  is  diffi- 
cultly soluble  in  cold  water,  but  readily  soluble  in  ammonia  and  caustic  soda, 
and  does  not  give  an  insoluble  sodium  compound.  It  is  readily  soluble  in  dilute 
acids,  and  it  crystallizes  from  its  acetic-acid  solution  in  thin,  generally  hexagonal 
plates.  The  chloride  is  decomposed  into  the  base  and  hydrochloric  acid  by 
water.  1-methylxanthine  gives  crystalline  double  salts  with  platinum  and  gold. 
It  is  not  precipitated  by  basic  lead  acetate,  nor  when  pure  by  basic  lead  acetate 
and  ammonia.  With  ammonia  and  silver  nitrate  it  gives  a  gelatinous  precipitate. 
The  silver-nitrate  compound  crystallized  from  nitric  acid  forms  rosettes  of  united 
needles.  With  the  xanthine  test  with  nitric  acid  it  gives  an  orange  coloration 
on  the  addition  of  caustic  soda.     It  gives  Weidel's  reaction  (according  to  Fischer) 


beautifully. 


CH3.X— CO 


Paraxanthine,     C7H8N4O2,     1.7-dimethylxanthine,      OC    C.XCH3,     urotheo- 

HX-C.X/'CH 
bromine  (Thudichum),  was  first  isolated  from  the  urine  by  Thudichum  and 

1  Skand.  Arch.  f.  Physiol.,  18. 

2  Salkowski's  Festschrift,  Berlin,  1904. 

•  Kruger,  Arch.  f.   (Anat.  u.)  Physiol.,   1894;    Kruger  and  Salomon,  Zeitschr.  f. 
physiol.  Chem.,  24. 


714  URINE. 

Salomon.1  It  crystallizes  beautifully  in  six-sided  plates  or  in  needles.  The 
sodium  compound  crystallizes  in  rectangular  plates  or  prisms  and,  like  the  hetero- 
xanthine-sodium  compound,  is  insoluble  in  33-per  cent  caustic-soda  solution. 
The  sodium  compound  separates  in  a  crystalline  state  on  neutralizing  its  solution 
in  water.  The  chloride  is  readily  soluble  and  is  not  decomposed  by  water.  The 
chloroplatinate  crystallizes  very  beautifully.  Mercuric  chloride  precipitates  it 
only  when  added  in  excess  and  after  a  long  time.  The  silver-nitrate  compound 
separates  as  white  silky  crystals  from  hot  nitric  acid  on  cooling.  It  gives  Weidel's 
reaction,  but  not  the  xanthine  test,  with  nitric  acid  and  alkali. 

Episarkine  is  the  name  given  by  Balke  to  a  purine  body  occurring  in  human 
urine.  The  same  body  has  been  observed  by  Salomon  2  in  pigs'  and  dogs'  urine, 
as  well  as  in  urine  in  leucaemia.  Balke  gives  C4H6N30  as  the  probable  formula 
for  episarkine.  It  is  nearly  insoluble  in  cold  water,  dissolves  with  difficulty  in 
hot  water,  but  may  be  obtained  therefrom  as  long  fine  needles.  Episarkine  does 
not  give  the  xanthine  -  reaction  with  nitric  acid,  or  Weidel's  reaction.  With 
hydrochloric  acid  and  potassium  chlorate  it  gives  a  white  residue  which  turns 
violet  with  ammonia.  It  does  not  form  any  insoluble  sodium  comrjound.  The 
silver  compound  is  difficultly  soluble  in  nitric  acid. 

HN— CO 

II' 
Epiguanine,  CeHrNsO,    7-methylguanine,    H2N.C    C.N.CH3,  was   first  pre- 

II      II     \CH 

pared  from  the  urine  by  Kruger.3  It  is  crystalline  and  difficultly  soluble  in 
hot  water  or  ammonia.  It  crystallizes  from  a  hot  33-per  cent  caustic-soda  solu- 
tion on  cooling  in  broad  shining  crystals  and  dissolves  readily  in  hydrochloric  or 
sulphuric  acid.  It  gives  a  characteristic  chloroplatinate  crystallizing  in  six-sided 
prisms.  It  is  precipitated  neither  by  basic  lead  acetate  nor  by  basic  lead  ace- 
tate and  ammonia.  Silver  nitrate  and  ammonia  give  a  gelatinous  precipitate. 
It  responds  to  the  xanthine  test  with  nitric  acid  and  alkali.  It  acts  like  episarkine 
with  Weidel's  test  according  to  Fischer. 

In  preparing  alloxuric  bases  from  the  urine,  the  fluid  is  supersaturated  with 
ammonia  and  precipitated  by  a  silver-nitrate  solution.  The  precipitate  is  then 
decomposed  with  sulphuretted  hydrogen.  The  boiling-hot  filtrate  is  evaporated 
to  dryness  and  the  dried  residue  treated  with  3-per  cent  sulphuric  acid.  The 
purine  bases  are  dissolved,  while  the  uric  acid  remains  undissolved.  This  filtrate 
is  saturated  with  ammonia  and  precipitated  by  silver-nitrate  solution.  If  instead 
of  precipitating  with  silver  solution  we  desire  to  precipitate,  according  to  Kruger 
and  Wulff,  with  copper  suboxide,  the  urine  may  be  heated  to  boiling,  and  imme- 
diately are  added,  successively,  100  cc.  of  a  50-per  cent  sodium-bisulphite  solu- 
tion and  100  cc.  of  a  12-per  cent  copper-sulphate  solution  for  every  liter  of  urine. 
The  thoroughly  washed  precipitate  is  decomposed  with  hydrochloric  acid  and 
sulphuretted  hydrogen.  The  uric  acid  remains  in  great  part  on  the  filter.  Further 
details  in  regard  to  the  treatment  of  the  solution  of  the  hydrochloric-acid  com- 
pounds may  be  found  in  Kruger  and  Salomon.4 


1  Thudichum,  "  Grundziige  d.  anal.  med.  klin.  Chemie  "  (Berlin,  1886);  Salomon, 
Arch.  f.  (Anat.  u.)  Physiol.,  1882,  and  Ber.  d.  deutsch.  chem.  Gesellsch.,  16  and    18. 

2  Balke,  "  Zur  Kenntniss  der  Xanthinkorper  "  (Inaug.-Diss.,  Leipzig,  1893);  Salo- 
mon, Zeitschr.  f.  physiol.  Chem.,  18. 

3  Arch.  f.   (Anat.  u.)  Physiol.,   1894;    Kruger  and  Salomon,  Zeitschr.  f.  physiol. 
Chem.,  24  and  26. 

4  Zeitschr.  f.  physiol.  Chem.,  26,  and  also   Hoppe-Seyler-Thierf elder's  Handbuch, 
8.  Aufl.,  188. 


ESTIMATION  OF  PURINE  BASES.  715 

Quantitative  Estimation  of  Purine  Bases  according  to  Salkowski.1 
400-600  cc.  of  the  urine  free  from  protein  are  first  precipitated  by  mag- 
nesia mixture,  and  then  by  a  3-per  cent  silver-nitrate  solution  as  described 
on  page  710.  The  thoroughly  washed  silver  precipitate  is  decomposed 
by  sulphuretted  hydrogen  after  being  suspended  in  600-800  cc.  of  water 
with  the  addition  of  a  few  drops  of  hydrochloric  acid.  It  is  heated  to 
boiling  and  filtered  hot,  and  finally  evaporated  to  dryness  on  the  water- 
bath.  The  residue  is  extracted  with  20-30  cc.  of  hot  3-per  cent  sul- 
phuric acid  and  allowed  to  stand  twenty-four  hours;  the  uric  acid  is 
filtered  off,  washed,  the  filtrate  made  ammoniacal,  and  the  purine  bodies 
again  precipitated  by  silver  nitrate,  the  precipitate  collected  on  a  small 
chlorine-free  filter,  washed  thoroughly,  dried,  carefully  incinerated, 
the  ash  dissolved  in  nitric  acid,  and  titrated  with  ammonium  sulpho- 
cyanide  according  to  Volhard's  method.  The  ammonium-sulphocyanide 
solution  should  contain  1.2-1.4  grams  per  liter,  and  its  strength  should 
be  determined  by  a  silver-nitrate  solution:  1  part  silver  corresponds 
to  0.277  gram  nitrogen  x>f  purine  bases,  or  to  0.7381  gram  purine  bases. 
By  this  method  the  uric-acid  and  purine  bases  can  be  simultaneously 
determined  in  the  same  portion  of  urine.2 

Malfatti  3  determines  the  nitrogen  of  the  purine  bases  in  the  hydrochloric- 
acid  filtrate  from  the  separated  uric  acid.  This  filtrate  is  evaporated  with  mag- 
nesia until  all  the  ammonia  has  been  expelled  and  the  residue  used  for  the  Kjel- 
dahl  determination. 

The  nitrogen  of  the  purine  bases  is  also  determined  as  the  difference  between 
the  uric-acid  nitrogen  and  the  total  nitrogen  of  the  purine  bodies  of  the  silver 
precipitate  (Camerer,  Arxstein4).  Certain  objections  have  been  raised 
against  this  method  but  they  can  be  overcome  by  using  the  modified  method 
as  suggested  by  Kexxaway.5 

According  to  the  method  of  Kruger  and  Schmid  6  the  uric  acid  and  the 
purine  bases  are  precipitated  as  a  cuprous  compound  by  copper-sulphate  solu- 
tion and  sodium  bisulphite.  The  precipitate  is  decomposed  in  sufficient  water 
by  sodium  sulphide,  and  the  uric  acid  precipitated  from  the  concentrated  filtrate 
with  hydrochloric  acid,  and  the  purine  bases  again  precipitated  from  this  nitrate 
as  cuprous  or  silver  compounds.  Finally,  the  nitrogen  in  the  uric-acid  part  and 
the  part  containing  the  mixture  of  purine  bases  is  estimated. 

Oxaluric  Acid,  C3HA204  =  (COX2H3).CO.COOH.  This  acid,  whose  relation 
to  uric  acid  and  urea  has  been  spoken  of  above,  does  not  always  occur  in  the 
urine,  and  then  only  in  traces  as  the  ammonium  salt.  This  salt  is  not  directly 
precipitated  by  CaCla  and  XH3,  but  on  boiling  it  is  decomposed  into  urea  and 
oxalate.  In  preparing  oxaluric  acid  from  urine  the  latter  is  filtered  through 
animal  charcoal.  The  oxalurate  retained  by  the  charcoal  may  be  obtained  by 
boiling;  with  alcohol. 


1  Pfluger's  Arch.,  69. 

1  In  regard  to  the  details  we  refer  the  reader  to  the  original  paper. 

3  Centralbl.  f.  innere  Med.,  1897. 

4  Camerer,  Zeitschr.  f.  Biologie,  26  and  28;  Arnstein,  Zeitschr.  f.  physiol.  Chem.,  23. 
8  Journ.  of  Physiol.,  39. 

6  Zeitschr.   f.   physiol.   Chem.,   45,    and   Hoppe-Sevler-Thierf elder's  Handbuch,   8. 
Aufl.,  590. 


716  URINE. 

COOTT 
Oxalic  Acid,  C2H2O4,  or  •  ,  occurs  under  physiological  conditions 

COOH 

in  very  small  amounts  in  the  urine,  about  0.02  gram  in  twenty-four  hours 
(Furbringer  x).  According  to  the  generally  accepted  view  it  exists  in 
the  urine  as  calcium  oxalate,  which  is  kept  in  solution  by  the  acid  phos- 
phates present.  Calcium  oxalate  is  a  frequent  constituent  of  uninary 
sediments,  and  also  occurs  in  certain  urinary  calculi. 

The  origin  of  the  oxalic  acid  in  the  urine  is  not  well  known.  Oxalic 
acid  when  administered  is  eliminated  unchanged,  at  least  in  part,  by 
the  urine;2  and  as  many  vegetables  and  fruits,  such  as  cabbage,  spinach, 
asparagus,  sorrel,  apples,  grapes,  etc.,  contain  oxalic  acid,  it  is  possible 
that  a  part  of  the  oxalic  acid  of  the  urine  originates  directly  from  the 
food.  That  oxalic  acid  may  be  formed  in  the  animal  body  as  a  metabolic 
product  from  proteins  or  fats  follows  from  the  observations  of  Mills 
and  Luthje  and  others,  who  found  that  in  dogs  on  an  exclusively  meat 
and  fat  diet,  as  also  in  starvation,  oxalic  acid  was  eliminated  by  the  urine. 
The  oxalic  acid  which  is  eliminated  in  increased  quantity  with  a  diminished 
oxygen  supply  and  an  increased  protein  catabolism,  as  found  by  Reale 
and  Boeri,  and  also  by  Terray,  is  supposed  to  be  derived  partly  from 
the  greater  destruction  of  proteins.  Pure  protein  does  not,  accord- 
ing to  Salkowski  and  Wegrzynowski  3  increase  the  quantity  of  oxalic 
acid  eliminated;  on  the  contrary,  after  meat  feeding  the  amount  of  this 
acid  is  increased,  due  in  part  to  the  meat  containing  oxalic  acid  (Sal- 
kowski). Gelatin  and  gelatin-yielding  tissues  seem  to  increase  the 
excretion  of  oxalic  acid,  and  the  same  is  also  true  for  fats  or  at  least 
glycerin  (Wegrzynowski).  After  feeding  nucleins  no  constant  increase 
in  the  elimination  of  oxalic  acid  has  been  observed.  The  statements 
as  to  the  action  of  carbohydrates  are  contradictory.  The  production  of 
oxalic  acid  due  to  an  incomplete  combustion  of  the  carbohydrates  has  also 
been  suggested,  and  the  work  of  Hildebrandt  and  P.  Mayer  seems  to 
indicate  this  under  abnormal  conditions.  According  to  Darin,4  in  rabbits 
an  increased  elimination  of  oxalic  acid  occurs  after  the  introduction  of 
glycollic  or  glyoxylic  acids,  and  the  oxalic  acid  seems  in  many  cases  to 
be  an  intermediary  product  of  metabolism,  which  is  further  burnt.  We 
cannot  exclude  the  possibility  of  the  formation  of  oxalic  acid  in  the 
oxidation  of  uric  acid  in  the  animal  body,  yet  we  have  no  positive  proof 


1  Deutsch.  Arch.  f.  klin.  Med.,  18.     See  also  Dunlop,  Journ.  Path,  and  Bacterid.,  3. 

2  In  regard  to  the  behavior  of  oxalic  acid  in  the  animal  body,  see  page  773. 

3  Reale  and  Boeri,  Wien.  med.  Wochenschr.,  1895;  Terray,  Pfliiger's  Arch.,  65; 
Salkowski,  Berl.  klin.  Wochenschr.,  1900;  Wegrzynowski.  Zeitschr.  f.  physiol.  Chem., 
83  which  contains  the  literature. 

4  Journ.  of  biol.  Chem.,  3,  57. 


ALLANTOIN.  717 

of  such  a  formation.1  An  endogenous  as  well  as  an  exogenous  origin 
of  oxalic  acid  has  also  been  suggested. 

Oxalic  acid  is  best  detected  and  quantitatively  determined  according 
to  the  method  suggested  by  Salkowski:  Shaking  out  the  oxalic  acid  from 
the  acidified  urine  by  means  of  ether.  Detailed  account  of  this  can 
be  found  in  Wegrzynowski.2 

.NH.CH.HN.CO.NH2, 
Allantoin  (Glyoxyldiureide),  C4H6N4O3,  OC\ 

xNH.CO 

occurs,  it  is  claimed  by  earlier  writers,  in  the  urine  of  children  within  the 
first  eight  days  after  birth,  and  in  very  small  amounts  also  in  the  urine 
of  adults  (Gtjsserow,  Ziegler  and  Hermann).  It  is  found  in  rather 
abundant  quantities  in  the  urine  of  pregnant  women  (Gusserow;. 
According  to  Wiechowski  the  urine  of  adults,  if  it  contains  any  allan- 
toin at  all,  has  only  traces,  and  he  could  not  detect  any  in  the  urine  of 
nurslings  or  in  the  amniotic  fluid,  which  does  not  agree  with  previous 
reports.  Allantoin  has  also  been  found  in  the  urine  of  suckling  calves 
(Wohler),  in  urine  of  oxen  (Salkowski),  and  sometimes  in  the  urine  of 
other  animals  (Meissner).  Wiechowski  has  .found  it  in  relatively 
large  quantities  in  the  urine  of  the  dog,  cat,  rabbit  and  monkey,  and  he 
considers  that  allantoin  is  a  terminal  metabolic  product  in  these  ani- 
mals. It  is  also  found,  as  first  shown  by  Valquelin  and  Lassaigne,3 
in  the  allantoic  fluid  of  the  cow  (hence  the  name).  That  allantoin  is 
formed  from  the  uric  acid  in  mammalia  is  almost  certain,  and  the  inves- 
tigations on  which  this  is  based  have  already  been  given  in  discussing  the 
decomposition  of  uric  acid.4  The  allantoin  thus  originates  from  the 
purine  bodies,  and  consequently  in  clogs  and  other  animals  the  excretion 
of  allantoin  is  considerably  increased,  according  to  Minkowski,  Cohn, 
Salkowski,  and  Mendel  and  Brown.5  after  feeding  thymus  or  pan- 
creas. A  strong  allantoin  excretion  is  also  found  in  dogs  after  poisoning 
with  hydrazine  (Borissow),  hydroxylamine,  semicarbazide,  and  amino- 
guanidine    (Pohl),    and   this   increase   in   the   excretion   cf   allantoin   is 

1  See  Wiener,  Ergebn.  d.  Physiol.,  1;  Tomaszewski,  Zeitschr.  f.  exp.  Path.  u.  Ther.f 
7;  Pohl,  ibid.,  8;  Jastrowitz,  Bioch.  Zeitschr.,  28. 

2  Zeitschr.  f.  physiol.  Chem.,  83. 

3  Ziegler  and  Hermann,  see  Gusserow,  Arch.  f.  Gynakol..  3 — both  cited  from  Huppert- 
Neibauer,  Ham-Analyse,  10.  Aufl.,  377;  Wohler,  Anna!  d.  Chem.  u.  Pharm.,  70; 
Salkowski,  Zeitschr.  f.  physiol.  Chem.,  42;  Meissner.  Zeitschr.  f.  rat.  Me. I.  (3),  31; 
Lassaigne,  Annal.  de  Chim.  et  Phys.,  17;  Wiechowski,  Hofmeister's  Beitrage,  11,  and 
Arch.  f.  exp.  Path.  u.  Pharm.,  60,  and  Bioch.  Zeitschr.,  l',>  and  25. 

4  See  footnote  2,  page  706. 

5  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  41.  and  Centralbl.  f.  innere  Med.,  1S98; 
Cohn,  Zeitschr.  f.  physiol.  Chem.,  25;  Salkowski,  Centralbl.  f.  d.  med.  Wissensch., 
1898;  Mendel  and  Brown,  Amer.  Journ.  of  Physiol.,  3. 


718  URINE. 

connected  with  the  nuclein  metabolism.  Pohl  1  has  found,  in  dogs  on 
poisoning  with  hydrazine,  that  the  liver  contained  allantoin  and  that  other 
organs  contained  traces,  while  it  does  not  exist  in  the  organs  of  normal 
dogs,  and  he  has  also  detected  the  formation  of  allantoin  in  the  autolysis 
of  the  intestinal  mucosa,  liver,  thymus,  spleen  and  pancreas.  It  is  very 
probable  that  in  these  cases  we  are  dealing  with  a  destruction  of  cells 
and  an  enzymotic  uric  acid  formation  with  a  subsequent  uricolysis 
with  the  formation  of  allantoin.  Certain  food-stuffs  such  as  milk,  wheat 
bread,  peas  and  beans  contain,  according  to  Ackroyd,  small  amounts  of 
allantoin,  which  are  introduced  into  the  body.  Nothing  is  known  about 
how  these  traces  of  allantoin  behave  in  the  body.  According  to  Po- 
duschka  and  Minkowski,2  allantoin  introduced  into  dogs  appears  almost 
entirely  in  the  urine,  while  in  man  only  a  small  portion  of  the  ingested 
substance  is  eliminated  in  the  urine  and  seems  in  the  last  case  to  be  chiefly 
burned. 

Allantoin  is  a  colorless  substance  often  crystallizing  in  prisms,  dif- 
ficultly soluble  in  cold  water,  easily  soluble  in  boiling  water,  and  also  in 
warm  alcohol,  but  not  soluble  in  cold  alcohol  or  ether.  A  watery  alla- 
toin  solution  gives  no  precipitate  with  silver  nitrate  alone,  but  by  the 
careful  addition  of  ammonia  a  white  flocculent  precipitate  is  formed, 
C4HsAgN403,  which  is  soluble  in  an  excess  of  ammonia  and  which  con- 
sists after  a  certain  time  of  very  small,  transparent  microscopic  globules. 
The  dry  precipitate  contains  40.75  per  cent  silver.  A  watery  allantoin 
solution  is  precipitated  by  mercuric  nitrate.  On  continued  boiling 
allantoin  reduces  Fehling's  solution.  It  gives  Schiff's  furfurol  reac- 
tion less  rapidly  and  less  intensely  than  urea.  Allantoin  does  not  give 
the  murexide  test. 

Allantoin  is  most  easily  prepared  by  the  oxidation  of  uric  acid  with 
lead  peroxide  or  potassium  permanganate.  In  preparing  allantoin  from 
urine  we  must  proceed  differently  according  to  whether  we  are  using  the 
urine  of  animals  comparatively  rich  in  allantoin  or  whether  we  are  using 
human  urine,  which  is  very  poor  in  allantoin.  The  same  applies  to  the 
quantitative  estimation  of  allantoin.  As  the  methods  in  both  cases  are 
complicated  and  require  certain  percautions  we  cannot  here  enter  into  a 
detailed  description  of  them,  and  we  refer  to  the  works  of  Loewi  and 
Wiechowski3  and   to  the  complete   handbooks  for  details.     The  pre- 

1  Borissow,  Zeitschr.  f.  physiol.  Chem.,  19;  Pohl.  Arch.  f.  exp.  Path.  u.  Pharm., 
46;  Poduschka,  ibid.,  44.  According  to  Underhill  and  Kleiner,  Journ.  of  biol.  Chem., 
4,  hydrazine  has  no  other  action  on  the  excretion  of  allantoin  than  that  caused  by 
the  refusal  to  take  food  brought  about  by  the  poison. 

2  Ackroyd,  Bioch.  Journ.,  5;  Poduschka,  Arch.  f.  exp.  Path.  u.  Pharm.,  44;  Min- 
kowski, ibid.,  41. 

3  Loewi,  ibid.,  44;  Wiechowski,  Hofmeister's  Beitriige,  11,  and  Arch,  f.  exp.  Path, 
u.  Pharm.,  (50;  and  Bioch.  Zeitschr.,  19  and  25. 


HIPPUKIC  ACID.  719 

cipitation  of  allantoin  from  the  urine  can  be  accomplished  by  mercuric 
nitrate  and  by  mercuric  acetate  solutions,  in  the  presence  of  sodium 
acetate. 

Glyoxylic  Acid,  C;H404,  /v^tt      ,  is  produced  on  boiling  allantoin  as  well  as 

uric  acid  with  alkalies,  and  also  on  the  oxidation  of  many  substances,  among 
which  we  can  mention  creatine  and  creatinine.  It  is  also  of  interest  that  allantoin 
can  be  prepared  synthetically  from  glyoxylic  acid  and  urea  and  that  glyoxylic 
acid  yields  oxalic  acid  when  introduced  into  the  body.  The  reports  in  regard 
to  its  occurrence  in  the  urine  conflict,1  as  it  is  readily  destroyed  in  the  body,  and 
its  passage  into  the  urine  is  very  improbable,  or  at  least  only  seldom  occurs. 

Hippuric  Acid  (Benzoyl  amino  acetic  acid), 

C9H9XO3  =  (C6H5CO)HN  •  CH2COOH. 

This  acid  decomposes  into  benzoic  acid  and  glycocoll  on  boiling  with 
mineral  acids  or  alkalies,  and  also  in  the  putrefaction  of  the  urine.  The 
reverse  of  this  occurs  if  these  two  components  are  heated  in  a  sealed 
tube,  according  to  the  following  equation:  C6H5COOH  +  XH2.CH2.COOH 
=  C6H5.CO.XH.CHo.COOH+H20.  This  acid  may  be  synthetically 
prepared  from  benzamide  and  monochloracetic  acid,  CeH5.CO.NHa 
+CH2Cl.COOH  =  C6H5.CO.NH.CH2.COOH-r-HCl,  and  in  variou:  other 
ways,  but  most  simply  from  glycocoll  and  benzoyl  chloride  in  the  presence 
of  alkali. 

Hippuric  acid  occurs  in  large  amounts  in  the  urine  of  herbivora, 
but  only  in  small  quantities  in  that  of  carnivora.  The  quantity  of  hip- 
puric acid  eliminated  in  human  urine  on  a  mixed  diet  is  usually  less  than 
1  gram  per  day;  as  an  average  it  is  0.7  gram.  After  eating  freely  of  vege- 
tables and  fruit,  especially  such  fruit  as  plums,  the  quantity  may  be 
more  than  2  grams.  Hippuric  acid  is  also  found  in  the  perspiration,  the 
blood,  the  suprarenal  capsule  of  oxen,  and  in  ichthyosis  scales.  Noth- 
ing is  positively  known  in  regard  to  the  quantity  of  hippuric  acid  in  the 
urine  in  disease. 

The  Formation  of  Hippuric  Acid  in  the  Organism.  Benzoic  acid  and 
also  the  substituted  benzoic  acids  are  converted  into  hippuric  acid  and 
substituted  hippuric  acids  within  the  body.  Moreover,  those  bodies 
are  transformed  into  hippuric  acid  which  by  oxidation  (toluene,  cinnamic 
acid,  hydrocinnamic  acid)  or  by  reduction  (quinic  acid)  are  converted 
into  benzoic  acid.  The  question  of  the  origin  of  hippuric  acid  is  there- 
fore connected  with  the  question  of  the  origin  of  benzoic  acid;  the  for- 
mation of  the  second  component,  glycocoll,  from  the  protein  substances 
in  the  body  is  unquestionable. 

1  The  literature  on  the  occurrence  and  detection  of  glyoxylic  acid  in  the  urine  can 
be  found  in  Granstrom,  Hofmeister"s  Beitrage,  11. 


720  UKINE. 

Hippuric  acid  is  found  in  the  urine  of  starving  dogs  (Salkowski), 
also  in  dog's  urine  after  a  diet  consisting  entirely  of  meat  (Meissner 
and  Shepard,  Salkowski,  and  others1).  It  is  evident  that  the  benzoic 
acid  originates  in  these  cases  from  the  proteins,  and  it  is  generally  admitted 
that  it  is  produced  by  the  putrefaction  of  proteins  in  the  intestine. 
Among  the  products  of  the  putrefaction  of  protein  outside  of  the  body 
Salkowski  found  phenylpropionic  acid,  CeH5.CH2.CH2.COOH,  which 
is  oxidized  in  the  organism  to  benzoic  acid  and  eliminated  as  hippuric 
acid  after  combining  with  glycocoll.  Phenylpropionic  acid  seems  to  be 
formed  from  the  phenylalanine.  The  supposition  that  the  phenylpro- 
pionic acid  is  produced  from  tyrosine  by  putrefaction  of  the  intestine 
has  not  been  substantiated  by  the  researches  of  Baumann,  Schotten, 
and  Baas.2  The  importance  of  putrefaction  in  the  intestine  in  pro- 
ducing hippuric  acid  is  evident  from  the  fact  that  after  thoroughly  dis- 
infecting the  intestine  of  dogs  with  calomel  the  hippuric  acid  disappears, 
from  the  urine  (Baumann3). 

The  large  quantity  of  hippuric  acid  present  in  the  urine  of  herbivora 
is  partly  explained  by  the  specially  active  processes  of  putrefaction  going 
on  in  the  intestine  of  these  animals.  According  to  Vasiliu  4  this  can 
hardly  be  correct,  because,  as  he  has  found,  by  feeding  sheep  with  casein, 
this  would  require  a  too  intense  putrefaction  of  the  protein  (indeed  40 
per  cent  of  it).  This  author's  explanation  lies  in  part  that  in  the  her- 
bivora only  a  small  part  of  the  phenylalanine  is  burnt,  and  is  used  to  a 
greater  extent  in  the  formation  of  hippuric  acid  than  hi  man  and  car- 
nivora,  and  in  part  by  the  fact  that  the  food  of  herbivora  contains  larger 
quantities  of  a  non-nitrogenous  mother-substance  of  the  benzoic  acid. 
There  is  hardly  any  doubt  that  the  hippuric  acid  in  human  urine  after 
a  mixed  diet,  and  especially  after  a  diet  of  vegetables  and  fruits,  orig- 
inates in  part  from  the  aromatic  substances,  e.  g.,  quinic  acid. 

The  view  proposed  by  Weiss  and  others  that  a  parallelism  exists  between 
the  excretion  of  hippuric  acid  and  uric  acid  in  that  an  increase  in  the  first  is 
followed  by  a  diminution  in  the  second,  and  that,  for  example,  quinic  acid  pro- 
duces a  diminution  in  the  excretion  of  uric  acid  corresponding  to  the  increased 
formation  of  hippuric  acid  (Weiss,  Lewin),  cannot  be  considered  as  sufficiently 
proven  (Hupfer).5 


1  Salkowski,  Ber.  d.  deutsch.  chem.  Gesellsch.,  11;  Meissner  and  Shepard, 
Untersuch.  iiber  das  Entstehen  der  Hippursiiure  im  thierschen  Organismus.  Hanover, 
1886. 

2  E.  and  H.  Salkowski,  Ber.  d.  deutsch.  Chem.  Gesellsch.,  12;  Baumann,  Zeitschr. 
f.  physiol.  Chem.,  7;  Schotten,  ibid.,  8;  Baas,  ibid.,  11. 

3  Ibid.,  10,  131. 

4  Vasiliu,  Mitt.  d.  landwirt.  Inst.  Breslau,  Bd.  4,  1907. 

6  Weiss,  Zeitschr.  f.  physiol.  Chem.,  25,  27,  38;  Lewin,  Zeitschr.  f.  klin.  Med.,  42; 


FORMATION   OF  HIPPURIC  ACID.  721 

As  the  thorough  investigations  of  Wiechowski  teach,  the  synthesis 
of  hippuric  acid  does  not  stand  in  any  direct  relation  to  the  extent  of 
protein  metabolism;  it  varies,  on  the  contrary,  with  the  duration  of 
circulation  of  benzoic  acid  and  the  quantity  of  glycocoll  present  in 
the  body.  The  amount  of  the  latter  in  intermediary  metabolism  is  so 
great  that  in  rabbits,  on  the  administration  of  benzoic  acid,  more  than 
one-half  of  the  total  urine  nitrogen  may  exist  as  glycocoll.  Magnus- 
Levy  l  found  in  rabbits  and  sheep  up  to  27.8  per  cent  of  the  total  nitro- 
gen as  hippuric-acid  nitrogen,  and  both  investigators  have  found  so  much 
hippuric-acid  nitrogen  that  it  could  not  be  accounted  for  by  the  glycocoll 
preformed  from  the  proteins,  which  amounts  to  about  4-5  per  cent  of 
the  total  nitrogen  of  the  protein  of  the  food  and  body. 

In  carnivora  (dog)  and  man  the  conditions  are  different,  according 
to  Brugsch  and  R.  Hirsch,  Feigin  and  Brugsch,  as  in  these  cases  there 
is  no  more  glycocoll  available  for  hippuric  acid  formation  than  is  split 
off  from  the  proteins  en  hydrolysis.  According  to  the  investigations  of 
Lewinski  2  this  does  not  seem  to  be  correct,  at  least  not  for  man.  After 
abundant  introduction  of  benzoic  acid  in  man  about  34  per  cent  of  the 
total  nitrogen  may  be  excreted  as  hippuric  acid  and  in  a  recent  investiga- 
tion he  was  able  to  obtain  50.5  grams  pure  crystalline  hippuric  acid 
from  the  24-hour  urine  of  a  man  after  feeding  sodium  benzoate. 

The  abundant  production  of  hippuric  acid  in  herbivora  induced  Abder- 
halden,  Gigon  and  Strauss  to  investigate  the  comparative  supply  of 
certain  amino-acids  in  carnivora  and  herbivora,  and  they  found  in  cats, 
rabbits  and  hens  that  the  percentage  quantity  of  glycocoll  split  off  from 
the  entire  organism  (with  the  exception  of  the  intestinal  contents  and 
fat  and  feathers)  by  hydrolysis  was  the  same,  namely  2.33  to  3.34  per 
cent  of  the  proteins.  In  order  to  account  for  the  large  quantity  of 
glycocoll  which  can  be  eliminated  as  hippuric  acid,  we  must  admit  of  a 
formation  of  glycocoll.  That  this  occurs  in  animals  fed  with  benzoic 
acid  has  been  recently  proved  by  Abderhalden  and  Hirsch  by  very 
conclusive  experiments.  It  can  be  assumed  that  the  benzoic  acid  com- 
bines with  higher  amino-acids  and  that  the  hippuric  acid  is  formed  from 
this  combination.  The  investigations  of  Magnus-Levy  to  prove  this 
assumption,   where  he  used  benzoylated  higher  amino-acids,   have  not 


Hupfer,  Zeitschr.  f.  physiol.  Chem.,  37.     See  also  Wiener,  "  Die  Harnsiiure,"  Ergeb- 
nisse  der  Physiol.,  1,  Abt.  1. 

1  Wiechowski,  Hofmeister's  Beitrage,  7  (literature);  A.  Mangus-Levy,  Munch, 
med.  Wochenschr.,  1905;  Ringer,  Journ.  of  biol.  Chem.,  10;  Epstein  and  Bookman, 
ibid.,  10. 

2  Brugsch  and  Hirsch,  Zeitschr.  f.  exp.  Path.  u.  Therap..  3;  Brunch.  Maly's 
Jahresber.,  37,  621,  and  Bioch.  Centralbl.,  8,  336;  Feigin,  Maly's  Jahresber.,  86, 
631;  Lewinski,  Arch.  f.  exp.  Path.  u.  Pharm.,  58  and  61. 


•722  URINE. 

given  support  to  this  assumption;  Epstein  and  Bookman  1  found  never- 
theless in  experiments  with  rabbits  after  feeding  with  benzoyl-leucine 
that  a  great  elimination  of  hippuric  acid  occurred  which  they  consider 
as  a  formation  of  glycocoll  from  this  leucine.  Free  leucine  on  the  con- 
trary does  not  increase  the  hippuric  acid  elimination. 

The  kidneys  may  be  considered  in  dogs  as  special  organs  for  the  syn- 
thesis of  hippuric  acid  (Schmiedeberg  and  Bunge2).  In  other  animals 
as  in  rabbits,  the  formation  of  hippuric  acid  seems  to  take  place  in  other 
organs,  such  as  the  liver  and  muscles.  The  synthesis  of  hippuric  acid  is 
therefore  not  exclusively  limited  to  any  special  organ,  though  perhaps 
in  some  species  of  animals  it  may  be  more  abundant  in  one  organ  than  in 
another. 

Properties  and  Reactions  of  Hippuric  Acid.  This  acid  crystallizes  in 
semi-transparent,  long,  four-sided,  milk-white,  rhombic  prisms  or  columns, 
or  in  needles  by  rapid  crystallization.  They  dissolve  in  600  parts  cold 
water,  but  more  easily  in  hot  water.  They  are  easily  soluble  in  alcohol, 
but  with  difficulty  in  ether.  The  acid  dissolves  more  easily  (about  12 
;  times)  in  acetic  ether  than  in.  ethyl  ether.  Petroleum-ether  does  not 
dissolve  hippuric  acid. 

On  heating  hippuric  acid  it  first  melts  at  187.5°  C.  to  an  oily  liquid 
which  crystallizes  on  cooling.  On  continued  heating  it  decomposes, 
producing  a  red  mass  and  a  sublimate  of  benzoic  acid,  with  the  genera- 
tion, first,  of  a  peculiar  pleasant  odor  of  hay  and  then  an  odor  of  hydro- 
cyanic acid.  Hippuric  acid  is  easily  differentiated  from  benzoic  acid 
by  this  behavior,  also  by  its  crystalline  form  and  its  insolubility  in 
petroleum  ether.  Hippuric  acid  and  benzoic  acid  both  give  Lucre's 
reaction,  namely,  they  generate  an  intense  odor  of  nitrobenzene  when 
evaporated  to  dryness  with  nitric  acid  and  when  the  residue  is  heated 
with  sand  in  a  glass  tube.  Hippuric  acid  in  most  cases  forms  crystal- 
lizable  salts,  with  bases.  The  combinations  with  alkalies  and  alkaline 
earths  are  soluble  in  water  and  alcohol.  The  silver,  copper,  and  lead 
salts  are  soluble  with  difficulty  in  water;  the  ferric  salt  is  insoluble. 

Hippuric  acid  is  best  prepared  from  the  fresh  urine  of  a  horse  or  cow. 
The  urine  is  boiled  a  few  minutes  with  an  excess  of  milk  of  lime.  The 
liquid  is  filtered  while  hot,  concentrated  and  then  cooled,  and  the  hippuric 
acid  precipitated  by  the  addition  of  an  excess  of  hydrochloric  acid.  The 
crystals  are  pressed,  dissolved  in  milk  of  lime  by  boiling,  and  treated  as 
above;    the  hippuric  acid  is  precipitated  again  from  the  concentrated 

1  Abderhalden,  Gigon  and  Strauss,  Zeitschr.  f.  physiol.  Chem.,  51;  Abderhalden 
and  Hirseh,  ibid.,  78;  Mangus-Levy,  Bioch.  Zeitschr.,  6;  Epstein  and  Bookman, 
Journ.  of  biol.  Chem.,  13. 

2  Arch.  f.  exp.  Path.  u.  Pharm.,  6;  alpo  A.  Hoffmann,  ibid.,  7,  and  Kochs,  Pfluger's: 
Arch.,  20;  Bashford  and  Cramer,  Zeitschr.  f.  physiol.  Chem.,  35. 


PHENACETURIC  ACID.     BENZOIC  ACID.  723 

filtrate  by  hydrochloric  acid.     The  crystals  arc  purified  by  recrystalliza- 
tion  and  decolorized,  when  necessary,  by  animal  charcoal. 

The  quantitative  estimation  of  hippuric  acid  in  the  urine  may  be 
performed  by  the  following  method  (Binge  and  Schmiedeberg): 
The  urine  is  first  made  faintly  alkaline  with  soda,  evaporated  nearly  to 
dryness,  and  the  residue  thoroughly  extracted  with  strong  alcohol. 
After  the  evaporation  of  the  alcohol  the  residue  is  dissolved  in  water, 
the  solution  acidified  with  sulphuric  acid,  and  completely  extracted  by 
agitating  (at  least  five  times)  with  fresh  portions  of  acetic  ether.  The 
acetic  ether  is  then  repeatedly  washed  with  water,  which  is  removed  by 
means  of  a  separatory  funnel,  then  evaporated  at  a  medium  temperature 
and  the  dry  residue  treated  repeatedly  with  petroleum-ether,  which 
dissolves  the  benzoic  acid,  oxyacids,  fats,  and  phenols,  while  the  hippuric 
acid  remains  undissolved.  This  residue  is  ncrw  dissolved  in  a  little  warm 
water  and  evaporated  at  50-60°  C.  to  crystallization.  The  crystals  are 
collected  on  a  small  weighed  filter.  According  to  Henriques  and 
Sorensen  the  acidified  urine  can  be  directly  shaken  out  with  acetic  ether, 
the  residue  after  evaporation  of  the  acetic  ether  boiled  with  hydrochloric 
acid  in  order  to  split  the  hippuric  acid  into  benzoic  acid  and  glycocoll 
and  the  quantity  of  nitrogen  in  the  latter  determined  by  a  formol  titra- 
tion. Other  methods  have  recently  been  suggested  by  Folin  and 
Flanders,  by  Steenbock  and  by  Hryntschak.1 

Phenaceturic  Acid,  Ci0HnNO3=C6H5.CH..CO.XH.CH2.COOH.  This  acid, 
which  is  produced  in  the  animal  body  by  a  combination  of  glycocoll  with  the  phenyl- 
acetic  acid,  CeH5.CH2.COOH,  formed  in  the  putrefaction  of  the  proteins,  has 
been  prepared  from  horse's  urine  by  Salkowski,2  but  it  probably  also  occurs  in 
human  urine.  According  to  Vasiliu  3  it  is  just  as  important  a  constituent  of  the 
urine  of  herbivora  as  hippuric  acid  is. 

Benzoic  Acid,  CyHeOo  or  C6H5.COOH,  is  found  in  rabbit's  urine  and  sometimes, 
though  in  small  amounts,  in  dog's  urine  (Weyl  and  v.  Anrep).  According  to 
Jaarsveld  and  Stokvis  and  to  Kronecker  it  is  also  found  in  human  urine  in 
diseases  of  the  kidneys.  The  occurrence  of  benzoic  acid  in  the  urine  seems  to 
be  due  to  a  fermentative  decomposition  of  hippuric  acid.  Such  a  decomposi- 
tion may  very  easily  occur  in  an  alkaline  urine  or  in  one  containing  proteid(\'AN 
de  Velde  and  Stokvis).  In  certain  animals — pigs  and  dogs — the  kidneys, 
according  to  Schmiedeberg  and  Minkowski,4  contain  a  special  enzyme,  Schmiede- 
berg's  histozym, winch  splits  the  hippuric  acid  with  the  separation  of  benzoic 
acid. 

Ethereal  Sulphuric  Acids.  In  the  putrefaction  of  proteins  in  the 
intestine,  phenols — whose  mother-substance  is  considered  to  be  tyrosine — 
and  also  indol  and  skatol  are  produced.  These  phenols  directly,  and  the 
two  last-named  bodies  after  they  have  been  oxidized  respectively  into 

1  Bunge  and  Schmiedeberg, .  Arch.  f.  exp.  Path.  u.  Pharm.,  6;  Henriques  and 
Sorensen,  Zeitschr.  f.  physiol.  Chem.,  64;  Folin  and  Flanders,  Journ.  of  biol.  Chem.,  11; 
Steenbock,  ibid.,  11;   Hryntschak,  Bioch.  Zeitschr.,  43. 

2  Zeitschr.  f.  physiol.  Chem.,  9. 

s  Mitteil.  d.  landw.  Inst.,  Breslau,  4. 

4  Weyl  and  v.  Anrep,  Zeitschr.  f.  physiol.  Chem.,  4;  Jaarsveld  and  Stokvis,  Arch, 
f.  exp.  Path.  u.  Pharm.,  10;  Kronecker,  ibid.,  16;  Van  de  Velde  and  Stokvis,  ibid., 
17;  Schmiedeberg,  ibid.,  14,  379;  Minkowski,  ibid.,  17. 


724  UEINE. 

indoxyl  and  skatoxyl,  pass  into  the  urine  as  ethereal  sulphuric  acids 
after  uniting  with  sulphuric  acid.  The  most  important  of  these  ethereal 
acids  are  phenol-  and  cresol-sulphuric  acids — which  were  formerly  also 
called  phenol-forming  substances — indoxyl-  and  skatoxyl-sulphuric  acids. 
To  this  group  also  belong  pyrocatechin-sulphuric  acid,  which  occurs 
only  in  very  small  amounts  in  human  urine,  and  hydroquinone-sulphuric 
acid,  which  appears  in  the  urine  after  poisoning  with  phenol,  and  under 
physiological  conditions  perhaps  other  ethereal  acids  occur  which  have 
not  been  isolated.  The  ethereal  sulphuric  acids  of  the  urine  were  dis- 
covered and  specially  studied  by  Baumann.1  The  quantity  of  these 
acids  in  human  urine  is  small,  while  horse's  urine  contains  larger  quan- 
tities. According  to  the  determinations  of  v.  d.  Velden  the  quantity 
of  ethereal  sulphuric  acid  in  human  urine  in  twenty-four  hours  varies 
between  0.094  and  0.620  gram.  C.  Tollens  found  an  average  of  0.18 
gram.  The  relation  of  the  sulphate-sulphuric  acid  A  to  the  conjugated 
sulphuric  acid  B,  in  health,  is  on  an  average  10:1.  It  undergoes  such 
great  variations,  as  found  by  Baumann  and  Herter,2  and  after  them 
by  many  other  investigators,  that  it  is  hardly  possible  to  consider  the 
average  figures  as  normal.  After  taking  phenol  and  certain  other 
aromatic  substances,  as  well  as  when  putrefaction  within  the  organism 
is  general,  the  elimination  of  ethereal  sulphuric  acid  is  greatly  increased. 
On  the  contrary,  it  is  diminished  when  the  putrefaction  in  the  intestine 
is  reduced  or  prevented.  For  this  reason  it  may  be  greatly  diminished 
by  carbohydrates  and  exclusive  milk  diet.3  The  intestinal  putrefaction 
and  the  elimination  of  ethereal  sulphuric  acid  have  also  been  diminished 
in  some  cases  by  certain  therapeutic  agents  which  have  an  antiseptic 
action;  still  the  investigators  do  not  agree  in  their  reports.4 

Great  importance  has  been  given  to  the  relation  between  the  total 
sulphuric  acid  and  the  conjugated  sulphuric  acid,  or  between  the  con- 
jugated sulphuric  acid  and  the  sulphate-sulphuric  acid,  in  the  study 
of  the  intensity  of  the  putrefaction  in  the  intestine  under  different  con- 
ditions.    Several  investigators,  F.  Muller,  Salkowski,  and  v.  Noorden,5- 


1  Pfluger's  Arch.,  12  and  13. 

2  v.  d.  Velden,  Virchow's  Arch.,  70;  Tollens,  Zeitschr.  f.  physiol.  Chem.,  67;  Herter, 
Zeitschr.  f.  physiol.  Chem.,  1. 

3  See  Hirschler,  Zeitschr.  f.  physiol.  Chem.,  10;  Biernacki,  Deutsch.  Arch.  f.  klin. 
Med.,  49;  Rovighi,  Zeitschr.  f.  physiol.  Chem.,  16;  Winternitz,  ibid.,  and  Schmitz, 
ibid.,  17  and  19. 

*  See  Baumann  and  Morax,  Zeitschr.  f.  physiol.  Chem.,  10;  Steiff,  Zeitschr.  f. 
klin.  Med.,  16;  Rovighi,  1.  c;  Stern,  Zeitschr.  f.  Hyg.,  12;  and  Bartoschewitsch, 
Zeitschr.  f.  physiol.  Chem.,  17;  Mosse,  ibid.,  23. 

'  Muller,  Zeitschr.  f.  klin.  Med.,  12;  v.  Noorden,  ibid.,  17;  Salkowski,  Zeitschr. 
f.  physiol.  Chem.,  12. 


PHENOL-  AND  CKESOL-SULPHURIC  ACIDS.  725 

consider  correct  ly  that  this  relation  is  only  of  secondary  value,  and  that 
it  is  more  correct  to  consider  the  absolute  value.  It  must  be  remarked 
that  the  absolute  values  for  the  conjugated  sulphuric  acid  also  undergo 
great  variation,  so  that  it  is  at  present  impossible  to  give  the  upper  or 
lower  limit  for  the  normal  value. 

Phenol-  and  p-Cresol-sulphuric  Acids,  C6H5.O.SO2.OH  and 
().S02.OH 
CeH4<^  These  acids  are  found  as  alkali  salts  in  human  urine, 

VH3 
in  which  also  orthocresol  has  been  detected.  The  quantity  of  cresol- 
sulphuric  acid  is  considerably  greater  than  of  phenol-sulphuric  acid. 
In  the  quantitative  estimation  the  phenols  are  set  free  from  the  two 
ethereal  acids  and  determined  together  as  tribromphenol.  The  quan- 
tity of  phenols  which  are  separated  from  the  ethereal-sulphuric  acids 
of  the  urine  amounts  to  17-51  milligrams  in  the  twenty-four  hours 
(Munk).  In  nine  case  investigated  by  Siegfried  and  Zimmermann  1 
they  found  in  the  urine  of  healthy  students  in  1500  cc.  urine  an  average 
of  44.6  milligrams  phenols,  of  which  26  milligrams  was  cresol  and  18.6 
milligrams  was  phenol.  After  the  ingestion  of  carbolic  acid,  which  is 
in  great  part  converted  by  synthesis  within  the  organism  into  phenol- 
sulphuric  acid,  also  into  pyrocatechin-  and  hydroquinon-sulphuric  acid2 
or  when  the  amount  of  sulphuric  acid  is  not  sufficient  to  combine  with 
the  phenol,  it  forms  phenol-glucuronic  acid,3  the  quantity  of  phenols 
and  ethereal-sulphuric  acids  in  the  urine  is  considerably  increased  at  the 
expanse  of  the  sulphate-sulphuric  acid.  The  same  is  also  true  of  other 
phenols.  The  cresol  is  in  great  part  changed  into  phenol  in  dogs,  accord- 
ing to  Siegfried  and  Zimmermann.4 

An  increased  elimination  of  phenol-sulphuric  acids  occurs  in  active 
putrefaction  in  the  intestine  with  stoppage  of  the  contents  of  the  intes- 
tine, as  in  ileus,  diffused  peritonitis  with  atony  of  the  intestine,  or  tuber- 
culous enteritis,  but  not  in  simple  obstruction.  The  elimination  is  also 
increased  by  the  absorption  of  the  products  of  putrefaction  from 
purulent  wounds  or  abscesses.  An  increased  elimination  of  phenol  has 
been  observed  in  a  few  other  cases  of  diseased  conditions  of  the  body.5 


1  Munk,   Pfluger's  Arch.,    12;  Siegfried   and    Zimmermann,    Bioch.    Zeitschi\,    34. 

2  See  Baumann,  Pfluger's  Arch.,  12  and  13,  and  Baurnann  and  Preusse,  Zeitschr. 
f.  physiol.  Chem.,  3,  156. 

3  Schmiedeberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  11;  C.  Tollens,  Zeitschr.  f.  physiol. 
Chem.,  67. 

4  Bioch.  Zeitschr.,  46. 

6  See  G.  Hoppe-Seyler,  Zeitschr.  f.  physiol.  Chem.,  12  (this  contains  also  all  refer- 
ences to  the  literature  on  this  subject);  Fedeli,  Moleschott's  Untersuch.,  15. 


726  URINE. 

The  alkali  salts  of  phenol-  and  cresol-sulphuric  acids  crystallize  in 
white  plates,  similar  to  mother-of-pearl,  which  are  rather  freely  soluble 
in  water.  They  are  soluble  in  boiling  alcohol,  but  only  slightly  soluble 
in  cold  alcohol.  On  boiling  with  dilute  mineral  acids  they  are  decom- 
posed into  sulphuric  acid  and  the  corresponding  phenol. 

Phenol-sulphuric  acids  have  been  synthetically  prepared  by  Baumann 
from  potassium  pyrosulphate  and  potassium  phenolate  or  p-cresolate. 
For  the  method  of  their  preparation  from  urine,  which  is  rather  compli- 
cated, and  also  for  the  known  phenol  reactions,  the  reader  is  referred 
to  other  text-books.  The  quantitative  estimation  of  the  phenols  from 
these  etheral  sulphuric  acids  is  now  ordinarily  done  by  the  following 
methods: 

Kossler  and  Penny's  method  with  Neuberg's  x  modification.  The 
liquid  containing  phenol  is  treated  with  N/10  caustic  soda  until  strongly 
alkaline,  warmed  on  the  water-bath  in  a  flask  with  a  glass  stopper,  and 
then  treated  with  an  excess  of  N/10  iodine  solution,  the  quantity  being 
exactly  measured.  Sodium  iodide  is  first  formed  and  then  sodium 
hypoiodite,  which  latter  forms  tri-iodophenol  with  the  phenol  accord- 
ing to  the  following  equation: 

C6N5OH+3NaIO  =  C6H2I3.OH+3NaOH. 

On  cooling,  acidify  with  sulphuric  acid  and  determine  the  excess  of  iodine 
by  titration  with  N/10  sodium  thiosulphate  solution.  This  process  is 
also  available  for  the  estimation  of  paracresol.  Each  cubic  centimeter 
of  the  iodine  solution  used  is  equivalent  to  1.5670  milligrams  of  phenol 
or  1.8018  milligrams  of  cresol.  As  the  determination  does  not  give  any 
idea  as  to  the  variable  proportions  of  the  two  phenols,  the  quantity  of 
iodine  used  must  be  calculated  as  one  or  the  other  of  the  two  phenols. 
Before  such  a  determination  is  carried  out,  the  concentrated  urine  is 
first  distilled  after  acidification  with  sulphuric  acid  and  the  distillate 
purified  by  precipitation  with  lead,  and  distilled  again  (Neuberg). 
Mooser  has  raised  objections  against  the  use  of  sulphuric  acid  and  rec- 
ommends instead  the  use  of  phosphoric  acid.  In  regard  to  the  dispute 
which  has  arisen  between  Neuberg  and  Mooser  2  as  well  as  to  the  details 
of  Neuberg's  method  we  must  refer  to  the  original  publications  and  to 
larger  handbooks. 

For  the  separate  estimation  of  phenol  and  p-cresol  in  the  urine  a 
special  method  has  been  suggested  by  Siegfried  and  Zimmermann.3 
The  principle  of  the  method  consists  in  the  two  following  estimations: 
1.  The  quantity  of  bromine  necessary  to  convert  the  phenol  and  cresol 
into  tribromphenol  and  tribromcresol  is  determined.  2.  The  quantity 
of  bromine  necessary  to  convert  the  phenol  into  tribromphenol  and  the 


1  Kossler  and  Penny,  Zeitschr.  f.  physiol.  Chem.,  17;  Neuberg,  ibid.,  27. 

2  Mooser,  Zeitschr.    f.  physiol.  Chem.,   63,  with    Liechti,  ibid.,   73;  Neuberg  and 
Hildesheimer,  Bioch.  Zeitschr.,  28;   Marie  Hensel,  Zeitschr.  f.  physiol.  Chem.,  78. 

3  Siegfried  and  Zimmermann,  Bioch.  Zeitschr.,  29,  34  and  38;  see  also  Ditz  and  Bar- 
dach,  ibid.,  37  and  42. 


PYR0CATE0HIN-8ULPHURIC  ACID.  727 

cresol  into  dibromcresol  under  exactly  observed  conditions  is  deter- 
mined  and  from  the  quantities  of  bromine  by  weight  (Bi  and  B2) 
quantities  by  weight  of  phenol  and  cresol  can  be  calculated.  In  regard  to 
the  procedure  as  well  as  to  the  necessary  solutions  we  refer  to  the  original 
publication. 

The  methods  for  the  separate  determination  of  the  conjugated  sul- 
phuric acid  and  the  sulphate-sulphuric  acid  will  be  spoken  of  later  in 
connection  with  the  determination  of  the  sulphuric  acid  of  the  urine. 

Pyrocatechin-sulphuric  Acid.  This  acid  was  first  found  in  horse's  urine  in 
rather  large  quantities  by  Baumann.  It  occurs  in  human  urine  only  in  the  very 
smallest  amounts,  and  perhaps  not  constantly,  but  it  is  present  abundantly  in 
the  urine  after  taking  phenol,  pyrocatechin,  or  protocatechuic  acid. 

With  an  exclusively  meat  diet  this  acid  does  not  occur  in  the  urine,  and  it 
therefore  must  originate  from  vegetable  food.  It  probably  originates  from  the 
protocatechuic  acid,  which,  according  to  Preusse,  passes  in  part  into  the  urine 
as  pyrocatechin-sulphuric  acid.  This  acid  may  also  perhaps  be  formed  by  the 
oxidation  of  phenol  within  the  organism  (Baumann  and  Preusse  1). 

Pyrocatechin,  or  o-Dioxybenzene,  CeBuCOH).,  was  first  observed  in  the  urine 
of  a  child  (Ebstein  and  J.  Muller).  The  reducing  body  alcapton,  first  found 
by  Bodeker  -  in  human  urine  and  which  was  considered  for  a  long  time  as  iden- 
tical with  pyrocatechin,  is  in  most  cases  probably  homogentisic  acid   (see  below). 

Pyrocatechin  crystallizes  in  prisms  which  are  soluble  in  alcohol,  ether,  and 
water.  It  melts  at  102-104°  C,  and  sublimes  in  shining  plates.  The  watery 
solution  becomes  green,  brown,  and  finally  black  in  the  presence  of  alkali 
and  the  oxygen  of  the  air.  If  very  dilute  ferric  chloride  is  treated  with  tartaric 
acid  and  then  made  alkaline  with  ammonia,  and  this  added  to  a  watery  solution  of 
pyrocatechin,  we  obtain  a  violet  or  cherry-red  liquid  which  becomes  green  on 
adding  excess  of  acetic  acid.  Pyroeateehin  is  precipitated  by  lead  acetate.  It 
reduces  an  ammoniacal  silver  solution  at  the  ordinary  temperature,  and  with 
heat  reduces  alkaline  copper-oxide  solutions  but  does  not  reduce  bismuth  oxide. 

A  urine  containing  pyrocatechin,  if  exposed  to  the  air,  especially  when  alkaline, 
quickly  becomes  dark  and  reduces  alkaline  copper  solutions  when  heated.  In 
detecting  pyrocatechin  in  the  urine  it  ie  concentrated  when  necessary,  filtered, 
boiled  with  the  addition  of  sulphuric  acid  to  remove  the  phenols,  and  repeatedly 
shaken,  after  cooling,  with  ether.  The  ether  is  distilled  from  the  several  ethereal 
extracts,  the  residue  neutralized  with  barium  carbonate  and  shaken  again  with 
ether.  The  pyrocatechin  which  remains  after  evaporating  the  ether  may  be 
purified  by  recrystallization  from  benzene. 

Hydroquinone,  or  p-Dioxybenzene,  C6H4(OH)2,  often  occurs  in  the  urine  after 
the  use  of  phenol  (Baumann  and  Preusse).  The  dark  color  which  certain  urines, 
so-called  "  carbolic  urines,"  assume  in  the  air  is  due  to  decomposition  products. 
Hydroquinone  does  not  occur  as  a  normal  constituent  of  urine,  but  only  after 
the  administration  of  hydroquinone;  and  according  to  Lewin,3  it  may  be  found 
in  the  urine  of  rabbits  as  an  ethereal-sulphuric  acid,  being  a  decomposition  product 
of  arbutin. 

Hydroquinone  forms  rhombic  crystals  which  are  readily  soluble  in  water, 
alcohol,  and  ether.  It  melts  at  169°  C.  Like  pyrocatechin,  it  easily  reduces 
metallic  oxides.  It  acts  like  pyrocatechin  with  alkalies,  but  is  not  precipitated 
by  lead  acetate.     It  is  oxidized  into  quinone  by  ferric  chloride  and  other  oxidiz- 


1  Baumann  and  Herter,  Zeitschr.  f.  physiol.  Chem.,  1;  Preusse,  ibid.,  2;  Baumann, 
ibiii.,  3. 

-  Ebstein  and  Muller,  Virchow's  Arch.,  62;  Bodeker,  Zeitschr.  f.  rat.  Med.   (3),  7. 
3  Lewin,  Virchow's  Arch.,  92;   Bass,  Zeitschr.  f.  exp.  Path.  u.  Ther.,  10. 


7'28  URINE. 

ing  agents,  and  quinone  can  be  detected  by  its  peculiar  odor.  Hydroquinone- 
sulphuric  acid  is  detected  in  the  urine  by  the  same  methods  as  pyrocatechin  sul- 
phuric acid. 

C.O.SO2.OH 
Indoxyl-sulphuric  Acid,  C8H7NS04,  C6H4\  7CH  ,  also  called 


NH 

urine  indican,  formerly  called  uroxanthine  (Heller),  occurs  as  an 
alkali-salt  in  the  urine.  This  acid  is  the  mother-substance  of  a  great 
part  of  the  indigo  of  the  urine.  The  quantity  of  indigo  which  can  be 
separated  from  the  urine  is  considered  as  a  measure  of  the  quantity  of 
indoxyl-sulphuric  acid  (and  indoxyl-glucuronic  acid)  contained  in  the 
urine.  This  amount,  according  to  Jaffe,  for  man  is  5-20  milligrams 
per  twenty-four  hours,  and  0.9-37.6  milligrams  according  to  Maillard.1 
Horse's  urine  contains  about  twenty-five  times  as  much  indigo-forming 
substance  as  human  urine. 

Indoxyl-sulphuric  acid  is  derived,  as  previously  mentioned  (page  515), 
from  indol,  which  is  first  oxidized  in  the  body  into  indoxyl  and  is  then 
conjugated  with  sulphuric  acid.  After  subcutaneous  injection  of  indol 
the  elimination  of  indican  is  considerably  increased  (Jaffe,  Baumann 
and  Brieger,  and  others).  It  is  also  increased  by  the  introduction 
in  the  animal  organism  of  orthonitrophenolpropiolic  acid  (G.  Hoppe- 
Seyler  2) .  Indol  is  formed  by  the  putrefaction  of  proteins.  The 
putrefaction  of  secretions  rich  in  protein  in  the  intestine  also  explains  the 
occurrence  of  indican  in  the  urine  during  starvation.  Gelatin,  on  the 
contrary,  does  not  increase  the  elimination  of  indican. 

An  abnormally  increased  elimination  of  indican  occurs  in  those 
diseases  where  the  small  intestines  are  obstructed,  causing  an  increased 
putrefaction  and  thus  producing  an  abundance  of  indol.  Such  an  increased 
elimination  of  indican  occurs  on  tying  the  small  intestine  of  a  dog,  but 
not  the  large  intestine  (Jaffe),  an  observation  which  has  been  recently 
confirmed  by  Eldnger  and  Prutz.3  They  removed  an  intestine  loop 
in  dogs  and  replaced  it  in  a  reversed  position,  the  distal  end  of  the  loop 
being  attached  to  the  proximal  end  of  the  intestine,  and  in  this  manner, 
by  the  inverted  peristalsis  so  obtained,  they  effected  a  disturbance  in 
the  movement  of  the  intestinal  contents.  It  was  shown  that  this  obstruc- 
tion in  the  small  intestine  caused  an  increased  elimination  of  indican, 
while  an  obstruction  in  the  large  intestine  showed  no  such  action. 


1  Jaffe,  Pfliiger's  Arch.,  3;  Maillard,  Journ.  de  Physiol,  et  de  Pathol.,  12. 

2  Jaffe,  Centralbl.  f.  d.  med.  Wissensch.,  1872;  Baumann  and  Brieger,  Zeitschr.  f. 
physiol.  Chem.,  3;  G.  Hoppe-Seyler,  ibid.,  7  and  8.  See  also  Porcher  and  Hervieux, 
Journ.  de  Phyisol.,  7. 

*  Jaffe,  Virchow's  Arch.,  70;  Ellinger  and  Prutz,  Zeitschr.  f .  physiol.  Chem.,  38. 


INDOXYL-SULPHURIC  ACID.  729 

The  putrefaction  of  proteins  in  other  organs  and  tissues  besides  the 
intestine  may  also  cause  an  increase  in  the  indican  of  the  urine.  Cer- 
tain investigators,  Blumenthal,  Rosenfeld,  and  Lewin,  claim  to 
have  shown  that  an  increased  excretion  of  indican  can  also  be  brought 
about  without  putrefaction  by  an  increased  destruction  of  tissue  in  starva- 
tion and  also  after  phlorhizin  poisoning;  but  these  statements  are  vehe- 
mently opposed  by  other  investigators,  such  as  P.  Mayer,  Scholz,  and 
Ellinger,  and  are  improbable.  The  indol,  it  seems,  is  not  formed  from 
the  tryptophane  (indolaminopropionic  acid)  as  intermediary  step  in  the 
demolition  of  the  proteins  in  the  animal  body,  but  rather  from  the 
putrefaction  of  the  tryptophane  in  the  intestine.  Gentzen  *  has  also 
shown  that  tryptophane  introduced  subcutaneously  or  per  os  into  the 
body  does  not  lead  to  an  indicanuria,  but  only  when  it  is  exposed  to  bac- 
terial decomposition  in  the  large  intestine.  The  reports  as  to  the  elimina- 
tion of  indican  after  oxalic-acid  poisoning  are  conflicting.  After  poison- 
ing with  oxalic  acid  Harnack  and  v.  Leyen  found  an  increased  indican 
elimination,  and  Moraczewski  believes  he  has  proven  a  certain  par- 
allelism between  the  quantity  of  indican  and  the  quantity  of  oxalic  acid 
in  diabetes.  Scholz,2  on  the  contrary,  obtained  no  increase  in  the 
excretion  of  indican  after  oxalic-acid  poisoning. 

The  excretion  of  indican  is,  as  above  stated,  increased  by  the  introduction  of 
indol,  but  also  by  indoxyl  or  indoxyl-c£rboxylic  acid.  Indol-carboxylic  acid, 
on  the  contrary,  does  not  yield  indican,  but,  according  to  Porcher  and  Her- 
vieux,  another  ehromogen.  Benedicenti  has  also  shown  that  indigo  blue  or 
analogous   blue   or  green   pigments  are  produced  only  from  those  derivatives 

CH  CH 

of  indol  which,  like  n-methyl  indol  C6H4\   /CH,  a-naphtindol,  GoH6\     /CH  or 


N.CH,  NH 

CH, 

n-methylindolin,  C6H4\'  y>CH2,do  not  have  the  hydrogen  atoms  of  the  two  methine 

X.CH3 
groups  substituted  by  alkyl.     From  those  derivatives  in  which  one  or  two  hydro- 
gen atoms  are  substituted  by  alkyl,  such  as  skatol,  a-methyl  indol,  dimethyl  indol, 
C.CH3  CH 

C6H4<J^C.CH3,  and  bz.  3,  p.  2-dimethyl  indol,  CH3.GH3<^\c.CH3,  red  pig- 
NH  XH 

1  Blumenthal,  Arch.  f.  (Anat.  u.)  Physiol.,  1901,  Suppl,  and  1902,  with  Rosenfeld, 
Charite  annalen,  27,  and  Hofmeister's  Beitrage,  5;  Lewin,  Hofmeister's  Beitrage,  1; 
Mayer,  Arch.  f.  (Anat.  u.)  Physiol.,  1902,  Zeitschr.  f.  klin.  Med.,  47,  and  Zeitschr.  f. 
physiol.  Chem.,  29,  32;  Scholz,  ibid.,  38;  Ellinger,  ibid.,  39;  Gentzen,  "  Ueber  die  Vor- 
stufen  des  Indols  bei  der  Eiweissfaulnis  im  Thierkorper,"  Inaug.-Dissert..  Konigsberg, 
1904. 

2  Harnack,  Zeitschr.  f.  physiol.  Chemie,  29;  Scholz,  1.  c,  Moraczewski,  CentralbL 
f.  innere  Med.,  1903. 


730  URINE. 

ments  are  produced,  a  behavior  which  Porcher  and  Hervieux  »  have  observed  in 
several  alkyl-substituted  indols. 

An  increased  elimination  of  indican  has  been  observed  in  many 
diseases,2  and  in  these  cases  the  quantity  of  phenol  eliminated  is  also 
generally  increased.     A  urine  rich  in  phenol  is  not  always  rich  in  indican. 

The  potassium  salt  of  indoxyl-sulphuric  acid,  which  was  prepared 
pure  by  Baumann  and  Brieger  from  the  urine  of  dog  fedj  on  indol, 
has  subsequently  been  prepared  synthetically  by  Baumann  and  Thesen,3 
by  fusing  phenyl-glycine-orthocarboxylic  acid  with  alkali  and  then  from 
this  producing  the  indoxylsulphate  by  means  of  potassium  pyrosulphate. 
It  crystallizes  in  colorless,  shining  plates  or  leaves  which  are  easily  soluble 
in  water,  but  less  readily  in  alcohol.  It  is  split  by  mineral  acids  into 
sulphuric  acid  and  indoxyl.  The  latter  without  access  of  air  passes  into 
a  red  compound,  indoxyl  red,  but  in  the  presence  of  oxidizing  reagents 
is  converted  into  indigo  blue:  2C8H7NO+20  =  Ci6HioN202+2H20.  The 
detection  of  indican  is  based  on  this  last  fact. 

For  the  rather  complicated  preparation  of  indoxyl-sulphuric  acid  as 
potassium  salt  from  urine  the  reader  is  referred  to  other  text-books. 
For  the  detection  of  indican  in  urine  in  ordinary  cases  the  following 
method  of  Jaffe-Obermayer,  which  also  serves  as  an  approximate  test 
for  the  quantity  of  indican,  is  sufficient. 

Jaffe-Obermayer's  Indican  Test.  Jaffe  uses  chloride  of  lime  as 
the  oxidizing  agent,  while  Obermayer  employs  ferric  chloride.  Other 
oxidizing  agents  have  been  suggested,  such  as  potassium  permanganate, 
potassium  dichromate,  alkali  chlorate,  and  hydrogen  peroxide  (the 
latter  suggested  by  Porcher  and  Hervieux  4) .  With  Obermayer's 
reagent  the  test  is  performed  as  follows: 

The  acid  urine  (if  alkaline  it  must  be  acidified  with  acetic  acid)  (Ellin- 
ger)  is  precipitated  with  basic  lead  acetate,  1  cc.  for  every  10  cc.  of 
the  urine.  20  cc.  of  the  filtrate  are  treated  in  a  test-tube  with  an  equal 
volume  of  pure  concentrated  hydrochloric  acid  (specific  gravity  1.19) 
which  contains  2-4  grams  ferric  chloride  to  the  liter,  and  2-3  cc.  chloro- 
form are  added  and  the  mixture  immediately  thoroughly  shaken.  The 
chloroform  is  thereby  colored  more  or  less  blue,  depending  upon  the 
amount  of  indican.  Besides  indigo  blue  we  may  also  have  indigo  red 
produced,  whose   formation  has  been  explained  in  various  ways.     The 


1  The  work  of  Porcher  and  Hervieux  can  be  found  in  Compt.  Rend.,  145,  Corapt. 
rend.  soc.  biol.,  62,  and  Bull.  soc.  chim.  (4),  1;  Benedicenti,  Zeitschr.  f.  physiol.  Chem., 
53  and  Arch.  f.  exp.  Path.  u.  Pharm.,  1908,  Suppl.  (Schmiedeberg's  Festschr.). 

2  See  Jaffe,  Pfliiger's  Arch.,  3;  Senator,  Centralbl.  f.  d.  med.  Wissensch.,  1877; 
G.  Hoppe-Seyler,  Zeitschr.  f.  physiol.  Chem.,  12  (contains  older  literature;  also  Berl., 
klin.  Wochenschr.,  1892. 

3  Baumann  with  Brieger,  Zeitschr.  f.  physiol.  Chem.,  3;  with  Thesen,  ibid.,  23. 

*  JafTc,  Pfliiger's  Arch.,  3;  Obermeyer,  Wien.  klin.  Wochenschr.,  1890;  Salkowski, 
Zeitschr.  f.  physiol.  Chem.,  57;  Porcher  and   Hervieux,  Zeitschr.  f.  physiol.  Chem.,  39. 


IN  I)  [CAN   TESTS.  731 

quantity  of  indigo  red  becomes  greater  the  more  slowly  the  oxidation 
takes  place,  and  especially  when  the  decomposition  takes  place  in  the 
warmth  (see  the  works  of  Rosin,  Bouma,  Wang,  Maillard,  Ellinger 
and  Hervieux  1). 

According  to  Ellingeb  the  source  of  the  indigo-red  formation  may  be  the 
isatin  that  is  produced  by  the  superoxidation  of  the  indoxyl  by  the  action  of 
the  reagent,  and  this  isatin  forms  indigo  red  with  the  indoxyl  in  the  hydrochloric- 
acid  solution.  Maillard,  on  the  contrary,  is  of  the  view  that  the  blue  substance 
which  is  taken  up  by  the  chloroform  from  the  urine  mixed  with  hydrochloric 
acid  is  not  indigotin  (indigo-blue),  but  another  substance,  called  by  him  hemi- 
indigotin,  which  in  alkaline  solution  polymerizes  immediately  into  indigotin, 
while  in  acid  reaction  it  is  converted  into  indirubin  (indigo  red). 

The  chloroform  solution  of  indigo  obtained  in  the  indican  test  may  be 
used  in  the  quantitative  colorimetric  determination  by  comparison  with 
a  solution  of  indigo  in  chloroform  of  known  strength  (Krauss  and  Adrian). 
Wang  and  others  convert  the  indigo  into  indigo-sulphonic  acid  by  con- 
centrated sulphuric  acid  and  titrate  with  potassium  permanganate. 
There  is  still  doubt  as  to  the  surest  and  most  trustworthy  method  for 
the  determination  of  indican,  and  especially  as  to  the  question  how  the 
indigo  residue  is  to  be  washed  (see  Wang,  Bouma,  Ellinger,  and  Sal- 
kowski  2) ,  and  for  this  reason  we  shall  refer  only  to  the  works  cited  above. 

Because  of  the  difficulty  arising  from  the  production  of  indirubin 
in  addition  to  indigotin,  Bouma  has  recommended  the  conversion  of 
all  the  indoxyl  into  indirubin  by  boiling  the  urine  with  hydrochloric 
acid  containing  isatin.  The  indirubin  can  be  taken  up  by  chloroform 
and  determined  by  titration  with  potassium  permanganate  and  sul- 
phuric acid  after  purification  of  the  chloroform  residue.  Oerum  3  has 
also  worked  out  a  colorimetric  method  of  estimation  based  upon  Bouma 's 
method. 

Indol  seems  also  to  pass  into  the  urine  as  a  glucuronic  acid,  inlnyl- 
glucuronic  acid  (Schmiedeberg).  Such  an  acid  has  been  found  in  the 
urine  of  animals  after  the  administration  of  the  sodium-salt  of  o-nitro- 
phenylpropiolic  acid  (G.  Hoppe-Seyler).  Porcher  and  Hervieux  4 
have  obtained  indoxyl  sulphuric  acid  in  dogs  and  asses  under  similar 
conditions. 

Free  indigo,  and  in  fact  indirubin  as  well  as  indigotin,  occur  in  rare  cases  in 
the  undecomposed  urine.     Grober  and  Wang  have  recently  observed  such 
According  to  Steensma  5  traces  of  free  indol  occur  always  in  the  urine. 

1  Rosin,  Yirchow's  Arch.,  123;  Bouma,  Zeitschr.  f.  physiol.  Chem.,  27,  30,  32, 
39;  Wang,  ibid.,  25,  27,  28;  Ellinger,  ibid.,  38  and  41;  -Maillard,  Bull.  soc.  chim., 
Paris  (3),  29,  and  Compt.  Rend.,  136;  also  L'indoxyle  urinaire  et  les  couleurs  qui  en 
derivent,  Paris,  1903,  and  Zeitschr.  f.  physiol.  Chem.,  41;  Hervieux,  see  Bioch. 
Centralbl.,  8,  54. 

2  Krauss,  Zeitschr.  f.  physiol.  Chem.,  18;  Adrian,  ibid.,  19;  Wang,  ibid.,  25;  Sal- 
kowski,  (bid.,  42. 

5  Bouma,  Zeitschr.  f.  physiol.  Chem.,  32;  Oerum,  ibid.,  45. 

*  Schmiedeberg,  Arch.  f.  exp.  Path.  u.  Pharm.,  14;  G.  Hoppe-Seyler,  Zeitschr.  f. 
physiol.  Chem.,  7  and  8;  Porcher  and  Hervieux,  Journ.  de  Physiol.,  7. 

5  Grober,  Munch,  med.  Wochenschr.,  1904;  Wang,  Salkowski's  Festschrift,  1904; 
Steensma,  Mary's  Jahresb.,  40,  314. 


732  URINE. 

C.CH3 
Skatoxyl-sulphuric  Acid,  C9H9NSO4,  C6H4<^Sc.O.S02.0H,  has  not 

NH 

been  positively  prepared  as  a  constituent  of  normal  urine,  but  Otto 
has  once  prepared  its  alkali  salt  from  diabetic  urine.  Perhaps  skatoxyl 
occurs  in  normal  urine  as  a  conjugated  glucuronate  (Mayer  and  Neu- 
berg1),  and  it  is  believed  that  the  urine  contains  a  skatol-chromogen 
from  which  red  and  reddish-violet  coloring-matters  are  obtained  by 
decomposition  with  strong  acids  and  an  oxidizing  agent. 

Skatoxyl-sulphuric  acid  originates,  if  it  exists  in  the  urine,  from 
skatol,  which  is  formed  by  putrefaction  in  the  intestine,  and  which  is 
then  conjugated  with  sulphuric  acid  after  oxidation  into  skatoxyl.  That 
skatol  introduced  into  the  bodj-  passes  partly  as  an  ethereal-sulphuric 
acid  into  the  urine  has  been  shown  by  Brieger.  Indol  and  skatol  act 
differently,  at  least  in  dogs,  indol  producing  a  considerable  amount 
of  ethereal-sulphuric  acid,  while  skatol  gives  only  a  small  quantity  (Mes- 
ter  2).     Reports  on  this  subject  are  at  variance. 

The  conditions  for  the  formation  of  indol  and  skatol  by  the  putrefaction  of 
proteins  in  the  intestine  are  decidedly  different,  according  to  Herter,  as  skatol 
is  produced  by  other  putrefaction  bacteria  than  indol.  For  example,  bacillus  coli 
communis  produces  indol,  but  only  traces  of  skatol,  while  skatol  is  formed  by 
certain  anaerobic  putrefactive  bacteria.  An  important  intermediary  step  in  the 
formation  of  skatol  is  the  indol  acetic  acid  (skatol  carboxylic  acid,  according  to 
Salkowski)  and  this  can  also  pass  into  the  urine  and  is  the  chromogen  of  the 
urorosein,  according  to  Herter.3 

The  potassium  salt  of  skatoxyl-sulphuric  acid  is  crystalline;  it  dis- 
solves in  water,  but  with  difficulty  in  alcohol.  A  watery  solution  becomes 
deep  violet  with  ferric  chloride.  The  solution  becomes  red  with  con- 
centrated hydrochloric  acid  with  the  separation  of  a  red  precipitate. 
This  precipitate  (skatol  red)  is,  after  washing  with  water,  insoluble  in 
ether  but  soluble  in  amyl  alcohol.  On  distillation  with  zinc-dust  the 
red  pigment  gives  a  strong  odor  of  skatol. 

Urines  containing  skatoxyl  are  colored  dark  red  to  violet  by  Jaffe's 
indican  test  even  on  the  addition  of  hydrochloric  acid  alone;  with  nitric 
acid  they  are  colored  cherry  red,  and  red  on  warming  with  ferric  chloride 
and  hydrochloric  acid.  A  red  coloration  of  the  urine  can  also  be  brought 
about  by  the  appearance  of  indigo  red  (indirubin)  and  a  confusion  of 
this  pigment  can  also  take  place.     Rosin4  is  of  the  opinion  that  no 

lOtto,  Pflliger'e  Arch.,  33;    Mayer  and  Neuberg,  Zeitschr.  f.  physiol.  Chem.,  29. 

2  Brieger,  Ber.  d.  deutsch.  chem.  Gesellsch.,  12,  and  Zeitschr.  f.  physiol.  Chem., 
4,  414;  Mester,  ibid.,  12. 

3  Journ.  of  biol.  Chem.,  4. 

*  Rosin,  Virchow's  Arch.,  123. 


INDOL  ACETIC   ACID.  733 

skatol  chromogen  exists  in  human  urine,  and  that  the  observations  made 
heretofore  were  due  to  a  confusion  of  skatol  red  with  indigo  red  or  uroro- 
sein.  It  cannot  be  disputed  that  derivatives  of  skatol  sometimes  occur  in 
human  urine,  and  to  prevent  confusion  with  indigo  red  it  must  be  borne 
in  mind  that  indigo  red  is  soluble  in  chloroform  as  well  as  in  ether,  while 
skatol  red  is  insoluble  in  these  solvents.  On  the  contrary  skatol  red 
is  soluble  in  amyl  alcohol,  and  this  solution  shows  absorption  bands  close 
to  the  line  D  between  it  and  E,  corresponding  to  X  =  577-550  (Porcher 
and  Hervieux  1). 

In  regard  to  a  confusion  of  skatol  red  for  urorosein  it  must  also  be 
remarked  that  urorosein  may  also  be  a  skatol  red.  The  chromogen  of 
urorosein,  as  Herter  has  shown  in  a  case,  is  identical  with  indol  acetic 
acid,  which  passes  into  skatol  on  splitting  off  carbon  dioxide.  According 
to  Herter2  urorosein  is  not  identical  with  skatol  red,  although  the 
investigations  of  Staal,  Grosser,  Porcher  and  Hervieux  3  indicate 
that  they  are  identical,  and  the  last  two  investigators  consider  them 
identical,  because  they  both  have  the  same  spectrum  and  the  same 
chemical  behavior. 

C.CH2COOH 

Indol  Acetic  Acid  (skatol-carboxylic  acid),  Ci0H9NO2,  C6H4\    /CH 


NH 

This  acid,  whose  occurrence  in  the  urine  was  first  shown  by  Salkowski,  is  found 
in  the  urine  in  special  putrefactive  processes  in  the  intestine  (Herter)  and  in 
various  diseases,  especially  in  cachectic  conditions.  This  is  of  course  dependent 
upon  the  fact  whether  indol  acetic  acid  is  the  actual  chromogen  of  urorosein,  and 
also  whether  the  experience  obtained  as  to  the  occurrence  of  urorosein  can  be 
applied  to  the  indol  acetic  acid.  According  to  Wechselmann  4  it  occurs  (more 
correctly  as  urorosein)  as  traces  in  normal  urine,  abundantly.in  horse  urine,  and 
in  especially  large  quantities  in  cow  urine.  When  introduced  into  the  animal 
body  it  appears  unchanged  in  the  urine. 

This  acid  crystallizes  in  leaves  which  melt  at  165°,  and  on  strongly  heating 
it  yields  skatol  with  the  splitting  off  of  carbon  dioxide.  The  solution,  acidified 
with  hydrochloric  acid,  when  treated  with  a  little  ferric  chloride  solution,  becomes 
cherry  red  on  boiling.  With  some  acid  and  a  little  nitrite  as  well  as  with  hydro- 
chloric acid  and  chloride  of  lime  the  solution  becomes  red,  then  cloudy,  and  a 
red  pigment  precipitates.  This  pigment  is  soluble  in  amyl  alcohol  and  gives  the 
above-mentioned  absorption  bands  between  D  and  E.  This  red  pigment  is 
urorosein. 

Urorosein  is  the  name  given  by  Nencki5  to  a  red  pigment  which  occurs  in 
the  urine  under  the  conditions  mentioned  under  indol  acetic  acid.     This  pig- 


1  Zeitschr.  f.  physiol.  Chem.,  45. 

2  Journ.  of  biol.  Chem.,  4. 

3  Staal,  Zeitschr.  f.  physiol.  Chem.,  46;  Grosser,  ibid.,  44;  Porcher  and  Hervieux, 
ibid.,  45;  Compt.  Rend.,  138,  and  Journ.  ile  Physiol.,  7. 

4  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  9;  Wechselmann,  cited  in  Bioch.  Centralbl., 
5,  784. 

5  Nencki  and  Sieber,  Journ.  f.  prakt.  Chem.,  (N.  F.),  26. 


734  URINE. 

ment  is  not  preformed  in  the  urine,  but  is  produced  from  its  chromogen  (indol 
acetic  acid)  when  the  urine  is  treated  with  ivydrochloric  acid  alone.  The  urine 
becomes  red.  Urorosein  differs  from  indirubin  essentially  by  the  same  properties 
as  skatol,  with  which,  according  to  some,  it  is  identical  (see  above). 

Nephrorosein  is  a  pigment  described  by  V.  Arnold  »  which  is  closely  related  to 
urorosein  and  which,  like  this,  is  produced  from  a  chromogen  when  the  urine  is 
treated  with  nitric  acid  or  with  concentrated  hydrochloric  acid  and  a  little  sodium 
nitrite  solution.  Xephrorosein  is  soluble  in  amyl  alcohol  and  gives  a  spectrum 
with  a  band  between  b  and  F,  reaching  from  b  to  a  little  beyond  the  middle  between 
b  and  F.  It  is  changed  by  the  action  of  light  and  finally  gives  a  band  between 
D  and  E,  near  E.  The  new  pigment  thus  obtained  is  called  ^-urorosein  to  dif- 
ferentiate it  from  the  ordinary  urorosein,  a-urorosein.  The  nephrorosein  has  not 
been  observed  in  normal  urines  but  only  in  certain  pathological  cases. 

The  pigment  obtained  by  de  Jager  by  precipitating  the  urine  with  HC1  and 
formol  seems  to  be  related  to  urorosein  and  nephrorosein.  According  to  Ellinger 
and  Flamand  "-  urorosein  belongs  probably,  like  skatol-red,  to  the  group  of  tri- 
indyl  methane  pigments  prepared  by  them  from  /3-indol  aldehyde  by  boiling  in 
pcid  solution.  Probably  the  leucobase  HCCCYHeN^,  which  gives  the  red  pigment, 
HO.C  :  (CsH6X)3  is  produced  by  condensation. 

Aromatic  Oxy acids.  In  the  putrefaction  of  proteins  in  the  intes- 
tine, paraoxyphenyl-o.cetic  acid  and  paraoxyphenyl-propionic  acid  are 
formed  from  tyrosine  as  an  intermediate  step,  and  these  in  great  part 
pass  unchanged  into  the  urine.  The  quantity  of  these  acids  is  usually 
very  small.  They  are  increased  under  the  same  conditions  as  the  phenols, 
especially  in  acute  phosphorus  poisoning,  in  which  the  increase  is  con- 
siderable. A  small  portion  of  these  oxyacids  is  also  combined  with 
sulphuric  acid. 

Besides  these  two  oxyacids  which  regularly  occur  in  human  urine 
we  sometimes  have  other  oxyacids  in  urines.  To  these  belong  homo- 
gentisic  acid  in  alcaptonuria,  oxyhydroparacomnaric  acid,  found  by  Blender- 
mann  in  the  urine  on  feeding  rabbits  with  tyrosine,  gallic  acid,  which, 
according  to  Baumann,3  sometimes  appears  in  horse's  urine,  and  kynu- 
renic  acid  (oxyquinolincarboxylic  acid),  which  up  to  the  present  time 
has  been  found  only  in  dog's  urine.  Although  all  these  acids  do  not 
belong  to  the  physiological  constituents  of  the  urine,  still  they  will  be 
treated  in  connection  with  these. 


Paraoxyphenylacetic  Acid,  CsHeOj,  C6H4\  ,  and  p-Oxyphenyl- 


/OK 

/OF 


propionic  Acid  (Hvdroparacoumaric  Acid),  C9H10O3,  CeH4\  ,  are 

^  \CH2.CH2COOH 
crystalline  and  are  both  soluble  in  water  and  in  ether.     The  one  melts  at  148°  C. 
ami  the  other  al   125°  C.     Both  give  a  beautiful  red  coloration  on  being  warmed 
with  Mil. 1  gent. 

1  Zeitschr.  f.  physiol.  Chem.,  61  and  71. 

2  de  Jager,  Zeitschr.  f.  physiol.  Chem.,  61;  Ellinger  and  Flamand,  ibid.,  62. 

3  Blendennann,  Zeitschr.  f.  physiol.  Chem.,  6,  267;  Baumann,  ibid.,  6,  193. 


HOMOGENTISIC  ACID.  735 

To  detect  the  presence  of  these  oxyacids  proceed  in  the  following  way  (Bau- 
mann):  Warm  the  urine  for  a  while  on  the  water-bath  with  hydrochloric  acid 
in  order  to  drive  off  the  volatile  phenols.  After  cooling  shake  three  times  with 
ether,  and  then  shake  the  ethereal  extracts  with  dilute  soda  Bollltion,  which  dis- 
solves the  oxyacids,  while  the  residue  of  the  phenols  which  are  soluble  in  ether 
remains.  The  alkaline  solution  of  the  oxyacids  is  now  faintly  acidified  with  sul- 
phuric acid,  shaken  again  with  ether,  the  ether  removed  and  allowed  to  evaporate 
the  residue  dissolved  in  a  little  water,  and  the  solution  tested  with  Millon's 
reagent.  The  two  oxyacids  are  best  differentiated  by  their  different  melting- 
points.  The  reader  is  referred  to  other  works  for  the  method  of  isolating  and 
separating  these  two  oxyacids. 

Homogentisic   Acid    (Dioxyphenylacetic   Acid),    CsHs'^  = 
/OH(l) 
CeH3^-OH(4)  .     This  acid,  which  was  discovered  by  Marshall1 

\CH2COOH(5) 
and  calle  1  by  him  glycosuric  acid,  was  isolated  in  larger  quantities  by 
Wolkow  and  Baumann  in  a  case  of  alcaptonuria  and  carefully  studied 
by  them.  They  called  it  homogentisic  acid  because  it  is  a  homologue 
of  gentisic  acid,  and  they  showed  that  the  peculiar  properties  of  so-called 
alcaptonuric  urine  in  this  case  were  due  to  this  acid.  This  acid  has  later 
been  found  in  many  cases  of  alcaptonuria.  Glycosuric  rcid,  isolated  from 
alcaptonuric  urine  by  Geyger,2  seems  to  be  identical  with  homogentisic 
acid. 

The  quantity  of  acid  eliminated,  which  varies  in  most  cases  between 
3  and  7  grams  per  twenty-four  hours,  and  which  is  higher — 14-16  grams — 
in  exceptional  cases,  is  increased  by  food  rich  in  protein.  On  the  inges- 
tion of  tyrosine  by  persons  with  alcaptonuria,  Wolkow  and  Baumann 
and  Embden  observed  a  greater  quantity  of  homogentisic  acid  in  the 
urine  and  this  has  been  substantiated  by  other  observers.  Since  Lang- 
stein  and  E.  Meyer  showed  in  a  case  of  alcaptonuria  that  the  quantity 
of  tyrosine  in  the  protein,  even  when  calculated  to  a  maximum,  was 
not  sufficient  to  account  for  the  quantity  of  homogentisic  acid,  and  that 
therefore  we  must  admit  of  another  source  (the  phenylalanine)  for  the 
alcapton,  Falta  and  Langstein  3  have  given  a  direct  proof  that  homo- 
gentisic acid  can  also  be  formed  from  phenylalanine.  Abderhalden, 
Bloch  and  Rona  4  have  shown  that  in  alcaptonurics  the  excretion  of 
homogentisic  acid  is  increased  by  the  introduction  of  tyrosine  or  phenyl- 


1  The  Medical  News,  Philadelphia,  January  8,  1887. 

2  Wolkow  and  Baumann,  Zeitschr.  f.  physiol.  Chem.,  15;  Geyger,  cited  from  Emb- 
den, 1.  c,  18.  The  literature  can  be  found  in  Fromherz,  Ueber  Alkaptonurie,  Inaug.- 
Dis.  Freiburg,  1908. 

3  Langstein  and  Meyer,  Deutsch.  Arch.  f.  klin.  Med.,  78;  Falta  and  Langstein, 
Zeitschr.  f.  physiol.  Chem.,  37;  Falta,  Der  Eiweiss-Stoffwechsel  bei  der  Alkaptonurie, 
Habilitationsschrift,  Naumburg,  a.  S.,  1904. 

4  Zeitschr.  f.  physiol.  Chem.,  52. 


736  URINE. 

alanine  in  the  form  of  polypeptides,  from  dipeptides  as  well  as  tripeptides. 
The  p-tyrosine  and  phenylalanine  are  quantitatively  converted  into  homo- 
gentisic  acid,  in  alcaptonuria  (Falta).  The  m-  and  o-tyrosine,  on  the 
contrary,  are  not  converted,  according  to  Blum,1  into  homogentisic  acid 
in  alcaptonurics,  and  the  dibromtyrosine  yields  just  as  little  homogentisic 
acid  as  the  bromine  or  iodine  derivatives  of  protein  bodies  (Falta). 
According  to  the  investigations  of  Langstein  and  Meyer,  and  especially 
of  Falta,  different  proteins  yield  varying  quantities  of  homogentisic 
acid  in  alcaptonuria,  and  accordingly  larger  amounts  in  proportion  as 
the  protein  is  rich  in  tyrosine  and  phenylalanine. 

On  this  account  the  quotient  H  (  =  homogentisic  acid):N  (nitrogen) 
is  variable  on  the  introduction  of  different  proteins.  For  example,  with 
casein  H :  N  is  on  an  average  much  higher  than  with  white  of  egg.  In 
most  of  the  cases  of  alcaptonuria  examined  the  H :  N  was  equal  to 
40-50:  100,  and  with  the  same  alcaptonuric,  when  no  essential  change 
in  the  diet  occurs,  the  quotient  is  relatively  constant. 

Wolkow  and  Baumann  explain  the  formation  of  homogentisic  acid 
from  tyrosine  by  an  abnormal  fermentation  in  the  upper  parts  of  the 
intestine,  but  this  view  has  now  been  generally  rejected.  The  observa- 
tions of  Aberhalden,  Bloch  and  Rona  2  that  glycyl-Z-tyrosine  on 
subcutaneous  injection  causes  an  increased  formation  of  homogentisic 
acid,  disproves  this  theory,  and  indicates  a  formation  of  homogentisic 
acid  in  the  tissues.  This  acid  is  also  burnt  in  the  healthy  organism  if 
not  too  large  quantities  of  the  acid  are  introduced  at  one  time,  and  it  is 
the  general  view  that  alcaptonuria  is  an  anomaly  in  the  protein  metabolism. 

In  order  to  understand  this  anomaly  and  the  origin  of  the  homogentisic 
acid  we  must  call  attention  to  the  fact  that  the  investigations  of  O. 
Neubauer  and  Falta,  Langstein  and  others3  show  that  only  such 
aromatic  acids  are  converted,  in  the  body,  into  homogentisic  acid,  which 
have  a  three-membered  side-chain  which  is  substituted  by  NH2,  OH 
or  0  in  the  a-position  to  the  carboxyl  group  and  not  in  the  ^-position, 
p-tyrosine,  phenylalanine,  phenyl-a-lactic  acid  and  phenyl-pyroracemic 
acid  are  such  acids.  It  can  be  admitted  with  Falta  that  the  phenyl- 
alanine in  the  body  by  deamidation  is  converted  into  phenyl-a-lactic 
acid,  CeH5.CH2.CHOH.COOH,  from  which  by  taking  up  two  hydroxyl 
groups,  dioxyphenyl-a-lactic  acid  (uroleucic  acid),  (OH)2CeH3.CH2. 
CHOH.COOH,  is  formed,  and  then  from  this  by  oxidation  dioxyphenyl- 
acetic  acid  (homogentisic  acid),  (OH)2C6H3.CH2.COOH,  is  produced. 
Tyrosine    is    also    supposed    to    undergo    an    analogous    transformation 


xArch.  f.  exp.  Path.  u.  Pharm.,  59. 
2  Zeitschr.  f.  physiol.  Chem.,  52. 
*  Ibid.,  42;  Frornherz,  1.  c. 


HOMOGENTISIC  ACID.  737 

whereby  a  removal  of  the  OH  group  in  the  para  position  must  be 
admitted. 

According  to  Neubauer,1  on  the  contrary,  the  tyrosine,  as  well  as 
the  other  amino-acids,  is  first  transformed  into  the  corresponding  keto- 
acid,  p-oxyphenyl  pyroracemic  acid,  OH.CeH4.CH2.CO.COOH,  which 
is  then  oxidized  into  the  corresponding  chinol  and  transformed  into 
hydroquinone  pyroracemic  acid,  (OHjoCeHs.Cr^.CO.COOH.  The  homo- 
gentisic  acid  is  derived  from  this  latter  by  the  splitting  off  of  carbon 
dioxide  by  oxidative  means.  Phenylalanine  is  either  changed  into 
phenyl  pyroracemic  acid  or  into  p-oxyphenyl  pyroracemic  acid  with 
tyrosine  as  intermediary  body  and  then  changed  as  above  stated. 

According  to  the  accepted  hypothesis  the  demolition  of  tyrosine 
and  phenylalanine  takes  place  into  homogentisic  acid,  and  the  anomaly 
in  the  metabolism  of  alcaptonurics  consists  in  that  in  these  the  demoli- 
tion stops  at  this  point  and  that  the  ability  to  rupture  the  benzene  ring 
is  absent,  in  the  organism,  in  alcaptonuria. 

,The  difficulties  in  accepting  the  assumption  of  a  transformation  of  tyrosine 
into  homogentisic  acid  due  to  the  different  positions  of  the  hydroxy!  groups  in 

the  side  chain  of  the  two  bodies,  as  shown  by  the  formulae  HO<f  yOH  (homo- 

CH-COOH 
OH 


gentisic  acid)  and  (  y  (tyrosine)  do  not  exist  now,  since  we  have 

CH,CHNH,COOH 

learnt  of  other  analogous  processes.    For  example,  the  oxidation,  by  Kumagai  and 

"Wolffensteix,2  of  paracresol  H3C\  /OH  with  potassium  persulphate  in 

OH 
acid  solution.     In  this  manner  the  expected  3.4  dioxytoluene  H3C< 


was  not  obtained,  but  instead  homohydroquinone  HO<f  /OH,  and  hence  a 

CH, 

transference  of  the  alkyl  group  must  have  occurred. 

Abderhalden  3  has  also  shown  in  healthy  human  beings  that  tyrosine 
may  cause  an  elimination  of  homogentisic  acid,  as  he  positively  detected 
a  small  quantity  of  homogentisic  acid  in  the  urine  of  a  man  who  had 
taken  50  grams  /-tyrosine  per  os  (of  which  44  grams  were  absorbed). 
In  the  urine  of  another  man  he  could  not  detect  either  homogentisic 

1  Cited  from  Centralbl.  f.  Physiol.,  23,  76. 
2Ber.  (1.  d.  Chem.  Gesellsch.,  41. 
'  Zeitschr.  f.  physiol.  Chem.,  77. 


738  URINE. 

acid  or  any  other  intermediary  product  of  the  cleavage  of  tyrosine  in 
the  urine  after  taking  150  grams  /-tyrosine  (of  which  141  grams  were 
absorbed) . 

Dakin  l  has  recently  opposed  the  above-mentioned  view  that  in  the 
cleavage  of  tyrosine  and  phenylalanine,  homogentisic  acid  is  always 
produced,  and  that  the  condition  of  alcaptonuria  consists  in  an  inability 
of  the  body  to  burn  this  intermediary  product  of  metabolism.  He  has 
found  that  tyrosin-methyl  ether,  which  cannot  form  any  quinone-like 
intermediary  product,  can  in  cats  be  just  as  completely  burnt  as  tyrosine, 
and  the  same  is  true  for  p-methylphenylalanine  and  for  p-methoxy- 
phenylalanine,  which  cannot  form  any  quinone  derivatives.  Still 
these  substances  can  be  completely  burnt  by  alcaptonurics  and  accord- 
ing to  Dakin  the  body  in  alcaptonuria  has  still  the  ability  to  completely 
burn  the  aromatic  nucleus  of  tyrosine  and  phenylalanine  when  the 
transformation  into  homogentisic  acid  is  prevented  by  a  proper  sub- 
stitution in  the  para-groups.  Dakin  therefore  considers  alcaptonuria 
as  a  condition  in  which  partly  the  formation  of  an  abnormal  metabolic 
product — the  homogentisic  acid— takes  place  and  where  partly  the  ability 
of  the  body  to  burn  this  product  is  diminished. 

Garrod,2  who  has  observed  several  cases  of  alcaptonuria,  has  also 
tabulated  a  large  number  of  cases  of  alcaptonuria  which  he  finds  in  the 
literature,  and  he  shows  that  the  anomaly  of  the  protein  metabolism 
occurs  oftener  in  males  than  in  females,  and  also  that  blood  relationship 
of  the  parents  (first  cousins)  predisposes  to  alcaptonuria. 

On  fusing  homogentisic  acid  with  alkali  it  yields  gentisic  acid  (hydro- 
quinone-carboxylic  acid)  and  hydroquinone.  When  introduced  into  the 
intestine  of  the  dog  a  part  is  converted  into  toluhydroquinone,  which 
is  eliminated  in  the  form  of  an  ethereal  sulphuric  acid.  Homogentisic 
acid  has  also  been  prepared  synthetically  by  Baumann  and  Frankel, 
starting  with  gentisic  aldehyde,  and  by  Neubauer  and  Flatow  3  from 
o-oxyphenylglyoxylic  acid  with  hydroquinone  glyoxylic  acid  and  hydro- 
quinone glycollic  acid  as  intermediary  bodies. 

Homogentisic  acid  crystallizes  with  1  mol.  of  water  in  large,  trans- 
parent prismatic  crystals,  which  become  non-transparent  at  the  tem- 
perature of  the  room  with  the  loss  of  water  of  crystallization.  They 
melt  at  146.5-147°  C,  and  are  soluble  in  water,  alcohol,  and  ether,  but 
nearly    insoluble    in    chloroform    and    benzene.      Homogentisic    acid    is 


1  Journ.  of  Biol.  Ohem.,  8  and  9,  with  Wakeman,  ibid.,  9. 

2  Med.  chirurti.  Transact.,  1899  (where  all  cases  up  to  that  time  are  tabulated); 
also  The  Lancet,  1901  and  1902;  Garrod  and  Hele,  Journ.  of  Physiol.,  33. 

3  Baumann  and  Friinkel,  Zeitsohr.  f.  physiol.  Chem.,  20;  Neubauer  and  Flatow., 
ibid.,  52. 


BOMOGENTISIC  ACID.  739 

optically  inactive  and  non-fermentable.  I^ts  watery  solution  has  the 
properties  of  so-called  alcaptonuric  urine.     It  becomes  greenish  brown 

from  the  surface  downward  on  the  addition  of  very  little  caustic  soda 
or  ammonia  with  access  of  oxygen,  and  on  shaking  it  quickly  becomes 
dark  la-own  or  black. 

If  alcaptonuric  mine  or  a  homogentisic  acid  solution  is  treated  with  10-40 
per  cent  ordinary  ammonia,  a  beautiful,  intensive  reddish-violet  coloration  is 
produced  on  access  of  air  according  to  C.  Moknkk,1  and  two  beautiful  pigments, 
a-  and  p-alcaptochrome,  are  formed.  The  first,  o-alcaptochrome,  is  crystalline  and 
has  a  beautiful  violel  color  In  alkaline  solution  and  is  without  fluorescence.  The 
(3-alcaptochrome  is  not  crystalline  and  its  alkaline  solution  has  a  more  reddish 
color  with  strong  fluorescence  in  the  yellowish-red. 

Homogentisic  acid  reduces  alkaline  copper  solutions  with  even  slight 
heat,  and  ammoniacal  silver  solutions  immediately  in  the  cold.  It 
does  not  reduce  alkaline  bismuth  solutions.  It  gives  a  lemon-colored 
precipitate  with  Millon's  reagent,  which  becomes  light  brick-red  on 
warming.  Ferric  chloride  gives  to  the  solution  a  blue  color  which  soon 
disappears.  On  boiling  with  concentrated  ferric-chloride  solution  an 
odor  of  quinone  develops.  With  benzoyl  chloride  and  caustic  soda  in 
the  presence  of  ammonia  we  obtain  the  amide  of  dibenzoylhomogentisic 
acid,  which  melts  at  204°  C,  and  which  can  be  used  in  the  isolation  of 
the  acid  from  the  urine,  and  also  for  its  detection  (Orton  and  Garrod). 
Among  the  salts  of  this  acid  must  be  mentioned  the  lead  salt  containing 
water  of  crystallization  and  34.79  per  cent  Pb.  This  salt  melts  at 
214-215° C. 

In  order  to  prepare  the  acid,  heat  the  urine  to  boiling,  add  5  grams 
of  lead  acetate  for  every  100  cc,  filter  as  soon  as  the  lead  acetate  has 
dissolved,  and  allow  the  nitrate  to  stand  in  a  cool  place  for  twenty-four 
hours  until  it  crystallizes  (Garrod).  The  dried,  powdered  lead  salt 
is  suspended  in  ether  and  decomposed  by  H2S.  After  the  spontaneous 
evaporation  of  the  ether  the  acid  is  obtained  in  almost  colorless  crystals 
(Orton  and  Garrod2). 

In  regard  to  the  quantitative  estimation  we  proceed  according  to  the  sug- 
gestion of  Baumann  by  titrating  the  acid  with  a  N/10  silver  solution.  For  details 
of  this  method  the  reader  is  referred  to  the  works  of  Baumann,  C.  Th.  Morner, 
Mittelbach,  Garrod  and  Hurtley.     Deniges  3  has  suggested  another  method. 

Uroleucic  acid,  C9Hi0O6,  is,  according  to  Htppert,  probablv  a  dioxvphenvl- 
lactic  acid,  C6H:i(OH)2.CH,.CH(OH).C()OH.  This  acid  was  first  prepared  by 
Kirk  from  the  urine  of  children  with  alcaptonuria,  which  also  contained  homo- 
gentisic acid.  Langstein  and  Meyer  4  have  also  found  a  small  amount  of  this 
acid  in  a  case  of  alcaptonuria  studied  by  them.     It  melts  at  130-133°  C.     Other- 

1  Zeitschr.  f.  physiol.  Chem.,  69. 

2  Orton  and  Garrod,  Journ.  of  Physiol.,  27;  Garrod,  ihid.,  23. 

3  Mittelbach,  Deutsch.  Arch.  f.  klin.  Med.,  71  (which  contains  the  work  of  Baumann 
and  Morner);  Garrod  and  Hurtley,  Journ.  of  Physiol.,  33;  Deniges,  Chem.  Centralbl., 
1897,  1,  338. 

4  Huppert,  Zeitschr.  f.  physiol.  Chem.,  23;  Kirk,  Brit.  Med.  Journ.,  1886  and  1888; 
Langstein  and  Meyer,  1.  c. 


740  URINE. 

wise,  in  regard  to  its  behavior  with  alkalies,  with  access  of  air,  and  also  with 
alkaline  copper  solutions  and  ammoniacal  silver  solutions,  and  also  Millon's 
reagent,  it  is  similar  to  homogentisic  acid. 

Neubaueb  and  Flatow,  who  have  prepared  dioxyphenyl-a-lactic  acid  syn- 
thetically, find  that  this  acid  has  entirely  different  properties  from  the  so-called 
uroleucic  acid.  Garrod  and  Hurtley  '  have  also  shown  that  an  impure  homo- 
gentisic acid  with  a  low  melting-point  is  easily  obtained,  and  they  suggest  the 
possibility  that  the  earlier  reports  in  regard  to  uroleucic  acid  are  due  to  an  error. 

CH     COH 

HC     C      C.COOH 
Kynurenicacid(y-ox}'-/3-quinohncarboxvhcacid),CioH7N03, 

HC      C      CH 

CH    N 

has  only  been  found  thus  far  in  dog's  urine,  but  not  always;  its  quantity  is  increased 
by  meat  feeding.  It  does  not  occur  in  the  urine  of  cats.  Ellinger  2  has  been 
able  to  show  positively  that  tryptophane  is  the  mother-substance  of  this  acid. 
By  the  introduction  of  tryptophane  in  the  organism  he  has  shown  the  formation 
of  a  kynurenic  acid  not  only  in  dogs  but  also  in  rabbits. 

The  acid  is  crystalline,  does  not  dissolve  in  cold  water,  rather  well  in  hot  alcohol, 
and  yields  a  barium  salt  which  crystallizes  in  triangular,  colorless  plates.  On 
heating  it  melts  and  decomposes  into  C02  and  kynurin.  On  evaporation  to  dry- 
ness on  the  water-bath  with  hydrochloric  acid  and  potassium  chlorate  a  reddish 
residue  is  obtained  which  on  adding  ammonia  becomes  first  brownish  green  and 
then  emerald  green  (Jaffe's  reaction  3). 

Urinary  Pigments  and  Chromogens.  The  yellow  color  of  normal 
urine  depends  perhaps  upon  several  pigments,  but  in  greatest  part  upon 
urochrome.  Besides  this  the  urine  seems  to  contain  a  very  small  quantity 
of  hoematoporphyrin  as  a  regular  constituent.  Uroerythrin  is  also  of 
frequent  occurrence  in  normal  urine,  but  not  always.  Finally,  the 
excreted  urine  when  exposed  to  the  action  of  light  regularly  contains  a 
yellow  pigment,  urobilin,  which  is  derived  from  a  chromogen,  urobilinogen, 
by  the  action  of  light  (Saillet)  and  air  (Jaffe,  Disque4)  and  others. 

Besides  this  chromogen,  urine  contains  various  other  bodies  from  which  color- 
ing matters  may  be  produced  by  the  action  of  chemical  agents.  Humin  sub- 
stances (perhaps  in  part  from  the  carbohydrates  of  the  urine)  may  be  formed 
by  the  action  of  acids  (v.  Udranszky)  without  regard  to  the  fact  that  such  sub- 
stances may  sometimes  originate  from  the  reagents  used,  as  from  impure  amyl 


1  Journ.  of  Physiol.,  30. 

*Ellinger,  Ber.  d.  d.  chem.  Gesellsch.,  37,  1804,  and  Zeitschr.  f.  physiol.  Chem., 
43.  The  older  literature  on  kynurenic  acid  may  be  found  in  Josephsohn,  Beitrage  zur 
Kenntnis  der  Kynurensaure  ausscheidung  beim  Hunde,  Inaug. -Dissert.,  Konigsberg, 
1898. 

'  Zeitschr.  f.  physiol.  Chem.,  7.  In  regard  to  kynurenic  acid,  see  also  Huppert- 
Neubauer,  10.  Aufl.,  and  Mendel  and  Jackson,  Amer.  Journ.  of  Physiol.,  2;  Mendel 
and  Schneider,  ibid.,  5;  Camps,  Zeitschr.  f.  physiol.  Chem.,  33. 

4  Jaffp,  Centralbl.  f.  d.  med.  Wissensch.,  1896  and  1869,  and  Virchow's  Arch.,  47; 
Disque,  Zeitschr.  f.  physiol.  Chem.,  2;  Saillet,  Revue  de  medecine,  17,  1897. 


DROCHROME.  741 

alcohol  (v.  Udraxszky  ')•  To  these  humin  bodies  developed  by  the  action  of 
acid  in  norma]  urine  when  exposed  bo  tin-  air  must  be  added  the  urophain  of  Heller, 
the  various  uromelanins  and  other  bodies  described  by  different  investigators 
(Pl6bz,  ThUDICHUM,  BchunCK,  DombbOWBKI  *).  Indigo  blue  (uroglaucin  of 
Heller,  vrocyanin,  cyanurin,  and  other  coloring  matters  of  earlier  investigators  3) 
is  split  off  from  the  indoxyl-sulphuric  acid  or  indoxyl-glucuronic  acid.  Red 
coloring  matter  may  be  formed  from  the  conjugated  indoxyl  and  skatoxyl  acids, 
and  urohodin  (Heller),  urorutrin  (Plosz),  urohcemabin  (Harley),  and  perhaps 
also  uroroscin  (Nencki  and  Sieber  4)  probably  have  such  an  origin. 

We  cannot  discuss  more  in  detail  the  different  coloring  matters  obtained 
as  decomposition  products  from  normal  urine.  Haematoporphyrin  has 
already  been  referred  to  in  a  previous  chapter  (V)  and  will  best  be 
described  in  connection  with  the  pathological  pigments.  It  only  remains 
to  describe  urochrome,  urobilin,  and  uroerythrin. 

Urochrome  is  the  name  given  by  Garrod  to  the  yellow  pigment  of 
the  urine.  Thudichum  5  had  previously  given  the  same  name  to  a  less 
pure  pigment  isolated  by  himself.  The  accounts  as  to  the  composi- 
tion and  properties  of  urochrome  differ  so  considerably  that  it  is  ques- 
tionable whether  anybody  has  ever  had  this  pigment  in  a  pure  form. 
Urochrome  is  free  from  iron,  but  contains  nitrogen.  Dombrowski 
found  11.15  per  cent  nitrogen,  Hohlweg  found  9.89  per  cent  nitrogen, 
and  Klemperer  found  only  4.2  per  cent  nitrogen.  According  to 
Dombrowski  urochrome  contains  about  5  per  cent  sulphur,  while 
other  investigators  like  Hohlweg,  Salomonsen,  and  Mancini  found 
that  it  was  free  from'  sulphur.6  According  to  Garrod  it  stands  in 
close  relation  to  urobilin  and  is  transformed  into  urobilin  by  the  action 
of  "  active  "  acetaldehyde,  while  Riva  7  claims  to  have  obtained  a  body 
similar  to  urochrome  by  the  oxidation  of  urobilin  by  permanganate. 
This  relation  of  the  two  pigments  is  denied  by  Dombrowski.  On  the 
contrary  it  is  the  unanimous  opinion  that  urochrome  under  certain  con- 
ditions may  yield  the  pyrrol  reaction.  Certain  investigators  such  as 
Bondzynski  and  Dombrowski  consider  urochrome  as  a  member  of  the 
oxyproteic  acid  group  (see  further  on),  a  view  which  does  not  seem  to 


1  v.  Udranszky,  Zeitschr.  f.  physiol.  Chem.,  11,  12,  and  13. 

*  P16sz,  Zeitschr.  f.  physiol.  Chem.,  8;  Thudichum,  Brit.  Med.  Journ.,  201,  and 
Journ.  f.  prakt.  Chem.,  104;  Schunck,  cited  from  Huppert-Neubauer,  10.  Aufl.,  509; 

Dombrowski,  Zeitschr.  f.  physiol.  Chem.,  62. 
s  See  Huppert-Neubauer,  161. 

*  In  regard  to  this  and  other  red  pigments,  see  Huppert-Neubauer,  593  and  597; 
Nencki  and  Sieber,  Journ.  f.  prakt.  Chem.  (2).  26. 

6  Garrod,  Proc.  Roy.  Soc,  55;  Thudichum,  1  c. 

•Dombrowski,  Zeitschr.  f.  physiol.  Chem.,  54  and  62;  Hohlweg,  Bioch.  Zeitschr., 
13;  Salomonsen.  ibid.,  13;  Mancini,  ibid.,  13;  Klemperer,  Berl.  klin.  Wochenschr., 
40. 

T  Garrod,  Journ.  of  Physiol.,  21  and  29;  Riva,  cited  from  Huppert-Neubauer,  524. 


742  URINE. 

have  sufficient  basis  and  in  fact  is  denied  by  others  such  as  Weisz.1  The 
above  disputed  statements  as  to  the  presence  or  absence  of  sulphur  in 
urochrome  as  well  as  the  nitrogen  content  of  urochrome  make  it  very- 
probable  that  the  preparation  of  pure  urochrome  has  not  thus  far  been 
accomplished. 

Urochrome,  as  obtained  thus  far,  is  amorphous,  brown,  readily  solu- 
ble in  water  and  ordinary  alcohol,  but  less  soluble  in  absolute  alcohol. 
It  dissolves  but  slightly  in  acetic  ether,  amyl  alcohol,  and  acetone,  while 
it  is  insoluble  in  ether,  chloroform,  and  benzene.  Urochrome  is  pre- 
cipitated by  copper  acetate,  lead  acetate,  silver  nitrate,  mercuric  acetate, 
phosphotungstic  and  phosphomolybdic  acids.  On  saturating  the  urine 
with  ammonium  sulphate  a  great  part  of  the  urochrome  remains  in  solu- 
tion. It  does  not  show  any  absorption-bands  and  does  not  fluoresce 
after  the  addition  of  ammonia  and  zinc  chloride.  Urochrome  is  very 
readily  decomposed,  by  the  action  of  acids,  with  the  formation  of  brown 
substances. 

Urochrome  can  be  prepared  according  to  a  rather  complicated  method  which 
is  based  upon  the  fact  that  the  substance  remains  in  great  part  in  solution  on 
saturating  the  urine  with  ammonium  sulphate.  If  the  proper  quantity  of  alcohol 
is  added  to  the  filtrate,  a  clear,  yellow  alcoholic  layer  forms  on  the  salt  solution, 
which  contains  the  urochrome  and  which  can  be  used  for  the  further  preparation 
of  the  latter  (Garrod,  0.  Bocchi  2).  Klemperer,  on  the  contrary,  removes  the 
pigment  from  the  urine  by  means  of  animal  charcoal,  washes  it  with  water  to 
remove  the  indican  and  other  bodies,  and  then  extracts  with  alcohol  and  uses 
this  alcoholic  extract  for  the  further  purification  according  to  Garrod.  Hohlweg, 
Salomosen  and  Mancini  also  remove  the  pigment  from  the  urine,  which  has 
previously  been  precipitated  by  calcium  or  barium  salts,  by  means  of  animal 
charcoal.  Dombrowski  uses  an  entirely  different  method  which  is  based  upon 
the  precipitation  of  the  urochrome  by  copper  acetate.  In  regard  to  the  details 
of  these  different  methods  we  refer  to  the  original  works. 

Dombrowski,  Browinski  and  Dombrowski  3  have  worked  out  a 
quantitative  method  for  estimating  urochrome,  but  its  value  is  dependent 
upon  a  further  investigation  as  to  the  purity  and  composition  of  the 
urochrome  obtained  by  them.  On  this  account  the  results  found  by  these 
investigators  will  not  be  given.  The  urochrome  can  be  quantitatively 
estimated,  according  to  Klemperer,  by  a  colorimetric  method,  using 
a  solution  of  true  yellow  G.  If  0.1  gram  of  this  dye  is  dissolved  in  1  liter 
of  water  and  5  cc.  of  this  solution  diluted  to  50  cc.  with  water,  then 
this  solution  has  the  same  color  and  shade  as  a  0.1  per  cent  urochrome 
solution.  The  urine  must  be  diluted  with  water  until  it  has  the  same 
depth  of  color.  The  comparison  is  performed  in  vessels  with  parallel 
walls.     The  value  of  this  method  cannot  be  judged  at  the  present  time. 

1  Dombrowski,  1.  c;  Bondzynski,  Chem.  Centralbl.,  1910,  Bd.  II;  Weisz,  Bioch. 
Zeitschr.,  30. 

2  Garrod,  1.  c;  Bocchi,  Hofmeister's  Beitrage,  11. 

'  Dombrowski,  Zeitschr.  f.  physiol.  Chem.,  54,  with  Browinski,  Bull.  Acad,  d.. 
d.  scien.  Cracovie,  1908;    Klemperer,  1.  c. 


UROBILIN.     UROBILINOIDS.  743 

Urobilin  is  the  pigment  first  isolated  from  the  urine  by  Jaff^:,1  and 
which  is  characterized  by  its  strong  fluorescence  and  by  its  absorption- 
spectrum.  Various  investigators  have  prepared, from  the  urine,  by  dif- 
ferent methods,  pigments  which  differed  slightly  from  each  other  but 
behaved  essentially  like  Jaffa's  urobilin.  Thus  different  urobilins  have 
been  suggested,  such  as  normal,  febrile,  physiological,  and  pathological 
urobilins.2  The  possibility  of  the  occurrence  of  different  urobilins  in 
the  urine  cannot  be  denied;  but  as  urobilin  is  a  readily  changeable  body 
and  difficult  to  purify  from  other  urinary  pigments,  the  question  as  to  the 
occurrence  of  different  urobilins  must  still  be  considered  open. 

In  the  perfectly  fresh  urine  of  healthy  human  beings  no  urobilin 
occurs,  as  first  suggested  by  Saillet,3  but  only  the  chromogen,  urobilino- 
gen, from  which  the  urobilin  is  readily  formed  by  the  action  of  light 
or  by  weak  oxidizing  agents.  Pathological  urines  contain  on  the  contrary 
preformed  urobilin. 

Urobilinoids,  i.e.,  bodies  which  are  similar  to  urobilin  in  that  they  fluoresce 
and  show  the  same  absorption  spectrum  have  been  prepared  from  bile-pig- 
ments (by  Maly  and  Stokvis)  and  from  hsematin  or  harnatoporphyrin  (by 
Hoppe-Seyler,  Le  Nobel,  Nencki  and  Sieber,  MacMunn4)  by  reduction 
as  well  as  by  oxidation.  According  to  H.  Fischer  and  Meyer-Betz  5  also  non- 
stable pyrrols,  which  contain  a  non-substituted  hydrogen  atom  in  a  ring  carbon 
atom,  pass  readily  in  the  animal  body  into  substances  which  give  the  character- 
istic urobilin  reactions.  These  reactions  are  also  given  by  bodies  of  different 
constitution,  but  which  probably  contain  the  same  ehromophore  groups,  and  it  is 
these  conditions  which  cause  the  above-mentioned  uncertainty  as  to  the  occur- 
rence of  different  urobilins. 

That  urobilin  is  identical  with  the  hydrobilirubin  of  Maly  (see  page 
428)  has  been  considered  for  a  long  time.  In  opposition  to  this  view 
we  find  that  both  bodies,  not  to  mention  other  small  differences,  have  an 
essentially  different  composition.  While  the  hydrobilirubin  contains 
9.22  per  cent  nitrogen,  according  to  Maly,  the  urobilin  contains  only 
4.09  per  cent  nitrogen,  according  to  Hopkins  and  Garrod,  and  5.93  per 
cent  nitrogen,  according  to  Fromholdt.  In  the  urobilin  of  the  feces, 
stercobilin,  which  is  identical  with  urobilin,  Hopkins  and  Garrod  found 


1  Centralbl.  f.  d.  med.  \\ 'issensch.,  1868  and  1869,  and  Virchow's  Arch.,  47. 

2  See  MacMunn,  Proc.  Roy.  Soc,  31  and  35;  Ber.  d.  deutsch.  chem.  Gesellsch.,  14, 
and'journ.  of  Physiol.,  6  and  10;  Bogomoloff,  Maly's  Jahresber.,  22;  Eichholz,  Journ. 
of  Physiol.,  14;  Ad.  Jolles,  Pfluger's  Arch.,  61. 

3  Revue  de  medecine,  1 7,  1897. 

4  Maly,  Ann.  d.  Chem.  u.  Pharm.,  163;  Disque,  Zeitschr.  f.  physiol.  Chem.,  2; 
Stokvis,  Centralbl.  f.  d.  med.  Wissensch.,  1873,  211  and  449;  Hoppe-Seyler,  Ber.  d. 
deutsch.  chem.  Gesellsch.,  7;  Le  Nobel,  Pfluger's  Arch.,  40;  Nencki  and  Sieber, 
Monatshefte  f.  Chem.,  9,  and  Arch.  f.  exp.  Path.  u.  Pharm.,  24;  MacMunn,  Proc.  Roy. 
Soc,  31. 

6  H.  Fischer  and  Meyer-Betz,  Zeitschr.  f.  physiol.  Chem.,  75. 


744  URINE. 

4  per  cent  nitrogen.  Still  H.  Fischer1  has  now  found  that  the  stercobilin 
has  a  lower  nitrogen  content  because  of  a  contamination  with  cholesterin 
or  bile  acids,  and  it  is  possible  that  also  the  low  nitrogen  content  of  the 
urine-urobilin  may  be  caused  by  contamination  with  non-nitrogenous 
substances. 

These  possibilities  have,  it  is  sure,  have  not  been  tested;  but  the 
unequal  nitrogen  content  of  the  two  pigments  does  not  positively  exclude 
the  identity  of  urobilin  and  hydrobilirubin.  Fischer  and  Myer-Betz 
have  in  fact  shown  that  the  hemibilirubin,  which  forms  about  one-half 
of  the  mixture  called  hydrobilirubin  (see  page  429)  is  identical  with  the 
urobilinogen  from  human  urine. 

The  possibility  of  the  formation  of  urobilinogen  and  of  urobilin  from 
bile  pigments  is  assured  and  many  physiological  as  well  as  clinical  observa- 
tions2 support  the  view  that  this  transformation  of  the  bile  pigments 
occurs  by  means  of  putrefactive  processes  in  the  intestine.  Of  these 
observations  we  must  mention  the  regular  appearance  in  the  intestinal 
tract  of  stercobilin,  undoubtedly  derived  from  the  bile-pigmets;  the 
absence  of  urobilin  in  the  urine  of  new-born  infants,  as  well  as  on  the  com- 
plete exclusion  of  bile  from  the  intestine,  and  also  the  increased  elimina- 
tion of  urobilin  with  strong  intestinal  putrefaction.  On  the  other  hand 
there  are  investigators  who,  basing  their  opinion  on  clinical  observations, 
deny  the  enterogenous  origin  of  urobilin  and  claim  that  the  urobilin  is 
derived  from  a  transformation  of  the  bilirubin  elsewhere  than  in  the 
intestine,  by  an  oxidation  of  the  bile-pigment  or  by  a  transformation  of 
the  blood-pigments.3 

Urobilin  or  urobilinogen  dees  not  occur  in  the  urine  of  all  animals, 
and  according  to  Fromholdt  it  is  absent  in  the  urine  of  rabbits.  The 
correctness  of  this  statement  is  denied  by  Gautier  and  Rosso.  In  nor- 
mal human  blood  they  seem  to  be  absent,  while  according  to  Biffi4 
urobilin  and  urobilinogen  occur  sometimes  in  disease  and  in  cadaver  blood. 


1  Hopkins  and  Garrod,  Journ.  of  Physiol.,  22;  Fromholdt,  Zeitschr.  f.  exp.  Path., 
u.  Ther.,  7;*H.  Fischer,  Zeitschr.  f.  physiol.  Chem.,  73. 

2  See  Fr.  Muller,  Schles.  Gesellsch.  f.  vaterl.  Kultur,  1892;  D.  Gerhardt,  "  Ueber 
Hydrobilirubin  und  seine  Bezieh.  zum  Ikterus  "  (Inaug.-Diss.,  Berlin,  1889);  Beck, 
Wien.  klin.  Wochenschr.,  1895;  Harley,  Brit.  Med.  Journ.,  1896;  Fischler,  Zeitschr. 
f.  physiol.  Chem.,  48. 

3  In  regard  to  the  various  theories  as  to  the  formation  of  -urobilin,  see  Harley, 
Brit.  Med.  Journ.,  1896;  A.  Katz,  Wien.  med.  Wochenschr.,  1891,  Nos.  28-32;  Grimm, 
Virchow's  Arch.,  132;  Zoja,  Conferenze  cliniche  italiane,  Ser.  la,  1;  Hildebrandt, 
Zeitschr.  f.  klin.  Med.,  59;  Biffi,  Boll.  d.  scienc.  med.  di  Bologna  (8),  anno  78,  7;  Troisier, 
Compt.  rend.  soc.  biol.,  66  and  Tsuschija,  Zeitschr.  f.  exp.  Path.  u.  Ther.,  7;  Fromholdt 
and  Nersessoff,  ibid.,  11. 

4  Fromholdt,  Zeitschr.  f.  physiol.  Chem.,  53;  CI.  Gautier  and  Russo,  Compt.  rend, 
soc.  biol.,  64;  Biffi,  Folia  hsematol.,  4,  and  1.  c.  Boll.,  78. 


UROBILIN.  745 

Tne  quantity  of  urobilin  in  the  urine  under  physiological  conditions 
varies  widely.  Saillet  found  30-130  milligrams  and  G.  Hoppe-Seyler 
80-  140  milligrams  in  one  day's  urine. 

There  are  numerous  observations  on  the  elimination  of  urobilin  or 
urobilinogen  in  disease,  especially  by  Jaffe,  Disque,  Gerhardt,  G. 
Hoppe-Seyler,1  and  others.  The  quantity  is  increased  in  hemorrhage 
and  in  disease  where  the  blood-corpuscles  are  destroyed,  as  is  the  case 
after  the  action  of  certain  blood-poisons,  such  as  antifebrin  and  anti- 
pyrine.  It  is  also  increased  in  fevers,  cardiac  diseases,  lead  colic,  atrophic 
cirrhosis  of  the  liver,  and  is  especially  abundant  in  so-called  urobilin 
icterus. 

The  properties  of  urobilin  may  vary,  depending  upon  the  method 
of  preparation  and  the  character  of  the  urine  used;  therefore  only  the 
most  important  properties  will  be  given.  Urobilin  is  amorphous,  brown, 
reddish  brown,  red,  or  reddish  yellow,  depending  upon  the  method 
of  preparation.  It  dissolves  readily  in  alcohol,  amyl  alcohol,  and 
chloroform,  but  less  readily  in  ether  or  acetic  ether.  It  is  less  soluble 
in  water,  but  the  solubility  is  augmented  by  the  presence  of  neutral 
salts.  It  may  be  completely  precipitated  from  the  urine  by  saturating 
with  ammonium  sulphate,  especially  after  the  addition  of  sulphuric  acid 
(Mehu2).  It  is  soluble  in  alkalies,  and  is  precipitated  from  the  alkaline 
solution  by  the  addition  of  acid.  It  is  partly  dissolved  by  chloroform 
from  an  acid  (watery-alcoholic)  solution;  alkali  solutions  remove  the 
urobilin  from  the  chloroform.  The  neutral  or  faintly  alkaline  solutions 
are  precipitated  by  certain  metallic  salts  (zinc  and  lead),  but  not  by  others, 
such  as  mercuric  sulphate.  Urobilin  is  precipitated  from  the  urine  by 
phosphotungstic  acid.  It  does  not  give  Gmelin's  test  for  bile-pigments. 
It  gives,  on  the  contrary,  a  reaction  which  may  be  mistaken  for  the  biuret 
test,  by  the  action  of  copper  sulphate  and  alkali.3 

Neutral  alcoholic  urobilin  solutions  are,  in  strong  concentration, 
brownish  yellow,  in  great  dilution  yellow  or  rose-colored.  They  have  a 
strong  green  fluorescence.  The  acid  alcoholic  solutions  are  brown, 
reddish  yellow,  or  rose-red,  according  to  concentration.  They  are  not 
fluorescent,  but  show  a  faint  absorption-band,  X,  between  b  and  F,  which 
borders  on  F.  The  absorption  maximum  lies  according  to  Lewin  and 
Stenger4  at  7  =  494-497.     The  alkaline  solutions  are  brownish  yellow, 

1  In  regard  to  the  literature  on  this  subject  we  refer  the  reader  to  D.  Gerhardt, 
"Ueber  Hydrobilirubin  und  seine  Beziehungen  zum  Ikterus  "  (Berlin,  1889),  and 
also  G.  Hoppe-Seyler,  Virchow's  Arch.,  124. 

2  Journ.  de  Pharm.  et  Chim.,  1878,  cited  from  Maly's  Jahresber.,  8. 

3  See  Salkowski,  Berlin,  klin.  Wochenschr.,  1897,  and  Stokvis,  Zeitschr.  f.  Biologie, 
34. 

4  Pfluger's  Arch.,  144. 


746  URINE. 

yellow,  or  (the  ammoniacal)  yellowish  green,  according  to  concentration. 
They  show  a  dark  band  7,  which  is  moved  somewhat  toward  the  red 
end  of  the  spectrum  and  lies  between  E  and  F.  The  absorption  max- 
imum lies  at  X  =  506-510.  If  some  zinc-chloride  solution  is  added  to 
an  ammoniacal  solution  of  the  pigment  it  becomes  red  and  shows  a 
beautiful  green  fluorescence  and  gives  the  same  absorption  bands.  If 
a  sufficiently  concentrated  solution  of  urobilin  alkali  is  carefully  acidified 
with  sulphuric  acid  it  becomes  cloudy  and  shows  a  second  band  exactly 
at  E,  and  connected  with  7  by  a  shadow  (Garrod  and  Hopkins,  Saillet  x). 
Urobilinogen  is  colorless  or  only  faintly  colored,  but  is  very  quickly 
changed  in  the  air  and  by  the  actionof  light  and  is  transformed  into  urobilin. 
The  urobilinogen,  which  is  identical  with  hemibilirubin,  can  be  obtained 
as  colorless  prisms  by  solution  in  hot  acetic-ether  and  treating  this  with 
ligroin,  and  evaporating.  Urobilinogen  is  soluble  in  ether,  acetic  ether, 
amyl  alcohol  and  in  chloroform,  and  can  in  part  be  removed  from  the 
urine  after  adding  sodium  bicarbonate  and  shaking  with  chloroform 
(Fischer  and  Meyer-Betz).  It  can  also  be  obtained  directly  from  the 
urine  or  from  the  acidified  urine  by  shaking  with  chloroform  or  ether, 
although  it  is  less  pure.  In  a  chloroform  solution  of  urobilin  and  urobilino- 
gen, according  to  Grimbert2,  only  urobilin  and  not  urobilinogen  is 
taken  up  by  a  sodium  diphosphate  solution,  which  is  not  colored  red  by 
phenolphthalein.  Like  urobilin,  it  is  precipitated  from  the  urine  on 
saturating  with  ammonium  sulphate.  When  free  from  urobilin  it  does 
not  give  auy  absorption  bands  and  no  fluorescence  with  ammonia  and 
zinc  salt.  For  the  detection  and  identification  of  urobilinogen  we  make 
use  of  Ehrlich's  reagent  (p-dimethylamino-benzaldehyde).  This 
reagent  consists  of  dissolving  2  grams  p-dimethylaminobenzaldehyde 
in  50  cc.  concentrated,  fuming  hydrochloric  acid  and  diluting  to  100 
cc.  with  water.  To  10  cc.  of  the  urine  add  1  cc.  of  the  reagent  and 
thoroughly  shake.  According  to  the  amount  of  urobilinogen,  the  solution 
becomes  pink  colored  or  intensely  red,  and  in  the  spectrum  we  find  a 
band  between  D  and  E.  The  red  color  can  be  taken  up  by  amyl  alcohol. 
Urobilin  does  not  give  this  reaction,  which  is  common  to  certain  haematin 
and  pyrrol  derivatives. 

The  preparation  of  urobilin  from  the  urine  can  be  done  according  to  the 
original  method  of  Jaffe,  or  according  to  the  method  suggested  by  Mehu, 
which  has  been  modified  somewhat  by  Garrod  and  Hopkins  3  (precipita- 
tion with  ammonium  sulphate)   or  according  to  Charnas*  suggestion. 

According  to  Charnas  4  the  preparation  is  best  from  urobilinogen, 

1  Garrod  and  Hopkins,  Journ.  of  Physiol.,  20;  Saillet,  1.  c. 

I    -'her  and  Meyer-Betz,  1.  c;  Grimbert,  Compt.  rend.  soc.  biol.,  70. 
»  Jaffe\  1.  c;  Mehu,  1.  c;  Garrod  and  Hopkins,  Journ.  of  Physiol.,  20. 
*  Charnas,  Bioch.  Zeitschr.,  20. 


DETECTION   AND   ESTIMATION  OF  UROBILIN.  747 

and  if  the  urine  contains  urobilin,  it  is  first  allowed  to  undergo  alkaline 
fermentation,  when  the  urobilin  is  converted  into  urobilinogen.  The 
urine  is  acidified  with  tartaric  acid  and  extracted  with  ether.  The 
foreign  pigments  are  precipitated  from  the  ethereal  solution  by  petroleum 
ether;  the  ether  solution  is  washed  with  water,  evaporated,  and  the  residue 
allowed  to  stand  with  water  for  several  hours  at  38°  C,  when  the  urobilino- 
gen is  transformed  into  urobilin.  The  urobilin  can  now  be  precipitated 
with  ammonium  sulphate,  and  the  dried  precipitate  extracted  with  absolute 
alcohol.  This  urobilin  has  about  three  times  as  much  extinction  ability 
as  Maly's  urobilin  (hydrobilirubin).  Other  methods  of  preparation 
have  been  suggested. 

The  urobilinogen  is  prepared  by  shaking  the  urine,  directly  after 
adding  sodium  bicarbonate,  with  chloroform  (Fischer  and  Meyer- 
Betz).     In  regard  to  details  we  must  refer  to  the  original  publication. 

The  detection  of  urobilin  can  sometimes  be  done  directly  on  the  urine. 
Otherwise  the  urine  is  shaken  with  ether,  amyl  alcohol  or  chloroform  and 
these  solutions  tested.  According  to  Schlesinger  l  the  urine  can  also  be 
precipitated  by  an  equal  volume  of  a  saturated  solution  of  zinc  acetate  in 
alcohol  and  the  filtrate  directly  tested  for  the  fluorescence  and  absorption. 
Grimbert  2  has  suggested  a  method  for  the  separate  testing  for  urobilin  and 
urobilinogen  by  using  the  chloroform,  after  shaking  the  urine  therewith. 
For  the  detection  of  urobilin  we  always  make  use  of  the  color  of  the  acid 
or  alkaline  solutions,  the  absorption  spectrum  and  the  beautiful  fluorescence 
of  the  ammoniacal  solution  containing  zinc  chloride.  For  the  detection 
of  urobilinogen  we  make  use  of  Erhlich's  reagent,  and  the  property  of 
the  colorless  solution  of  being  changed  into  urobilin  in  the  air  and  light. 

In  the  quantitative  estimation  of  urobilin  we  proceed  as  follows, 
according  to  G.  Hoppe-Seyler:3  100  cc.  of  the  urine  are  acidified  with 
sulphuric  acid  and  saturated  with  ammonium  sulphate.  The  precipitate 
is  collected  on  a  filter  after  some  time,  washed  with  a  saturated  solu- 
tion of  ammonium  sulphate,  and  repeatedly  extracted  with  equal  parts 
of  alcohol  and  chloroform  after  pressing.  The  filtered  solution  is  treated 
with  water  in  a  separatory  funnel  until  the  chloroform  separates  well 
and  becomes  clear.  The  chloroform  solution  is  evaporated  on  the  water- 
bath  in  a  weighed  beaker,  the  residue  dried  at  100°  C.,  and  then  extracted 
with  ether.  The  ethereal  extract  is  filtered,  the  residue  on  the  filter  dis- 
solved in  alcohol,  and  transferred  to  the  beaker  and  evaporated,  then 
dried  and  weighed.  According  to  this  method  G.  Hoppe-Seyler  found 
0.08-0.14  gram  of  urobilin  in  one  day's  urine  of  a  healthy  person,  or  an 
average  of  0.123  gram. 

The  urobilin  can  also  be  determined  according  to  the  method  sug- 
gested by  Charnas  for  its  preparation  and  urobilin  can  also  be  deter- 
mined spectroscopically  by  the  method  suggested  by  Saillet.4  Further 
details  will  be  found  in  the  original  publications  and  in  larger  handbooks. 

The  quantitative  estimation  of  urobilinogen  can  be  accomplished 
spectroscopically  by  means  of  Ehrmch's  reagent,  as  suggested  by  Charnas. 

1  Deutsch.  rued.  Wochenschr.,  1903. 

*  Compt.  rend.  soc.  biol.,  70. 

1  Virchow's  Arch.,  124. 

4  Charnas,  1.  c;  Saillet,  1.  c;  see  also  Tsuschija,  Zeitschr.  f.  exp.  Path.  u.  Ther.,  7. 


74.8  URINE. 

Uroerythrin  is  the  pigment  which  often  gives  the  beautiful  red  color  to 
the  urinary  sediments  (sedi?nentum  lateritium) .  It  also  frequently  occurs 
although  only  in  very  small  quantities,  dissolved  in  normal  urines.  The 
quantity  is  increased  after  great  muscular  activity,  after  profuse  perspira- 
tion, immoderate  eating,  or  partaking  of  alcoholic  drinks,  as  well  as  after 
digestive  disturbances,  fevers,  circulatory  disturbances  of  the  liver,  and 
in  many  other  pathological  conditions. 

Uroerythrin,  which  has  been  especially  studied  by  Zoja,  Riva,  and 
Garrod,1  has  a  pink  color,  is  amorphous,  and  is  very  quickly  destroyed 
by  light,  especially  when  in  solution.  The  best  solvent  is  amyl  alcohol; 
acetic  ether  is  not  so  good,  and  alcohol,  chloroform,  and  water  are  even 
less  valuable.  The  very  dilute  solutions  show  a  pink  color;  but  on  greater 
concentration  they  become  reddish  orange  or  bright  red.  They  do  not 
fluoresce  either  directly  or  after  the  addition  of  an  ammoniacal  solution 
of  zinc  chloride;  but  they  have  a  strong  absorption,  beginning  in  the 
middle  between  D  and  E  and  extending  to  about  F,  and  consisting  of  two 
bands  which  are  connected  by  a  shadow  between  E  and  b.  Concentrated 
sulphuric  acid  colors  a  uroerythrin  solution  a  beautiful  carmine  red; 
hydrochloric  acid  gives  a  pink  color.  Alkalies  make  its  solution  grass 
green,  and  often  a  play  of  colors  from  pink  to  purple  and  blue  is  observed. 
Porcher  and  Hervieux  2  claim  that  uroerythrin  is  a  skatol  pigment. 

In  preparing  uroerythrin  according  to  Garrod,  the  sediment  is  dissolved 
in  water  at  a  gentle  heat  and  saturated  with  ammonium  chloride,  which  pre- 
cipitates the  pigment  with  the  ammonium  urate.  This  is  purified  by  repeated 
solution  in  water  and  precipitation  with  ammonium  chloride  until  all  the  urobilin 
is  removed.  The  precipitate  is  finally  extracted  on  the  filter  in  the  dark  with 
warm  water,  filtered,  then  diluted  with  water,  any  haematoporpr^rin  remaining 
being  removed  by  shaking  with  chloroform;  the  precipitate  is  then  faintly  acidi- 
fied with  acetic  acid  and  shaken  with  chloroform,  which  takes  up  the  uroerythrin. 
The  chloroform  is  evaporated  in  the  dark  at  a  gentle  heat. 

Volatile  fatty  acids,  such  as  formic  acid,  acetic  acid,  and  perhaps  also  butyric 
acid,  occur  under  normal  conditions  in  human  urine  (v.  Jaksch),  also  in  that  of 
dogs  and  herbivora  (Schotten).  The  acids  poorest  in  carbon,  such  as  formic 
acid  and  acetic  acid,  are  more  stable  in  the  body  than  those  richer  in  carbon, 
and  therefore  the  relatively  greater  part  of  these  pass  unchanged  into  the  urine 
(Schotten).  Normal  human  urine  contains  besides  these  bodies  others  which 
yield  acetic  acid  when  oxidized  by  potassium  dichromate  and  sulphuric  acid 
(v.  Jaksch).  The  quantity  of  volatile  fatty  acids  in  normal  urine  calculated  as 
acetic  acid  is,  according  to  v.  Jaksch,  0.008-0.009  gram  per  twenty-four  hours; 
according  to  v.  Rokitansky,  0.054  gram;  and  according  to  Magnus-Levy 
0.060  gram.  The  quantity  is  increased  by  exclusively  farinaceous  food  (Roki- 
tansky), in  fever  and  in  certain  diseases,  while  in  others  it  is  diminished  (v. 
Jaksch,  Rosen feld).  Large  amounts  of  volatile  fatty  acids  are  produced  in  the 
alkaline  fermentation  of  the  urine,  and  the  quantity  is  6-15  times  as  large  as  in 

1  Zoja,  Arch.  ital.  di  clinica  med.,  1893,  and  Centralbl.  f.  d.  med.  Wissensch.,  1892; 
Riva,  Gaz.  med.  di  Torino,  Anno  43,  cited  from  Maly's  Jahresber.,  24;  Garrod,  Journ. 
of  Physiol.,  17  and  21. 

2  Journ.  de  Physiol.,  7. 


CARBOHYDRATES  AND   REDUCING   SUBSTANCES.  749 

normal  urine  (Salkowski).1  Non-volatile  fatty  acids  have  been  detected  as 
normal  constituents  of  urine  by  K.  Morneu  and  Hyhuinette.2 

Parallactic  Acid.  It  is  claimed  that  this  acid  occurs  in  the  urine  of  healthy 
persons  after  very  fatiguing  marches  (Colasanti  and  Moscatelli).  It  is  found 
in  larger  amounts  in  the  urine  in  acute  phosphorus-poisoning  or  acute  yellow 
atrophy  of  the  liver  (Schultzen  and  Riess),  in  pregnancy  (Undekhill),  and 
especially  abundant  in  eclampsia  (Zweifel  and  others).  According  to  the 
investigations  of  Hoppe-Seyler,  Araki,  and  v.  Terray,  lactic  acid  passes  into 
the  urine  as  soon  as  the  supply  of  oxj^gen  is  decreased  in  any  way,  and  this  probably 
explains  the  occurrence  of  lactic  acid  in  the  urine  after  epileptic  attacks  (Inouye 
and  Saiki).  Minkowski  3  has  shown  that  lactic  acid  occurs  in  the  urine  in  large 
quantities  on  the  extirpation  of  the  liver  of  birds. 

Glycerophosphoric  acid  occurs  as  traces  in  the  urine,4  and  it  is  probably  a 
decomposition  product  of  lecithin.  The  occurrence  of  succinic  acid  in  normal 
urine  is  a  subject  of  discussion. 

Carbohydrates  and  Reducing  Substances  in  the  Urine.  The  occurrence 
of  glucose,  as  traces,  in  normal  urine  is  highly  probable,  as  the  investiga- 
tions of  Brucke,  Abeles,  and  v.  UdrAnszky  show.  The  last  investigator 
has  also  shown  the  habitual  occurrence  of  carbohydrates  in  the  urine, 
and  their  presence  has  been  positively  proven  by  the  investigations  of 
Baumann  and  Wedenski,  and  especially  by  Baisch.  Besides  glucose 
normal  urine  contains,  according  to  Baisch,  another  not  well-studied 
variety  of  sugar,  according  to  Lemaire,  probably  isomaltose,  and  besides 
this  a  dextrin-like  carbohydrate  (animal  gum),  as  shown  by  Landwehr, 
Wedenski,  and  Baisch.  The  quantity  of  carbohydrates  eliminated  under 
normal  conditions  in  the  twenty-four  hours'  urine  and  determined  by 
the  benzoylation  method,  which  is  perhaps  not  sufficiently  trustworthy, 
varies  considerably  between  1.5  and  5.09  grams.5 

The  precipitate  obtained  from  concentrated  urine  by  the  aid  of  alcohol  and 
whose  nitrogen  (colloidal  nitrogen  according  to  Salkowski)  in  normal  urine 
amounts  to  2.34-4.08  per  cent  of  the  total  nitrogen,  and  in  pathological  urines  to 
8-9  per  cent,  and  in  a  case  of  acute  yellow  atrophy  of  the  liver  to  21.8  per  cent 
contains,    Salkowski  6   claims,    a   nitrogenous   carbohydrate   which   has   strong 


1  v.  Jaksch,  Zeitschr.  f.  physiol.  Chem.,  10;  Schotten,  ibid.,  7;  Rokitansky,  Wien. 
med.  Jahrbuch,  1887;  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  13;  Magnus-Levy, 
Salkowski's  Festschrift,  1004;  Rosenfeld,  Deutsch,  med.  Wochenschr.,  29. 

*Skand.  Arch.  f.  Physiol.,  7. 

3  Colasanti  and  Moscatelli,  Moleschott's  Untersuch.,  14;  Schultzen  and  Reiss, 
Chem.  Centralbl.,  1869;  Underhill,  Journ.  of  biol.  Chem.,  2;  Zweifel,  Arch.  f.  Gynakol., 
76;  Araki,  Zeitschr.  f.  physiol.  Chem.,  15,  16,  17,  19.  See  also  Irisawa,  ibid.,  17;  v. 
Terray,  PAu^er's  Arch.,  65;  Schutz,  Zeitschr.  f.  physiol.  Chem.,  19;  Inouye  and 
Saiki,  ibid.,  37;  Minkowski,  Arch.  f.  exp.  Path.  u.  Pharm.,  21  and  31. 

4  See  Pasqualis,  Maly's  Jahresber.,  24. 

5  Lemaire,  Zeitschr.  f.  physiol.  Chem.,  21;  Baisch,  ibid.,  18,  19,  and  20.  In  these 
as  well  as  in  Treupel,  ibid.,  16,  the  works  of  other  investigators  are  cited.  See  also 
v.  Alfthan,  Deutsch.  med.  Wochenschr.,  26. 

6  Berlin,  klin.  Wochenschr.,  1905.  In  regard  to  urinary  colloids  see  also  Lichtwitz, 
Zeitschr.  f.  physiol.  Chem.,  61  and  72,  with  Rosenbach,  ibid..  61. 


750       •  URINE. 

reducing  action  upon  alkaline  copper  solutions  after  cleavage  with  hydrochloric 
acid. 

Besides  traces  of  sugar  and  the  reducing  substances  previously  men- 
tioned, uric  acid  and  creatinine,  the  urine  contains  still  other  bodies  of  this 
character.  These  latter  are  partiy  conjugated  compounds  of  glucuronic 
acid,  C6N10O7,  which  is  closely  allied  to  sugar.  The  reducing  power  of( 
normal  urine  corresponds,  according  to  various  investigators,  to  1.5-5.96 
p.  m.  glucose.  That  portion  of  the  reduction  belonging  to  glucose 
alone  is  equal  to  0.1-0.6  p.  m.  Laveson  *  believes  that  of  the  total 
reduction  17.8  per  cent  is  due  to  sugar,  26.3  per  cent  to  creatinine,  7.8 
per  cent  to  uric  acid,  and  the  remainder,  nearly  50  per  cent,  is  caused 
by  chiefly  unknown  bodies. 

Conjugated  glucuronates  occur,  as  indicated  by  Fluckiger  and  first 
positively  shown  by  Mayer  and  Neuberg,  in  an  exact  manner,  in  very 
small  amounts  in  normal  urine.  They  occur  chiefly  as  phenol-  and  only 
very  small  amounts  of  indoxyl-  or  skatoxylglucuronates.  The  quantity 
of  glucuronic  acid  obtained  from  the  conjugated  glucuronates  is  estimated 
as  0.01  p.  m.  by  Mayer  and  Neuberg,  and  by  C.  Tollens  and  Fr.  Stern,2 
on  the  contrary  it  was  found  to  be  2.5  p.  m.  or  0.37  gram  per  day.  Besides 
these  conjugated  glucuronates  perhaps  the  urine  sometimes  contains  the 
urea  glucuronic  acid,  the  ureidoglucuronic  acid  prepared  synthetically 
by  Neuberg  and  Niemann.3 

Very  large  amounts  of  these  conjugated  glucuronates  occur  in  the 
urine,  on  the  other  hand,  after  partaking  of  various  therapeutic  agents 
aDd  other  substances,  such  as  chloral  hydrate,  camphor,  naphthol,  borneol, 
turpentine,  morphine,  and  many  other  substances.  The  elimination 
of  glucuronic  acid  may  be  markedly  increased  in  severe  disturbances  of 
the  respiration,  severe  dyspnoea,  in  diabetes  mellitus,  and  by  the  direct 
introduction  of  large  amounts  of  glucose.  According  to  P.  Mayer, 
in  the  oxidation  of  glucose  a  part  of  it  forms  glucuronic  acid,  hence  it  is 
to  be  expected  that  the  glucuronic  acid  can  in  part  be  derived  from  the 
glucose.  As  a  conjugation  of  the  glucuronic  acid  with  other  bodies, 
such  as  aromatic  atomic  complexes,  prevents  the  combustion  of  this 
acid  in  ,the  animal  body,  it  ought  to  follow  that  after  the  introduction 
of  such  an  atomic  complex  in  the  body  during  a  glycosuria  a  correspond- 
ing reduction  of  the  glucose  elimination  would  take  place  with  the 
increased  excretion  of  conjugated  glucuronates.  In  order  to  prove 
this   possibility,   O.    Loew'i  4   fed   dogs  with  camphor  during  phlorhizin 

1  Fluckiger,  Zeitschr.  f.  physiol.  Chem.,  9;  Laveson,  Bioch.  Zeitschr.,  4. 

2  Fluckiger,  1.  c;  Mayer  and  Neuberg,  Zeitschr.  f.  physiol.  Chem.,  29;  Tollens  and 
Stern,  Zeitschr.  f.  physiol.  Chem.,  67. 

'  Zeitschr.  f.  physiol.  Chem.,  44. 
*  Arch.  f.  exp.  Path.  u.  Pharm.,  47. 


CONJUGATED  GLUCURONIC  ACIDS.  751 

diabetes  and  found  that  the  above  expectation  was  not  realised.  Although 
large  quantities  of  cainpho-gluciironic  acid  were  excreted,  the  sugar 
excretion  was  only  slightly  diminished  and  not  in  proportion  to  the 
quantity  of  conjugated  glucuronatc  excreted.  These  negative  results 
are  contradicted  by  the  positive-  results  obtained  by  Paul  Mayer.1 
Rabbits  normally  convert  almost  all  the  camphor  introduced  into  con- 
jugated glucuronic  acid.  Mayer  claims  that  if  we  allow  a  rabbit  to 
starve  several  days,  the  animal  becomes  so  poor  in  the  mother-substance 
(glycogen)  yielding  the  glucuronic  acid  that  the  introduction  of  camphor 
only  brings  about  an  elimination  of  small  quantities  of  glucuronic  acid. 
By  the  simultaneous  administration  of  camphor  and  glucose  while 
starvation  is  going  on,  the  elimination  of  glucuronic  acid  rises  again  to 
the  same  height  as  it  was  before  the  starvation  period.  This  shows  that 
the  sugar  had  conjugated  itself  with  the  camphor  as  glucuronic  acid. 
Hildebrandt  2  has  also  made  experiments  showing  that  glucuronic  acid 
can  very  likely  be  formed  from  sugar.  The  observations  of  Mayer  are 
not  substantiated  by  the  recent  investigations  of  Fenyvessy,3  and  the 
observers  do  not  agree  on  this  question. 

The  conjugated  glucuronic  acids  are  formed,  based  upon  the  investi- 
gations of  Sundwik,  Fischer  and  Piloty,4  by  a  combination  taking 
place  first  between  the  conjugator  and  the  glucose  by  means  of  the  alde- 
hyde group,  and  then  the  end  alcohol  group,  CHoOH,  is  oxidized  to  COOH. 
The  conjugated  glucuronic  acids,  at  least  in  most  cases,  seem  to  be  con- 
structed after  the  glucoside  type,  a  view  which  has  received  further 
support  by  the  synthesis  of  phenolglucuronic  acid  and  euxanthonglu- 
curonic  acids  by  Neuberg  and  Neimann.5  Based  upon  their  cleavage 
(as  far  as  they  have  been  investigated)  by  kephir  lactase  and  emulsin, 
but  not  by  yeast  lactase  (Neuberg  and  Wohlgemuth  6),  the  conjugated 
glucuronic  acids  must  belong  to  the  /3-series  of  glucosides.  We  also 
know  of  certain  conjugated  glucuronates  that  are  constructed  upon  the 
ester  type,  namely,  the  dimethylaminobenzoicglucuronate,  discovered 
by  Jaff:e  and  also  the  benzoicglucuronic  acid,  after  feeding  benzoic 
acid  (Magnus-Levy  1 7 

According  to  the  body  with  which  they  are  conjugated  the  glucuronates 
vary  in  behavior.     On  taking  up  water  they  split  into  glucuronic  acid 

1  Zeitschr.  f.  klin.  Med..  47. 

2  Arch.  f.  exp.  Path.  u.  Pharm.,  44. 
8  See  Maly's  Jahresber.,  34. 

4E.  Sundwik,  Akademische  Abhandlung  Helsingfors,  18S6;  see  also  Maly's  Jahr- 
esber., 16,  76;  Fischer  and  Piloty,  Ber.  d.  d.  chem.  Gesellsch.,  24. 
B  Zeitschr.  f.  physiol.  Chem.,  44. 

6  See  Neuberg,  Ergebnisse  der  Physiologic,  Bd.  3,  Abt.  1,  444. 

7  Jaffe,  Zeitschr.  f.  physiol.  Chem.,  43;  Magnus-Levy,  Bioch.  Zeitschr.,  6. 


752  URINE. 

and  the  conjugated  group  and  this  is  brought  about  by  boiling  with 
a  dilute  mineral  acid.  They  are  precipitated  by  basic  lead  acetate  or 
by  basic  lead  acetate  and  ammonia.  Most  of  the  conjugated  glucuronic 
acids  do  not  have  a  direct  reducing  action  but  are  reducing  after  hydrolysis. 
Certain  of  them,  and  to  this  group  belong  especially  those  acids  of  the 
ester  type,  reduce  copper  oxide  and  certain  other  metallic  oxides  in  alkaline 
solution  directly,  and  hence  cause  errors  in  the  investigations  of  the 
urine  for  sugar.  The  conjugated  acids  of  the  glucoside  type  rotate  the 
plane  of  polarized  light  to  the  left,  while  the  glucuronic  acid  itself  is  dextro- 
rotatory. The  conjugated  acids  of  the  ester  type,  which  as  a  rule  are 
less  stable,  rotate  the  ray  of  polarized  light  to  the  right.  As  the  detection 
of  conjugated  glucuronic  acids  is  connected  with  the  tests  for  sugar  in 
the  urine,  we  will  treat  of  this  in  connection  with  these  tests. 

Organic  combinations  containing  sulphur  of  unknown  kind,  which  may 
in  small  part  consist  of  sulphocyanides,  0.04  (Gscheidlen)  to  0.11  p.  m.- 
(I.  Munk),1  cystine  or  bodies  related  to  it,  taurine  derivatives,  chon- 
droitin-sulphuric  acid  and  protein  bodies,  but  in  greater  part  are  made  up 
of  antoxyproteic  acid,  oxyproteic  acid,  alloxyproteic  acid,  and  uroferric 
acid,  are  found  in  human  as  well  as  in  animal  urines.  The  sulphur  of 
these  mostly  unknown  combinations  has  been  called  "  neutral,"  to  dif- 
ferentiate it  from  the  "  acid  "  sulphur  of  the  sulphate  and  ethereal-sul- 
phuric acid  (Salkowski  2) .  The  neutral  sulphur  in  normal  urine  is 
13-24  per  cent  of  the  total  sulphur.3  In  anaemia,  cachetic  conditions, 
pulmonary  tuberculosis  and  especially  in  carcinoma  the  quantity  is 
greatly  increased  (Weiss).  In  general  it  can  be  said  that  the  quantity 
is  increased  by  an  increased  catabolism  of  protein  and  therefore  an  increase 
in  the  neutral  sulphur  has  been  found  in  starvation  (Fr.  Muller),  with 
insufficient  oxygen  supply  (Reale  and  Boeri,  Harnack  and  Kleine) 
and  after  chloroform  narcosis  (Kast  and  Mester).  After  the  introduc- 
tion of  free  sulphur  the  quantity  of  neutral  sulphur  is  increased,  accord- 
ing to  Presch  and  Yvon  and  to  Maillard.*  The  quantity  of  neutral 
sulphur  varies,  according  to  Benedict,  within  rather  narrow  limits 
and  especially,  according  to  Folin,  it  is  dependent  to  a  less  degree  than 
the  sulphate  excretion  upon  the  extent  of  the  protein  metabolism.  The 
relation  between  the  neutral  and  acid  sulphur  depends  in  the  first  place 


1  Gscheidlen,  Pfliiger's  Arch.,  14;  Munk,  Virchow's  Arch.,  69. 

2  Ibid.,  58,  and  Zeitschr.  f.  physiol.  Chem.,  9. 

'Salkowski,  1.  c;  Stadthagen,  Virchow's  Arch.,  100;  Lepine,  Compt.  Rend.,  91 
and  97;  Harnack  and  Kleine,  Zeitschr.  f.  Biologie,  37;  Mor.  Weiss,  Bioch.  Zeitschr.,  27. 
4  Weiss,  1.  c;  Fr.  Muller,  Berl.  klin.  Wochenschr.,  1887;  Reale  and  Boeri,  Maly's 
Jahresber.,  24;  Harnack  and  Kleine,  1.  c;  Kast  and  Mester,  Zeitschr.  f.  klin.  Med., 
18;  Presch.,  Virchow's  Arch.,  119;  Yvon,  Arch,  de  Physiol.  (5),  10;  Maillard,  Compt. 
Rend.,  152. 


ORGANIC  SULPHUR  COMBINATIONS.  753 

upon  the  extent  of  the  sulphuric-acid  excretion.  According  to  Harnack 
and  Kleine,1  the  relation  of  the  oxidized  sulphur  to  the  total  sulphur 
changes  always  in  the  same  way  as  the  relation  of  the  nitrogen  of  the 
urea  to  the  total  nitrogen.  The  more  unoxidized  sulphur  is  eliminated 
the  more  abundant  are  the  nitrogen  compounds,  not  urea,  in  the  urine — 
a  statement  which  coincides  with  recent  observations  showing  that  the 
neutral  sulphur  originates  chiefly  from  the  different  proteic  acids,  and 
the  uroferric  acid. 

According  to  Lepine,  a  part  of  the  neutral  sulphur  is  more  readily  oxidized 
(directly  with  chlorine  or  bromine)  into  sulphuric  acid  than  the  other,  which  is 
only  converted  into  sulphuric  acid  after  fusing  with  potash  and  saltpeter.  The 
investigations  of  W.  Smith  2  show  that  it  is  probable  that  the  difficultly  oxidizable 
part  of  the  neutral  sulphur  occurs  as  sulpho-acids.  An  increased  elimination  of 
neutral  sulphur  has  been  observed  in  various  diseases,  such  as  pneumonia,  cystin- 
uria,  and  especially  where  the  flow  of  bile  into  the  intestine  is  prevented. 

The  total  quantity  of  sulphur  in  the  urine  is  determined  by  fusing  the  solid 
urinary  residue  with  saltpeter  and  caustic  alkali  or  sodium  peroxide,  or  by  oxida- 
tion with  nitric  acid.3  The  quantity  of  neutral  sulphur  is  determined  as  the 
difference  between  the  total  sulphur  and  the  sulphur  of  the  sulphate  and  ethereal- 
sulphuric  acids.  The  readily  oxidizable  part  of  the  neutral  sulphur  is  determined 
by  oxidation  with  bromine  or  potassium  chlorate  and  hydrochloric  acid  (Lepine, 
Jerome  4). 

Sulphuretted  hydrogen  occurs  in  the  urine  only  under  abnormal  conditions 
or  as  a  decomposition  product.  This  compound  may  be  produced  from  the 
neutral  sulphur  of  the  organic  substances  of  the  urine  by  the  action  of  certain 
bacteria  (Fr.  Muller,  Salkowski  5).  Other  investigators  have  given  hypo- 
sulphites as  the  source  of  the  sulphuretted  hydrogen.  The  occurrence  of  hypo- 
sulphites in  normal  human  urine,  which  is  asserted  by  Heffter,  is  disputed  by 
Salkowski  and  Presch.6  Hyposulphites  occur  constantly  in  cat's  urine  and, 
as  a  rule,  also  in  clog's  urine. 

Antoxyproteic  acid  is  a  nitrogenous  acid  containing  sulphur  which 
Bondzynski,  Dombrowski,  and  Panek  7  have  isolated  from  human 
urine.  The  composition  of  the  acid  was:  C  43.21,  H  4.91,  N  24.4,  S  0.61, 
and  O  26.23  per  cent.  A  part  of  the  sulphur  can  be  split  off  by  alkali. 
This  acid  is  soluble  in  water,  is  dextrorotatory,  and  is  only  precipitated 
from  concentrated  solution  by  phosphotungstic  acid.  It  does  not  give 
the  protein  color  reactions,  but  gives  Ehrlich's  diazo  reaction  (see  below). 


1  Benedict,  Zeitschr.  f.  klin.  Med.,  36;  Harnack  and  Kleine,  1.  c;  Folin,  Amer. 
Journ.  of  Physiol.,  13. 

2  Lepine,  1.  c;  Smith,  Zeitschr.  f.  physiol.  Chem.,  17. 

3  See  Abderhalden  and  Funk,  Zeitschr.  f.  physiol.  Chem.,  58  and  59,  which  also 
cites  other  methods.  See  S.  R.  Benedict,  Journ.  of  biol.  Chem.,  6  and  8;  Denis,  ibid., 
8;  Gill  and  Grindley,  Journ.  Amer.  Chem.  Soc,  31;  Folin,  ibid.,  31. 

4  Jerome,  Pfluger's  Arch.,  60. 

5  Fr.  Muller,  Berlin,  klin.  Wochenschr.,  1887;  Salkowski,  ibid.,  1888. 

6  Heffter,  Pfluger's  Arch.,  38;  Salkowski,  ibid.,  39;  Presch,  Virchow'a  Arch.,  119. 

7  Zeitschr.  f.  physiol.  Chem.,  46. 


754  URINE. 

The  salts  with  the  alkalies,  barium,  calcium,  and  silver  are  soluble  in 
water,  and  of  these  salts  that  with  barium  and,  to  a  still  higher  degree, 
the  silver  salt  are  soluble  with  difficulty  in  alcohol.  The  free  acid  and 
its  salts  are  precipitated  by  mercuric  nitrate  and  acetate,  and  by  this 
last  reagent  even  from  solutions  strongly  acidified  with  acetic  acid.  Basic 
lead  acetate  does  not  precipitate  the  pure  acid. 

Oxyproteic  acid  is  the  name  given  by  Bondzynski  and  Gottlieb  * 
to  a  nitrogenous  acid  containing  sulphur,  and  which  they  prepared  from 
human  urine,  which  has  recently  been  further  studied  by  Bondzynski, 
Dombrowski  and  Panek.  This  acid  contained  C  39.62,  H  5.64,  N 
18.08,  S  1.12,  and  0  35.54  per  cent,  and  also  contains  sulphur  which  could 
be  split  off.  On  cleavage  it  yields  no  tyrosine,  nor  does  it  give  Ehrlich's 
diazo  reaction,  the  xanthoproteic  nor  the  biuret  reaction.  It  gives  a 
faint  indication  of  a  Millon  reaction  and  is  not  precipitated  by  phos- 
photungstic  acid,  hence  it  leads  to  an  error  in  the  Pfluger-Bohland 
method  for  estimating  urea.  The  acid  soluble  in  water  is  precipitated 
by  mercuric  nitrate  and  acetate  in  neutral  solutions,  but  is  not  precipitated 
by  basic  lead  acetate.  The  salts  of  this  acid  are  readily  soluble  in  water 
and  more  soluble  in  alcohol  than  the  corresponding  salts  of  antoxy- 
proteic  acid. 

The  acid  which  is  found  in  large  quantities,  especially  in  the  urine  of 
dogs  poisoned  with  phosphorus  (Bondzynski  and  Gottlieb),  is  considered 
like  the  preceding  acid  as  an  intermediary  oxidation  product  of  the  pro- 
teins, and  oxyproteic  acid  seems  to  represent  a  higher  state  of  oxidation 
or  a  demolition  of  the  proteins  than  the  antoxyproteic  acid. 

The  acid  called  uroproteic  acid  by  Cloetta  is  probably  a  mixture  of  several 
bodies,  according  to  the  recent  investigations  of  Bondzynski,  Dombrowski,  and 
Panek.  The  same  applies  also  to  the  barium  oxyproteate  prepared  by  Pregl  2 
from  the  urine. 

Alloxyproteic  acid  is  a  third  acid  related  to  the  above,  which  was 
first  isolated  by  Bondzynski  and  Panek  3  from  the  urine  and  then  care- 
fully studied  with  Dombrowski.  The  composition  is:  C  41.33,  H 
5.70,  N  13.55,  S  2.19,  and  0  37.23  per  cent,  based  upon  new  investigations. 
The  free  acid  is  soluble  in  water.  It  gives  neither  the  biuret  reaction 
nor  Ehrlich's  reaction,  and  is  not  precipitated  by  phosphotungstic 
acid.  Differing  from  the  other  acids,  it  is  precipitated  by  basic  lead 
acetate,  and  its  salts  are  only  slightly  soluble  in  alcohol.    According  to 


'Centralbl.  f.  d.  med.  Wissensch.,  1897,  No.  33. 

2  Cloetta,  Arch.  f.  pxp.  Path.  u.  Pharm.,  40;  Pregl,  Pfliiger's  Arch.,  75. 

*  Ber.  d.  d.  chem.  Gesellsch.,  35. 


OXYPROTEIC  ACIDS.  755 

Liebermann  x  this  acid  is  not  a  unit  substance,  and  contains  a  part  of 
its  sulphur  as  ethereal  sulphuric  acid,  and  it  also  contains  uroferric  acid. 

Tho  urochrome,  which  has  been  specially  studied  by  Dombrowski,2  is  con- 
eidered  by  him  and  Bondzynski  us  belonging  to  the  oxyproteic  acid  group.  I 
contains  about  5  per  cent  sulphur,  is  precipitated  by  copper  acetate  and  yields 
melanin-like  substances  on  its  decomposition.  No  positive  proofs  arc  at  hand  in 
regard  to  the  purity  of  this  urochrome  and  the  reports  as  to  its  composition, 
which  are  very  contradictory,  do  not  exclude  the  possibility  that  this  is  a  mixture 
of  a  yellow  pigment  with  another  substance  (see  page  741). 

Browinski  and  Dombrowski  3  have  carried  out  investigations  on  the  nitrogen 
tit  ratable  with  forniol  on  the  oxyproteic  acids  before  and  after  acid  hydrolysis. 
They  found  that  the  antoxyproteic  acid  and  the  oxyproteic  acid  did  not  contain 
any  nitrogen  split  off  as  NH3  by  MgO  before  hydrolysis,  while  the  alloxyproteic 
acid  as  well  as  the  urochrome  yielded  about  3  per  cent  of  the  total  nitrogen  in  this 
form.  After  acid  hydrolysis  all  gave  about  the  same  quantity  of  ammonia.  The 
two  first-mentioned  acids,  especially  oxyproteic  acid,  were  before  hydrolysis 
considerably  richer  in  amino  groups,  tit  ratable  with  formol,  than  the  others. 
This  indicates  that  these  two  acids  are  produced  from  the  proteins  by  a  deeper 
cleavage  than  is  the  alloxyproteic  acid.  The  large  amount  of  free  amino  groups, 
which  occur  especially  in  the  oxyproteic  acid  and  which  amount  to  38.8  per  cent 
of  the  total  nitrogen,  is  nevertheless  remarkable. 

The  preparation  of  the  three  above-mentioned  acids  is  based  in  part 
upon  the  fact  that  alloxyproteic  acid  alone  is  precipitated  by  basic  lead 
acetate  and  that  the  two  other  acids  can  be  precipitated  from  the  fil- 
trate by  mercuric  acetate,  the  antoxyproteic  acid  in  acetic  acid  solution 
and  the  oxyproteic  acid  in  neutral  solution.  The  preparation  is  never- 
theless very  tedious  and  complicated  and  therefore  we  must  refer  to  the 
original  works  for  details. 

Uroferric  acid  is  an  acid  isolated  by  Thiele  4  from  the  urine,  according  to 
Siegfried's  method  for  preparing  pure  peptone.  It  also  contains  sulphur,  3.46 
per  cent,  and  has  the  formula  CssHseXsSOig.  The  acid  forms  a  white  powder 
which  is  readily  soluble  in  water,  saturated  ammonium-sulphate  solution,  and 
methyl  alcohol.  It  is  soluble  with  difficulty  in  absolute  alcohol,  insoluble  in  benzene, 
chloroform,  ether,  and  acetic  ether.  About  one-half  of  the  sulphur  can  be  split 
off  as  sulphuric  acid  on  boiling  with  hydrochloric  acid.  The  acid  gives  neither 
the  biuret  test  nor  Millon's  or  Adamkiewicz's  reactions.  It  is  precipitated  by 
mercuric  nitrate  and  sulphate,  and  also  by  phosphotungstic  acid.  This  acid  is 
hexabasic,  and  its  specific  rotation  at  18°  C.  (a)o  =  —32.5°.  On  cleavage  it 
yields  melanine  substances,  sulphuric  acid,  aspartic  acid,  but  no  hexone  bases. 
The  existence  of  this  acid  is  disputed  by  Bondzynski,  Dombrowski  and  Panek. 
The  investigations  of  Ginsberg  also  contradict  the  occurrence  of  such  an  acid, 
because  no  sulphuric  acid  could  be  split  off  from  the  mixture  of  the  oxyproteic 
acids  by  hydrolysis. 

Methods  for  the  quantitative  estimation  of  the  total  oxyproteic 
acids  have  been  suggested  by  Ginsberg  and  by  Gawinski.5      Accord- 


1  Zeitschr.  f.  physiol.  Chem.,  52. 

2  Ibid.,  46  and  62. 

3  Ibid.,  77. 

4  Zeitschr.  f.  physiol.  Chem.,  37. 

5  Ginsberg,  Hofmeister's  Beitrage,  10;  Gawinski,  Zeitschr.  f.  physiol.  Chem.,  58. 


756  URINE. 

ing  to  their  determinations,  in  man  with  a  mixed  diet,  the  nitrogen  of  the 
oxyproteic  acids  represented  3-6.8  per  cent  of  the  total  nitrogen,  and  with 
a  milk  diet  it  sinks  to  about  one-half  of  this  (Gawinski).  In  dogs  it 
amounts  to  2  per  cent  of  the  total  nitrogen  (Ginsberg).  In  disease  it 
may  rise,  and  in  typhoid  cases  it  may  rise  to  14.69  per  cent  of  the  total 
nitrogen  (Gawinski).  In  phosphorus  poisoning  this  nitrogen  fraction 
is  also  markedly  increased  according  to  several  observations.  The 
oxyproteic  acids  are  considered,  as  above  remarked,  as  intermediary 
products  of  the  protein  metabolism,  and  Gawinski  holds  that  the 
elimination  of  their  nitrogen  runs  parallel  with  the  elimination  of  neutral 
sulphur,  so  that  this  latter  may  serve  as  an  approximate  measure  of  the 
elimination  of  these  acids. 

Abderhalden  and  Pregl  »  have  shown  that  human  urine  normally  contains 
compounds  which  stand,  perhaps,  in  close  relation  to  the  polypeptides,  and 
which  on  hydrolysis  with  acids  yield  at  least  a  part  of  the  moities  existing  in  the 
protein  molecule.  In  the  case  investigated  they  obtained  abundant  glycocoll, 
also  leucine,  alanine,  glutamic  acid,  phenylalanine,  and  probably  also  aspartic 
acid.  The  relation  between  these  polypeptide-like  bodies  and  the  above-men- 
tioned proteic  acids  and  uroferric  acid  has  not  been  investigated. 

Henriques  and  Sorensen  2  have  given  further  proof  for  the  occurrence  of 
nitrogen  in  peptide  combinations  in  the  urine.  They  have  shown  by  formol 
titration  that  in  normal  urine  amino-acid  nitrogen  occurs.  It  must  be  remarked 
that  they  consider  as  amino-acid  nitrogen  not  only  the  nitrogen  occurring  as  amino- 
acids  but  also  the  urine  nitrogen  directly  titratable  with  formol,  therefore  also  the 
titratable  amino-nitrogen  in  the  oxyproteic  acids,  polypeptides  or  more  com- 
plicated protein  derivatives.  They  have  further  shown  that  after  boiling  with 
acid  that  the  quantity  of  titratable  nitrogen  increases,  and  this  increase  which 
in  man  may  be  8.9-28.3  per  cent  of  the  amino-acid  nitrogen,  they  consider  as  pep- 
tide-like  nitrogen.  We  have  abundant  literature  3  on  the  ways  and  means  of 
carrying  out  the  formol  titration  in  urines,  considering  the  presence  of  ammonia. 

Amino-acids  may,  when  they  are  introduced  into  the  body  in  large  amounts, 
also  pass  in  part  into  the  urine.  This  has  been  shown  for  r-alanine  by  R.  Hirsch 
in  the  dog,  and  by  Plaut  and  Reese  in  dog  and  man,  and  for  r-leucine  by 
Abderhalden  and  Samuely4  in  rabbits  and  by  others  using  different  amino-acids. 
Embden  and  Reese,  Forssner,  Abderhalden  and  Schittenhelm,  Samuely, 
Embden  and  Marx  6  were  able,  by  means  of  the  naphthalene  sulpho- 
chloride  method  to  detect  glycocoll  in  normal  human  urine,  and  this  glycocoll 
must  occur  in  the  urine  in  a  combination  which  is  readily  split  by  alkali.     Although 


1  Zeitschr.  f.  physiol.  Chem.,  46. 

2  Henriques,  Zeitschr.  f.  physiol.  Chem.,  60;  Henriques  and  Sorensen,  ibid.,  63 
and  64. 

3  See  Henriques  and  S6rensen,  1.  c;  Malfatti,  Zeitschr.  f.  physiol.  Chem.,  61  and 
66;  de  Jager,  ibid.,  62  and  65;   Fry  and  Gigon,  Bioch.  Zeitschr.,  22. 

4  R.  Hirsch,  Zeitschr.  f.  exp.  Path.  u.  Therap.,  1;  Plaut  and  Reese,  Hofmeister's 
Beitrage,  7;  Abderhalden  and  Samuely,  Zeitschr.  f.  physiol.  Chem.,  47. 

5  Forssner,  Zeitschr.  f.  physiol.  Chem.,  47;  Abderhalden  and  Schittenhelm,  ibid., 
47;  Samuely,  ibid.,  47;  Embden  and  Reese,  Hofmeister's  Beitrage,  7,  with  Marx, 
ihi'l..  11,  which  also  cites  the  rather  conflicting  deductions  of  Neuberg  and  Wolgemuth 
and  of  Hirschstein. 


ORGANIC   PHOSPHORUS  COMBINATIONS.  757 

there  have  been  numerous  investigations,  no  amino-acids  besides  glycocoll  could 
be  detected  in  normal  human  urine,  while,  on  the  contrary,  in  pathological  con- 
ditions other  amino-acids  have  been  found  several  times.  The  amino-acid  frac- 
tion of  the  urine  seems  to  be  increased  in  starvation  and  in  high  altitudes  (  Loewt  ')• 
The  conclusions  of  various  investigators  2  in  regard  to  the  behavior  of  amino- 
acids  in  diseases  such  as  gout,  disagree. 

Non-dialyzable  substances,  the  so-called  adialyzable  bodies,  or  bodies  that 
dialyze  with  difficulty,  also  occur  in  the  urine.  They  consist  in  part  of  chon- 
droit in-sulphuric  acid  whose  daily  amount,  according  to  Pons,  is  0.08-0.09  gram, 
and  also  of  nucleic  acid,  mucoids,  the  colloidal  nitrogenous  bodies  (see  Balkowski, 
page  749)  and  unknown  bodies.  Sasaki  found  0.218-0.68  gram  of  such  bodies 
per  liter  of  normal  urine,  and  Ebbecke  found  1.44  grams  in  men.  In  pregnant 
women  Savare  found  somewhat  higher  results  (0.G  gram  per  liter)  than  in  non- 
pregnant women  (0.4  gram).  The  quantity  is  increased  in  fevers,  in  pneumonia 
(Ebbecke),  in  nephritis,  and  especially  in  eclampsia,  where  Savare  •  indeed  in 
one  ease  found  13.84  grams  per  liter.  The  adialyzable  bodies  occurring  in  eclampsia 
are  toxic. 

Organic  combinations  containing  phosphorus  such  as  glycerophosphoric  acid, 
phosphocarnic  acid  (Rockwood),  etc.,  which  yield  phosphoric  acid  on  fusing  with 
saltpeter  and  caustic  alkali,  are  also  found  in  urine  (Lepine  and  Eymonnet, 
Oertel).  With  a  total  elimination  of  about  2.0  grams  total  P«05,  Oertel  found 
on  an  average  about  0.05  gram  P205  as  phosphorus  in  organic  combination.  Accord- 
ing to  Kondo  the  quantity  of  organic  phosphorus  is  increased  by  taking  phos- 
phatides and  nucleins  but  not  to  the  same  extent  as  the  quantity  of  phosphoric 
acid.  According  to  Sy.m.mers  4  the  organic  combined  phosphorus  ma}-  in  many 
pathological  conditions  be  25-50  per  cent  of  the  total  phosphoric  acid.  In  lym- 
phatic leucaemia,  and  especially  in  degenerative  diseases  of  the  nervous  system, 
the  quantity  may  increase. 

Enzymes  of  various  kinds  have  been  isolated  from  the  urine.  Among  these 
may  be  mentioned  pepsin,  diastolic  enzyme  and  lipase.6 

Mucin.  The  nubecula  consists,  as  shown  by  K.  Morxer,6  of  a  mucoid  which 
contains  12.74  per  cent  N  and  2.3  per  cent  S.  This  mucoid,  which  apparently 
originates  in  the  urinary  passages,  may  pass  to  a  slight  extent  into  solution  in 
the  urine.  In  regard  to  the  nature  of  the  mucins  and  nucleoalbumins  otherwise 
occurring  in  the  urine  we  refer  the  reader  to  the  pathological  constituents  of  the 
urine. 

Ptomaines  and  leucomaines,  or  poisonous  substances  of  an  unknown  kind, 
which  are  often  described  as  alkaloidal  substances,  occur  in  normal  urine,  as  shown 
by  earlier  investigations  (Pouchet,  Bouchard,  Aducco  and  others  7)  and  also 
by  recent  researches  of  Kutscher,  Lohmann  and  Engeland.     The  trimethyl- 


1  Deutsch.  med.  Wochenschr.,  1905;  see  also  Signorelli,  Bioch.  Zeitschr.,  39. 

2  See  Jastrowitz,  Arch.  f.  exp.  Path.  u.  Pharm.,  59;  Walker  Hall,  Bioch.  Journ., 
1;  Brugsch  and  Sehittenhelm,  Zeitschr.  f.  exp.  Path.  u.  Therap.,  4. 

3  Pons,  Hofmeister's  Beitrage,  9;  Sasaki,  ibid.,  9;  Savare,  ibid.,  9  and  11;  Ebbecke, 
Bioch.  Zeitschr.,  13. 

4  Rockwood,  Arch.  f.  (Anat.  u.)  Physiol.,  1895;  Oertel,  Zeitschr.  f.  physiol.  Chem., 
26,  which  cites  the  other  works.  See  also  Keller,  Zeitschr.  f.  physiol.  Chem.,  29; 
Mandel  and  Oertel,  N.  Y.  Univ.  Bull.  Med.  Sciences,  1;  and  Maly's  Jahresber.,  31; 
Symmers,  Journ.  of  Path,  and  Bact,  10. 

5  In  regard  to  the  literature  on  enzymes  in  the  urine,  see  Huppert-Xeubauer,  599. 
In  regard  to  trypsin  in  the  urine  see  Johansson,  Zeitschr.  f.  physiol.  Chem.,  85. 

6  Skand.  Arch,  f .  Physiol,  6. 

7  A  complete  bibliography  on  the  ptomaines  and  leucomaines  of  the  urine  is  found 
in  Huppert-Xeubauer,  403. 


758  URINE. 

amine,  which  originates  from  the  phosphatides,  and  was  first  detected  by  de  Filippi 
and  later  by  K.  Baler,  belong  to  the  leucomanir.es  and  also  the  bases  found  by 
Kutscher  and  by  Kutscher  and  Lohmann,  namely,  methyl  guanidine  (also 
found  by  Achelis),  dimcthylguanidine,  novain  (previously  found  by  Dombrowski), 
reductonoraitt,  C7H17NO2,  gynesin,  C19H23N3O3  (from  female  urine)  mingin,  Ci3Hi8N202, 
vitiatin  (Chapter  X)  and  methylpyridine  chloride,  which  is  not  a  leucomaine,  but 
is  probably  derived  from  smoking  tobacco  or  from  drinking  coffee.  The  imidazole 
derivatives  histidine  and  imidazolamino-acetic  acid  found  by  Kutscher  and 
Engeland  also  belong  to  this  group  and  the  urohypertensin  and  urohypotensin 
of  Abelous  and   Bardier.1 

Under  pathological  conditions  the  quantity  of  leucomaines  and  other  bodies 
may  be  increased  (Bouchard,  Lepine  and  Geurin,  Villiers,  Griffiths,  Albu, 
and  others).  Within  the  last  few  years  the  poisonous  properties  of  urine  have 
been  the  subject  of  more  thorough  investigation,  especialby  by  Bouchard.  He 
found  that  the  night  urine  is  less  poisonous  than  the  day  urine,  and  that  the 
poisonous  constituents  of  the  da}'  and  night  urine  have  not  the  same  action. 
In  order  to  be  able  to  compare  the  toxic  power  of  the  urine  under  different  con- 
ditions, Bouchard  determines  the  urotoxic  coefficient,  which  is  the  weight 
of  rabbit  in  kilos  that  is  killed  by  the  quantity  of  urine  excreted  in  twenty-four 
hours  by  1  kilo  of  the  person  experimented  upon.2 

Many  substances  have  been  observed  in  animal  urine  which  are  not  found  in 
human  urine.  To  these  belong  the  above-described  kynurenic  acid,  urocanic  acid, 
which  according  to  Hunter  is  imidazolacrylic  acid,  also  found  in  dog's  urine; 
damaluric  acid  and  damolic  acid  (according  to  Schotten,3  probably  a  mixture 
of  benzoic  acid  with  volatile  fatty  acids),  obtained  by  the  distillation  of  cow's 
urine;  and  lastly  lithuric  acid,  found  in  the  urinary  concrements  of  certain 
animals. 


HI.     INORGANIC    CONSTITUENTS    OF  URINE. 

Chlorides.  The  chlorine  occurring  in  the  urine  is  undoubtedly  com- 
bined with  the  bases  contained  in  this  excretion;  the  chief  part  is  in 
combination  with  sodium.  In  accordance  with  this,  the  quantity  of 
chlorine  in  the  urine  is  generally  expressed  as  NaCl. 

The  question  as  to  whether  a  part  of  the  chlorine  contained  in  the 
urine  exists  as  organic  combinations,  as  considered  by  Berlioz  and 
Lepinois,  is  still  disputed,  although  recently  Baumgarten  4  has  supported 
this  view. 

The  quantity  of  chlorine  combinations  in  the  urine  is  subject  to  con- 
siderable variation.     In  general  the  amount  from  a  healthy  adult  on  a 


1  de  Filippi,  Zeitschr.  f.  physiol.  Chem.,  49;  Bauer,  Hofmeister's  Beitrage,  11; 
Kutscher,  Zeitschr.  f.  physiol.  Chem.,  51,  with  Lohmann,  ibid.,  48  and  49;  Achelis, 
ibid.,  50;  England,  ibid.,  57,  and  Munch,  med.  Wochenschr.,  55;  Abelous  and  Bardier, 
Maly's  Jahresb.,  39  and  40. 

2  See  footnote  7,  page  757. 

3  Hunter,  Journ.  of  biol.  Chem.,  11;  Schotten,  Zeitschr.  f.  physiol.  Chem.,  7. 

*  Berlioz  and  Lepinois,  see  Chem.  Centralbl.,  1894,  1,  and  1895,  1;  also  Petit  and 
Terrat,  ibid.,  1894,  2,  and  Vitali,  ibid.,  1897,  2;  Ville  and  Moitessier,  Maly's  Jahres- 
ber.,  31;  Meillere,  ibid.;  Bruno,  ibid.,  452;  Baumgarten,  Zeitschr.  f.  exp.  Path.  u. 
Therap.,  5. 


CHLORIDES.  759 

mixed  diet  is  10-1.")  grams  of  NaCl  per  twenty-four  hours.  The  quantity 
of  common  salt  in  the  urine  depends  chiefly  upon  the  amount  of  sail  in  the 
food,  with  which  the  elimination  of  chlorine  increases  and  decreases. 
The  free  drinking  of  water  also  increases  the  elimination  of  chlorine, 
which  is  greater  during  activity  than  during  rest  (at  night).  Certain 
organic  chlorine  combinations,  such  as  chloroform,  may  increase  the 
elimination  of  inorganic  chlorides  by  the  urine  (Zeller,  Kast  1). 

In  diarrhoea,  in  quick  formation  of  large  transudates  and  exudates, 
also  in  specially  marked  cases  of  acute  febrile  diseases  at  the  time  of  the 
crisis,  the  elimination  of  NaCl  is  materially  decreased.  The  excretion 
of  chlorine  may  vary  considerably  in  disease,  but  still  the  NaCl  taken  with 
the  food  has  here,  as  in  physiological  conditions,  a  great  influence  on  the 
NaCl  excretion.2 

The  quantitative  estimation  of  chlorine  in  the  urine  is  most  simply  per- 
formed by  titration  with  silver-nitrate  solution.  The  urine  must  not 
contain  either  proteid  (which  if  present  must  be  removed  by  coagulation) 
or  iodine  or  bromine  compounds. 

In  the  presence  of  bromides  or  iodides  evaporate  a  measured  quantity  of  the 
urine  to  dryness,  fuse  the  residue  with  saltpeter  and  soda,  dissolve  the  fused 
mass  in  water,  and  remove  the  iodine  or  bromine  by  the  addition  of  dilute  sul- 
phuric acid  and  some  nitrite,  and  thoroughly  shake  with  carbon  disulphide. 
The  liquid  thus  obtained  may  now  be  titrated  with  silver  nitrate  according  to 
Volhard's  method.  The  quantity  of  bromide  or  iodide  is  calculated  as  the 
difference  between  the  quantity  of  silver-nitrate  solution  used  for  the  titration 
of  the  solution  of  the  fused  mass  and  the  quantity  used  for  the  coresponding 
volume  of  the  original  urine. 

The  otherwise  excellent  titration  method  of  Mphr,  according  to  which 
we  titrate  with  silver  nitrate  in  neutral  liquids,  using  neutral  potassium 
chromate  as  an  indicator,  cannot  be  used  directly  on  the  urine  in  careful 
work.  Organic  urinary  constituents  are  also  precipitated  by  the  silver 
salt,  and  the  results  are  therefore  somewhat  high  for  the  chlorine.  If 
this  method  is  to  be  employed,  the  organic  urinary  constituents  must  be 
destroyed,  by  incineration  after  the  addition  of  saltpeter  free  from 
chlorine. 

According  to  Bang  and  Larsson  3  the  disturbing  substances  which  react 
with  AgX03  can  be  removed  by  shaking  with  blood-charcoal.  The  value  of  this 
surest  ion  is  essentially  diminished,  because  every  blood-charcoal  cannot  be  used, 
and  therefore  a  special  testing  of  the  blood-charcoal  must  be  done. 


teller.  Zehschr.  f.  physiol.  Chem.,  8;  Kast,  ibid.,  11;  Vitali,  Chem.  Centralbl., 
1899.  2. 

2  On  the  elimination  of  chlorine  in  disease,  see  Albu  and  Neuberg,  Physiol,  u.  Pathol. 
des  MineralsotTwe chsels,  Berlin,  1906. 

3  Bioch.  Zeitschr.,  49. 


760  URINE. 

The  silver-nitrate  solution  may  be  a  N/10  one.  It  is  often  made  of 
such  a  strength  that  each  cubic  centimeter  corresponds  to  0.006  gram 
CI  or  0.01  gram  NaCl.  This  last-mentioned  solution  contains  29.075 
grams  of  AgXC>3  in  1  liter. 

Freund  and  Toepfer,  as  well  as  Bodtker,1  have  suggested  modifica- 
tions of  Mohr's  method. 

Volhard's  Method.  Instead  of  the  preceding  determination,  Vol- 
hard's  method,  which  can  be  performed  directly  on  the  urine,  may  be 
employed.  The  principle  is  as  follows:  All  the  chlorine  from  the  urine 
acidified  with  nitric  acid  is  precipitated  by  an  excess  of  silver  nitrate, 
filtered,  and  in  a  measured  part  of  the  filtrate  the  quantity  of  silver  added 
in  excess  is  determined  by  means  of  a  sulphocyanide  solution.  This 
excess  of  silver  is  completely  precipitated  by  the  sulphocyanide,  and  a 
solution  of  some  ferric  salt,  which,  as  is  well  known,  gives  a  blood-red 
reaction  with  the  smallest  quantity  of  sulphocyanide,  is  used  as  an  indicator. 

We  require  the  following  solutions  for  this  titration:  1.  A  silver- 
nitrate  solution  which  contains  29.075  grams  of  AgNOs  per  liter,  and  of 
which  each  cubic  centimeter  corresponds  to  0.01  gram  NaCl  or  0.00607 
gram  CI.  2.  A  saturated  solution  at  the  ordinary  temperature  of  chlorine- 
free  iron  alum  or  ferric  sulphate.  3.  Chlorine-free  nitric  acid  of  a  specific 
gravity  of  1.2.  4.  A  potassium-sulphocyanide  solution  which  contains 
8.3  grams  KCNS  per  liter,  and  of  which  2  cc.  corresponds  to  1  cc.  of  the 
silver-nitrate  solution. 

About  9  grams  of  potassium  sulphocyanide  are  dissolved  in  water  and  diluted 
to  1  liter.  The  quantity  of  KCNS  contained  in  this  solution  is  determined  by  the 
silver-nitrate  solution  in  the  following  way:  Measure  exactly  10  cc.  of  the  silver 
solution  and  treat  it  with  5  cc.  of  nitric  acid  and  1-2  cc.  of  the  ferric-salt  solu- 
tion and  dilute  with  water  to  about  100  cc.  Now  the  sulphocyanide  solution 
is  added  from  a  burette,  constantly  stirring  until  a  permanent  faint-red  colora- 
tion of  the  liquid  takes  place.  The  quantity  of  sulphocyanide  found  in  the  solu- 
tion by  this  means  indicates  how  much  it  must  be  diluted  to  be  of  the  proper 
strength.  Titrate  once  mere  with  10  cc.  of  AgNOs  solution  and  correct  the  sul- 
phocyanide solution  by  the  careful  addition  of  water  until  20  cc.  exactly  cor- 
responds to  10  cc.  of  the  silver  solution. 

The  determination  of  the  chlorine  in  the  urine  is  performed  by  this 
method  in  the  following  way:  Exactly  10  cc.  of  the  urine  are  placed  in  a 
flask  which  has  a  mark  corresponding  to  100  cc.  and  which  is  provided 
with  a  stopper;  5  cc.  of  nitric  acid  are  added;  dilute  with  about  50  cc. 
of  water  and  then  allow  exactly  20  cc.  of  the  silver-nitrate  solution  to 
flow  in.  Close  the  flask  with  the  stopper  and  shake  well,  remove  the  stop- 
per and  wash  it  with  distilled  water  into  the  flask,  and  fill  the  flask  to  the 
100-cc.  mark  with  distilled  water.  Close  again  with  the  stopper,  care- 
fully mix  by  shaking,  and  filter  through  a  dry  filter.  Measure  off  50 
cc.  of  the  filtrate  by  means  of  a  dry  pipette,  add  3  cc.  of  ferric-salt  solu- 
tion, and  allow  the  sulphocyanide  solution  to  flow  in  until  the  liquid 
above  the  precipitate  has  a  permanent  red  color.  The  calculation  is 
very   simple.     For   example,    if  4.6   cc.   of  the   sulphocyanide   solution 

xFreund  and  Toepfer,  see  Maly's  Jahresber.,  22;  Bodtker,  Zeitschr.  f.  physiol. 
Chem.,  20. 


PHOSPHATES.  761 

was  necessary  to  produce  the  final  reaction,  then  for  100  cc.  of  the  filtrate 
(=10  cc.  urine)  9.2  cc.  of  this  solution  arc  necessary.  9.2  cc.  of  the 
Bulphocyanide  solution  corresponds  to  4.6  cc.  of  the  silver  solution, 
and  since  20—4.6  =  15.4  cc.  of  the  Bilver  solution  was  necessary  to  com- 
pletely precipitate  the  chlorine  in  10  cc.  of  the  urine,  then  10  cc.  con- 
tains 0.154  gram  of  NaCl.  The  quantity  of  sodium  chloride  in  the  urine 
is  therefore  1.54  per  cent,  or  15.4  p.  m.  If  we  always  use  10  cc.  for  the 
determination,  and  always  20  cc.  of  AgNCb  solution,  and  dilute  with 
water  to  100  cc,  the  quantity  of  Na(  '1  in  1000  parts  of  the  urine  is  found 
by  subtracting  from  20  the  number  of  cubic  centimeters  of  sulphocyanide 
(R)  required  with  50  cc.  of  the  filtrate.  The  quantity  of  NaCl  p.  m. 
therefore    under   these    circumstances  =  20  —  R,    and    the    percentage    of 

NaCl  =  ^. 

If  it  is  necessary  to  destroy  the  organic  urinary  constituents  before  titration, 
this  can  be  best  performed,  according  to  Dehn,1  by  evaporating  the  urine  (10  cc.) 
to  dryness  on  the  water-bath  after  the  addition  of  a  small  amount  of  sodium  per- 
oxide, then  faintly  acidifying  with  nitric  acid  and  then  titrating  according  to 
Volhard.     Incineration  is  unnecessary. 

For  the  approximate  estimation  of  chlorine  in  the  urine  Ekehorn  has  made 
use  of  Volhard's  titration  method  by  using  for  the  determination  a  glass  tube 
closed  at  one  end  and  divided  into  half  cubic  centimeters  and  called  the  chlorom- 
eter.  The  reagents  necessary  are:  (a)  a  mixture  of  20  cc.  silver-nitrate  solution 
(according  to  Volhard),  5  cc.  nitric  acid  and  water  to  100  cc;  (b)  40  cc.  sul- 
phocyanide solution  and  60  cc.  of  a  ferric  alum,  chlorine  free  and  saturated  at 
the  ordinary  temperature.  The  silver-nitrate  solution,  of  which  each  cubic 
centimeter  corresponds  to  0.002  gm.  NaCl,  is  equivalent  to  the  iron  sulphocyanide 
solution.  First  2  cc.  of  the  urine  are  placed  in  the  graduated  tube  and  then  0.5 
cc.  sulphocyanide  solution,  and  the  silver-nitrate  solution  gradually  added  (shak- 
ing the  tube  closed  with  a  rubber  stopper)  until  the  coloration  of  the  sulphocyanide 
just  disappears.  0.5  cc.  is  subtracted  from  the  silver  solution  for  the  0.5  cc.  of 
the  sulphocyanide;  the  tube  is  so  graduated  that  the  quantity  of  XaCl  in  the 
urine  in  parts  per  thousand  is  read  off  directly  on  the  tube.  The  difference 
between  these  results  and  those  obtained  by  Volhard's  titration  method  amounts 
only,  according  to  C.  Th.  Mobner,2  to  0.25  to  at  most  0.5  p.  m. 

The  approximate  estimation  of  chlorine  in  the  urine  (which  must  be  free  from 
protein)  is  made  by  strongly  acidifying  with  nitric  acid  and  then  adding  to  it, 
drop  by  drop,  a  concentrated  silver-nitrate  solution  (1:  8).  In  a  normal  quantity 
of  chlorides  the  drop  sinks  to  the  bottom  as  a  rather  compact  cheesy  lump.  In 
diminished  quantity  of  chlorides  the  precipitate  is  less  compact  and  coherent, 
and  in  the  presence  of  very  little  chlorine  a  fine  white  precipitate  or  only  a 
cloudiness  or  opalescence  is  obtained. 

Phosphates.  Phosphoric  acid  occurs  in  acid  urines  partly  as  dihydro- 
gen,  MH2PO4,  and  partly  as  monohydrogen,  M2HPO4,  phosphates, 
both  of  which  may  be  found  in  acid  urines  at  the  same  time.  The 
proportion  of  these  may  vary  eonsiderabby ;  acid  urines  contain  chiefly 
dihydrogen  phosphate  and  in  many  cases  the  urine  seems  to  contain  only 
dihydrogen  phosphate  and  sometimes  indeed  only  a  small  quantity  of  phos- 


1  Zeitschr.  f.  physiol.  Chem.,  44. 

2  Ekehorn,  Hygiea,  Stockholm,  1906;    Morner,  Upsala  Lakaref.  Forh.  (N.  F.),  11. 


762  UKINE. 

phoric  acid.  The  total  quantity  of  phosphoric  acid  varies  and  depends  on 
the  character  and  the  quantity  of  food.  The  average  quantity  of  P2O5 
is  in  round  numbers  2.5  grams,  with  a  variation  of  1-5  grams  per  day. 
A  small  part  of  the  phosphoric  acid  of  the  urine  originates  from  the 
burning  of  organic  compounds,  such  as  nuclein,  and  phosphatides 
within  the  organism;  on  exclusive  feeding  with  substances  rich  in  nuclein 
or  pseudonuclein  the  quantity  of  phosphates  is  essentially  increased; 
still  it  is  undecided  to  what  extent  the  excretion  of  phosphoric  acid  is 
a  measure  of  the  absorption  and  decomposition  of  these  bodies.1  The 
greater  part  originates  from  the  phosphates  of  the  food,  and  the  quan- 
tity of  phosphoric  acid  eliminated  is  greater  when  the  food  is  rich  in 
alkali  phosphates  in  proportion  to  the  quantity  of  lime  and  magnesium 
phosphates.  If  the  food  contains  much  lime  and  magnesia,  large  quan- 
tities of  earthy  phosphates  are  eliminated  by  the  excrement;  and  even 
though  the  food  contains  considerable  amounts  of  phosphoric  acid  in  these 
cases,  the  quantity  excreted  by  the  urine  is  small.  This  is  especially 
true  of  herbivora,  in  which  the  kidneys  are  the  chief  organs  for  the 
excretion  of  alkali  phosphates.  In  man,  according  to  Ehrstrom,  the 
content  of  lime  in  the  food  seems  to  play  no  important  role,  as  in  his  exper- 
iments about  one-half  of  the  phosphoric  acid  taken  as  CaHP04  was 
absorbed,  still  the  extent  of  phosphoric-acid  excretion  through  the  urine 
depends  in  man  not  only  upon  the  total  quantity  of  phosphoric  acid  in 
the  food,  but  also  upon  the  relative  amounts  of  the  alkaline  earths  and  the 
alkali  salts  of  the  food.  In  carnivora,  in  which  phosphate  injected  sub- 
cutaneously  is  eliminated  by  the  intestine  (Bergmann),  the  urine  is 
habitually  poor  in  phosphates.2 

As  the  extent  of  the  elimination  of  phosphoric  acid  is  mostly  dependent 
upon  the  character  of  the  food  and  the  absorption  of  the  phosphates 
in  the  intestine,  it  is  apparent  that  the  relation  between  the  nitrogen  and 
phosphoric-acid  excretion  cannot  run  parallel.  This  is  in  fact  so,  and, 
according  to  Ehrstrom,  the  organism  has  the  power  of  accumulating 
large  quantities  of  phosphorus  for  a  relatively  long  time  independent  of 
the  condition  of  the  nitrogen  balance.  With  a  certain  regular  food  the 
relation  between  nitrogen  and  phosphoric  acid  in  the  urine  can  be  kept 
almost  constant.  Thus  on  feeding  with  an  exclusive  meat  diet,  as 
observed  by  Voit3   in  dogs,   when  the  nitrogen  and  phosphoric  acid 

1  See  A.  Gumlich,  Zeitschr.  f.  physiol.  Chem.,  18;  Roos,  ibid.,  21;  Weintraud, 
Arch.  f.  (Anat.  u.)  Physiol.,  1895;  Milroy  and  Malcolm,  Journ.  of  Physiol.,  23;  Roh- 
mann  and  Steinitz,  Pfluger's  Arch.,  72;  Loewi,  Arch.  f.  exp.  Path.  u.  Pharm.,  44  and  45. 

2  Ehrstrom,  Skand.  Arch.  f.  Physiol.,  14;  Bergmann,  Arch.  f.  exp.  Path.  u.  Pharm., 

47. 

3  Physiologie  des  allgemeinen  Stoffwechsels  und  der  Ernahrung  in  L.  Hermann's 
Handbuch,  (J,  Thill.  1,  7'.). 


PHOSPHATES.  763 

(P2O5)  of  the  food  exactly  reappeared  in  the  urine  and  feces,  the  rela- 
tion was  8.1:1.  In  starvation,  as  shown  by  the  compilation  of  R.  Tiger- 
stedt,1  the  phosphorized  constituents  of  the  body  are  destroyed  to  a 
much  greater  extent  than  when  food  very  poor  in  phosphorus  is  given. 
In  starvation  this  relation  is  changed,  namely,  relatively  more  phosphoric 
acid  is  eliminated,  which  seems  to  indicate  that  besides  flesh  and  related 
tissues  another  tissue  rich  in  phosphorus  is  largely  destroyed.  The 
starvation  experiments  show  that  this  is  the  bone-tissue.  According  to 
Preysz,  Olsavszky,  Klug,  I.  Munk  and  Maillard  2  the  elimination 
of  phosphoric  acid  is  considerably  increased  by  intense  muscular  work. 
As  the  phosphoric  acid  is  in  part  derived  from  the  nucleins,  it  would 
be  expected  that  in  those  diseases  in  which  the  excretion  of  purine 
bodies  was  increased  the  phosphoric  acid  would  also  be  augmented. 
This  is  not  the  case,  and  indeed  we  have  observed  cases  with  an  increased 
elimination  of  purine  bodies  with  a  diminution  in  the  phosphoric-acid 
excretion.  Cases  of  leucaemia  have  been  observed  in  which  the  phos- 
phoric-acid excretion  was  reduced,  although  there  was  a  pronounced 
increase  in  the  number  of  leucocytes.  In  these  cases  there  may  be  a 
subsequent  excretion  or  a  retention  of  phosphoric  acid.  This  last  condition 
also  occurs  in  inflammatory  and  renal  diseases.  The  earthy  phosphates 
of  the  urine  sometimes  have  the  tendency  of  precipitating  either  spon- 
taneously or  after  warming,  and  this  has  been  called  phosphaturia.  We 
are  here  dealing  with  a  diminished  acidity  and,  it  seems,  with  a  dimin- 
ished excretion  of  phosphoric  acid  and  an  increased  elimination  of  lime, 
or  at  least  an  essentially  different  relation  between  the  phosphoric  acid 
and  the  alkaline  earths  of  the  urine,  as  compared  with  the  normal  (Panek 
Iwanoff,  Soetber  and  Krieger3). 

Quantitative  Estimation  of  the  Total  Phosphoric  Acid  in  the  Urine. 
This  estimation  is  most  simply  performed  by  titrating  with  a  solution 
of  uranium  acetate.  The  principle  of  the  titration  is  as  follows:  A 
warm  solution  of  phosphates  containing  free  acetic  acid  gives  a  whitish- 
yellow  precipitate  of  uranium  phosphate  with  a  solution  of  a  uranium 
salt.  This  precipitate  is  insoluble  in  acetic  acid,  but  dissolves  in  mineral 
acids,  and  on  this  account  there  is  always  added,  in  titrating,  a  certain 
quantity  of  sodium-acetate  solution.  Potassium  ferrocyanide  is  used 
as  the  indicator,  which  does  not  act  on  the  uranium-phosphate  precipitate, 
but  gives  a  reddish-brown  precipitate  or  coloration  in  the  presence  of  the 

1  Skand.  Arch.  f.  Physiol.,  16. 

2  Preysz,  see  Maly's  Jahresber.,  21;  Olsavszky  and  Klug,  Pfluger's  Arch.,  54; 
Munk,  Arch.  f.  (Anat.  u.)  Physiol.,  1895;  Maillard,  Journ.  de  Phvsiol.  et  de  Path. 
10  and  11. 

3  Panek,  see  Maly's  Jahresber.,  30,  112;  Iwanoff,  Biochem.  Centralbl.,  1,  710; 
Soetber^and  Krieger,  Deutsch.  Arch.  f.  klin.  Me.!.,  "2;  Cainpaui,  Biochem.  Centralbl., 
3,  616;  Tobler,  Arch.  f.  exp.  Path.  u.  Phann.,  52. 


764  URINE. 

smallest  amount  of  soluble  uranium  salt.  The  solutions  necessary  for 
the  titration  are:  1.  A  solution  of  a  uranium  salt  of  which  each  cubic 
centimeter  corresponds  to  0.005  gram  P2O5  and  which  contains  20.3 
grams  of  uranium  oxide  per  liter.  20  cc.  of  this  solution  corresponds 
to  0.100  gram  P2O5.  2.  A  solution  of  sodium  acetate.  3.  A  freshly 
prepared  solution  of  potassium  ferrocyanide. 

The  uranium  solution  is  prepared  from  uranium  nitrate  or  acetate.  Dissolve 
about  35  grams  uranium  acetate  in  water,  add  some  acetic  acid  to  facilitate  solu- 
tion, and  dilute  to  1  liter.  The  strength  of  this  solution  is  determined  by  titrat- 
ing withasolution  of  sodium  phosphate  of  known  strength  (10.085  grams  crystallized 
salt  in  1  liter,  which  corresponds  to  0.010  gram  P206  in  50  cc).  Proceed  in  the 
same  way  as  in  the  titration  of  the  urine  (see  below),  and  correct  the  solution  by 
diluting  with  water,  and  titrate  again  until  20  cc.  of  the  uranium  solution  cor- 
responds exactly  to  50  cc.  of  the  above  phosphate  solution. 

The  sodium-acetate  solution  should  contain  10  grams  sodium  acetate  and 
10  grams  cone,  acetic  acid  in  100  cc.  For  each  titration  5  cc.  of  this  solution 
is  used  with  50  cc.  of  the  urine. 

In  performing  the  titration,  mix  50  cc.  of  filtered  urine  in  a  beaker 
with  5  cc.  of  the  sodium  acetate,  cover  the  beaker  with  a  watch-glass, 
and  warm  over  the  water-bath.  Then  allow  the  uranium  solution  to 
flow  in  from  a  burette,  and  when  the  precipitate  does  not  seem  to  increase, 
place  a  drop  of  the  mixture  on  a  porcelain  plate  with  a  drop  of  the  potas- 
sium-ferrocyanide  solution.  If  the  amount  of  uranium  solution  added 
has  not  been  sufficient,  the  color  will  remain  pale  yellow  and  more 
uranium  solution  must  be  added;  but  as  soon  as  the  slightest  excess  of 
uranium  solution  has  been  used  the  color  becomes  a  faint  reddish  brown. 
When  this  point  has  been  obtained,  warm  the  solution  again  and  add 
another  drop.  If  the  color  remains  of  the  same  intensity,  the  titration 
is  ended;  but  if  the  color  varies,  add  more  uranium  solution,  drop  by 
drop,  until  a  permanent  coloration  is  obtained  after  warming,  and  now 
repeat  the  test  with  another  50  cc.  of  the  urine.  The  calculation  is  so 
simple  that  it  is  unnecessary  to  give  an  example. 

In  the  above  manner  one  determines  the  total  quantity  of  phosphoric 
acid  in  the  urine.  If  we  wish  to  know  the  phosphoric  acid  combined 
with  alkaline  earths  and  with  alkalies,  we  first  determine  the  total  phos- 
phoric acid  in  a  portion  of  the  urine  and  then  remove  the  earthy  phos- 
phates in  another  portion  by  ammonia.  The  precipitate  is  collected  on 
a  filter,  washed,  transferred  into  a  beaker  with  water,  treated  with  acetic 
acid,  and  dissolved  by  warming.  This  solution  is  now  diluted  to 
50  cc.  with  water,  and  5  cc.  sodium-acetate  solution  added,  then 
titrated  with  uranium  solution.  The  difference  between  the  two  deter- 
minations gives  the  quantity  of  phosphoric  acid  combined  with  the 
alkalies.  The  results  obtained  are  not  quite  accurate,  as  a  partial  trans- 
formation of  the  monophosphates  of  the  alkaline  earths  and  also  calcium 
diphosphate  into  triphosphates  of  the  alkaline  earths  and  ammonium 
phosphate  takes  place  on  precipitating  with  ammonia,  and  the  method 
gives  too  high  results  for  the  phosphoric  acid  combined  with  alkalies  and 
remaining  in  solution. 

Sulphates.  The  sulphuric  acid  of  the  urine  originates  only  to  a  very 
small    extent    from    the    sulphates    of   the   food.     A    disproportionately 


SULPHATES.  765 

greater  part  is  formed  by  the  burning  within  the  body  of  the  proteins 
which  contain  sulphur,  and  it  is  chiefly  this  formation  of  sulphuric  acid 
from  the  proteins  which  gives  rise  to  the  previously  mentioned  excess 
of  acids  over  the  bases  in  the  urine.  The  quantity  of  sulphuric  acid 
eliminated  by  the  urine  amounts  to  about  2.5  grams  H2SO4  per  day. 
As  the  sulphuric  acid  chiefly  originates  from  the  proteins,  it  follows  that 
the  elimination  of  sulphuric  acid  and  the  elimination  of  nitrogen  runs 
almost  parallel,  and  the  relation  Nit^SO*  is  about  5:1.  A  complete 
parallelism  can  hardly  be  expected,  as  in  the  first  place  a  part  of  the  sul- 
phur is  always  eliminated  as  neutral  sulphur,  and  secondly  because  the 
small  proportion  of  sulphur  in  different  protein  bodies  undergoes  greater 
variation  as  compared  with  the  large  proportion  of  nitrogen  contained 
therein.  In  general  the  elimination  of  nitrogen  and  sulphuric  acid  under 
normal  and  under  diseased  conditions  seems  to  run  parallel.  Sulphuric 
acid  occurs  in  the  urine  partly  preformed  (sulphate-sulphuric  acid)  and 
partly  as  ethereal-sulphuric  acid.  The  first  is  designated  as  A-  and  the 
other  as  ^-sulphuric  acid. 

The  quantity  of  total  sulphuric  acid  is  determined  in  the  following 
way,  but  at  the  same  time  the  precautions  described  in  other  works 
must  be  observed.  100  cc.  of  filtered  urine  is  treated  with  5  cc.  of  con- 
centrated hydrochloric  acid  and  boiled  for  fifteen  minutes.  While 
boiling  precipitate  with  2  cc.  of  a  saturated  BaCb  solution,  and  warm  for 
a  little  while  until  the  barium  sulphate  has  completely  settled.  The 
precipitate  must  then  be  washed  with  water  and  also  with  alcohol  and 
ether  (to  remove  resinous  substances),  and  then  treated  according  to 
the  usual  method. 

The  separate  determination  of  the  sulphate-sulphuric  acid  and  the 
ethereal-sulphuric  acid  may  be  accomplished,  according  to  Baumann's 
method,  by  first  precipitating  the  sulphate-sulphuric  acid  by  BaCb  from 
the  urine  acidified  with  acetic  acid,  then  decomposing  the  ethereal- 
sulphuric  acid  by  boiling  after  the  addition  of  hydrochloric  acid,  and 
finally  determining  the  sulphuric  acid  set  free  as  barium  sulphate.  A 
still  better  method  is  the  following,  suggested  by  Salkowski  l : 

200  cc.  of  urine  are  precipitated  by  an  equal  volume  of  a  barium  solu- 
tion, which  consists  of  2  vols,  barium  hydrate  and  1  vol.  barium  chloride 
solution,  both  saturated  at  the  ordinary  temperature.  Filter  through 
a  dry  filter,  measure  off  100  cc.  of  the  filtrate  which  contains  only  the 
ethereal-sulphuric  acid,  treat  with  10  cc.  of  hydrochloric  acid  of  a  specific 
gravity  1.12,  boil  for  fifteen  minutes,  and  then  warm  on  the  water-bath 
until  the  precipitate  has  completely  settled  and  the  supernatant  liquid 
is  entirely  clear.  Filter  and  wash  with  warm  water  and  with  alcohol 
and  ether,  and  proceed  according  to  the  generally  prescribed  method. 
The  difference  between  the  ethereal-sulphuric  acid  found  and  the  total 
quantity  of  sulphuric  acid  as  determined  in  a  special  portion  of  urine 
is  taken  to  be  the  quantity  of  sulphate-sulphuric  acid. 

1  Baumann,  Zeitschr.  £.  physiol.  Chem.,  1;  Salkowski,  Virchow's  Arch.,  79. 


766  URINE. 

Folin  a  has  suggested  a  method  for  estimating  the  sulphate-sul- 
phuric acid  as  well  as  the  ethereal-sulphuric  acid,  and  also  the  totaL 
sulphur,  which  is  somewhat  different  from  the  ordinary  methods. 

Nitrates  occur  in  small  quantities  in  human  urine  (Schonbein),  and  they 
probably  originate  from  the  drinking-water  and  the  food.  According  to  Weyl 
and  Citron,2  the  quantity  of  nitrates  is  smallest  with  a  meat  diet  and  greatest 
with  vegetable  food.     The  average  amount  is  about  42.5  milligrams  per  liter. 

Potassium  and  Sodium.  The  quantity  of  these  bodies  eliminated 
by  the  urine  by  a  healthy  adult  on  a  mixed  diet  is,  according  to  Salkow- 
ski,3  3-4  grams  K2O  and  5-8  grams  Na20,  with  an  average  of  about  2-3 
grams  K2O  and  4-6  grams  Xa20.  The  proportion  of  K  to  Na  is  ordinarily 
3:5.  The  quantity  depends  above  all  upon  the  food.  In  starvation 
the  urine  may  become  richer  in  potassium  than  in  sodium,  which  results 
from  the  lack  of  common  salt  and  the  destruction  of  tissue  rich  in  potas- 
sium. The  quantity  of  potassium  may  be  relatively  increased  during 
fever,  while  after  the  crisis  the  reverse  is  the  case. 

The  quantitative  estimation  of  these  bodies  is  made  by  the  gravi- 
metric methods  as  described  in  works  on  quantitative  analysis.  In 
the  determination  of  the  total  alkalies  new  methods  have  been  devised 
by  Pribram  and  Gregor,  and  for  the  potassium  alone  a  method  by 
Autenrieth  and  Bernheim.4 

Ammonia.  Some  ammonia  is  habitually  found  in  human  urine  and 
in  that  of  carnivora.  The  quantity  in  human  urine  on  a  mixed  diet  is 
an  average  of  0.7  gram,  according  to  Neubauer.  Maillard  5  found 
higher  values  for  soldiers,  namely  1.11  grams.  The  ammonia  nitrogen 
relative  to  the  total  nitrogen  is,  on  a  mixed  diet,  3.6-5.8  per  cent. 

As  above  stated  (page  685),  on  the  formation  of  urea  from  ammonia, 
this  quantity  may  represent  the  small  amount  of  ammonia  which  is 
excluded  from  the  synthesis  to  urea  by  being  combined  with  acids  formed 
in  excess  by  combustion  and  not  united  with  the  fixed  alkalies.  This 
view  is  confirmed  by  the  observation  that  the  elimination  of  ammonia  was 
smaller  on  a  vegetable  diet  and  larger  on  a  rich  meat  diet  than  on  a  mixed 
diet.  After  abundant  meat  feeding  Bouchez  found,  for  example,  1.35- 
1.67  gram  NH?  in  twenty-four  hours.  The  relationship  of  the  ammonia 
elimination  to  the  acid  formation  in  the  animal  body  corresponds  also 
to  the  unquestioned  relation  between  the  hydrochloric  acid  content  of  the 

1  Journ.  of  Biol.  Chem.,  1,  and  Amer.  Journ.  of  Physiol.,  13. 

5  Schonbein,  Journ.  f.  prakt.  Chem.,  92;  Weyl,  Virchow's  Arch.,  96,  with  Citron, 
iUd.,  101. 

'  Ibid.,  53. 

4  Pribram  and  Gregor,  Zeitschr.  f.  analyt.  Chem.,  38;  Autenreith  and  Bernheim, 
Zeitschr.  f.  phyaol.  f'hem.,  37. 

1  Journ.  de  Physiol,  et  de  Path.,  10. 


AMMONIA.  767 

gastric  juice  and  the  ammonia  elimination.  Thus  Hchittenhelm  found 
that  with  a  rise  in  the  hydrochloric  acid  content  the  percentage  of  ammonia 
in  the  urine  was  raised  and  also  the  reverse.  A.  Loeb  and  v  Jammfltoft  l 
have  also  observed  a  fall  in  the  ammonia  elimination  a  few  hours  ufter  a 
meal,  although  no  satisfactory  explanation  of  this  behavior  has  been  given. 
That  ammonia  plays  the  role  of  a  neutralization  medium  for  the  acids 
produced  in  the  body  or  introduced  therein  has  been  shown  by  various 
observations. 

In  man  and  certain  animals  the  elimination  of  ammonia  is  increased 
by  the  introduction  of  mineral  acids;  and,  as  shown  by  Jolin,2  organic 
acids,  such  as  benzoic  acid,  which  are  not  destroyed  in  the  body  act  in  a 
similar  manner.  The  ammonia  set  free  in  the  protein  destruction  is  in 
part  used  in  the  neutralization  of  the  acids  introduced,  and  in  this  way 
a  destructive  removal  of  fixed  alkalies  is  prevented.  ■■■■■  ~^ 

Acids  formed  in  the  destruction  of  proteins  in  the  body  act  on  the 
elimination  of  ammonia  like  those  introduced  from  without.  For  this 
reason  the  quantity  of  ammonia  in  human  urine  is  increased  under  such 
conditions  and  in  such  diseases  where  an  increased  formation  of  acid 
takes  place,  because  of  an  increased  metabolism  of  proteins.  This  is  the 
case  with  a  lack  of  oxygen  in  fevers  and  diabetes.  In  the  last-mentioned 
disease,  organic  acids — /3-oxybutyric  acid  and  acetoacetic  acid — are  pro- 
duced, which  pass  into  the  urine  combined  with  ammonia.3 

The  liver  forms  urea  from  the  ammonia  supplied  to  it  by  the  blood 
and  it  would  therefore  be  expected  that  in  certain  diseases  of  the  liver 
or  with  insufficient  liver  function  that  a  diminished  urea  formation  and  an 
increased  ammonia  elimination  should  take  place.  This  condition  has 
already  been  mentioned  above  (page  685),  and  as  there  remarked  we 
must  consider  whether  the  abnormal  production  of  acid  with  increased 
elimination  of  neutralization  ammonia  is  primary  or  whether  it  is  a 
diminished  synthetic  activity  of  the  liver. 

In  close  relation  to  what  has  been  said  stands  the  question  whether  all 
of  the  ammonia  occurring  in  the  urine  under  normal  conditions  is  to  be 
considered  as  neutralization  ammonia.  If  this  were  so  then  probably 
by  introducing  large  amounts  of  alkali  it  would  be  possible  to  cause  the 
disappearance  of  ammonia  from  the  urine.     In  Stadelmann  and  Beck- 


:  Bouchez,  Journ.  de  Physiol,  et  de  Path.,  14;  Schittenhelm,  Deutsch.  Archiv.  f. 
klin.  Med.,  77;  Adam  Loeb,  Zeitschr.  f.  klin.  Med.,  56,  and  Zeitschr.  f.  Biol.,  55;  Gam- 
meltoft,  Zeitschr.  f.  physiol.  Chem.,  75. 

2  Jolin,  Skand.  Arch.  f.  Physiol.,  1.  In  regard  to  the  behavior  of  ammonium  salts 
in  the  animal  body,  see  Rumpf  and  Kleine,  Zeitschr.  f.  Biologic,  54;  Kowalewski  and 
Markewicz,  Bioch.  Zeitschr.,  4,  and  the  works  cited  on  pages  682,  683. 

s  On  the  elimination  of  ammonia  in  disease,  see  the  works  of  Rumpf,  Virchow's 
Arch.,  143;  Hallervorden,  ibid. 


768  URINE. 

mann's  experiments  this  was  not  possible,  still  in  recent  experiments  of 
Janney  !  it  was  possible,  by  introducing  large  quantities  of  sodium  citrate, 
which  was  burned  in  the  body  into  carbonate,  to  reduce  the  ammonia 
elimination  to  very  insignificant  quantities. 

The  detection  and  quantitative  estimation  of  ammonia  used  to  be  performed 
according  to  the  method  suggested  by  Schlosing.  The  principle  of  this  method 
is  that  the  ammonia  from  a  measured  amount  of  urine  is  set  free  by  lime-water 
in  a  closed  vessel  and  absorbed  by  a  measured  amount  of  N/10  sulphuric  acid. 
After  the  absorption  of  the  ammonia  the  quantity  is  determined  by  titrating  the 
remaining  free  sulphuric  acid  with  a  N/10  caustic-alkali  solution.  This  method 
gives  low  results,  and  in  exact  work  we  must  proceed  as  suggested  by  Bohland.2 

The  recent  methods  for  estimating  the  ammonia  are  all  based  upon 
the  distillation  of  the  ammonia,  after  the  addition  of  lime,  magnesia, 
or  alkali  carbonate,  at  low  temperatures  either  by  the  aid  of  vacuum 
(Nencki  and  Zaleski,  Wurster,  Kruger,  Reich  and  Schittenhelm 
and  Schaffer)  or  by  the  aid  of  a  current  of  air  (Folin)  and  then  collect- 
ing it  in  a  standard  acid. 

According  to  the  methods  suggested  by  Kruger,  Reich  and  Schitten- 
helm 3  25  cc.  of  the  urine  are  placed  in  a  distillation-flask  with  about 
10  grams  of  NaCl  and  1  gram  of  Na2CC>3,  and  this  distilled  at  43°  C. 
and  a  pressure  of  30-40  millimeters  Hg  with  the  aid  of  an  air-pump. 
Alcohol  is  added  to  prevent  foaming.  The  ammonia  is  absorbed  in 
N/10  acid  contained  in  a  Peligot  tube  surrounded  by  ice-water,  and 
when  the  distillation  is  finished  the  acid  is  retitrated,  making  use  of 
rosolic  acid  as  indicator.  In  regard  to  details,  see  the  original  publica- 
tions. Instead  of  alkali  carbonate  a  one-half  normal  solution  of  barium 
hvdrate  in  methyl  alcohol  can  be  used.  According  to  Folin's  4  method, 
25-50  cc.  of  the  urine  are  treated  in  a  wash-bottle  with  1-2  grams  soda  and 
8-10  grams  sodium  chloride  and  some  petroleum,  in  order  to  prevent 
frothing,  and  then  a  current  of  air  is  passed  through  and  this  passed 
through  a  second  wash-bottle  containing  N/10  acid.  It  has  also  been 
suggested  (Ronchese,  Malfatti  and  others)  to  determine  the  ammonia 
by  the  formol  titration.  This  method  is  based  upon  the  fact  that  an 
ammonium  salt  yields  hexamethylentetramine  and  free  acid  with  for- 
maldehyde according  to  the  equation  4NH4Cl-f-6HCOH==C6Hi2N4 
+  6H20+4HC1.  This  acid  is  determined  by  titration  after  the  addition 
of  formol.  Folin  5  also  recently  suggested  a  method  for  the  quantitative 
colorimetric  estimation  of  ammonia  by  the  use  of  Nessler's  reagent. 

Calcium  and  Magnesium  occur  in  the  urine  chiefly  as  phosphates. 
The  quantity  of  earthy  phosphates  eliminated  daily  is  somewhat  more 

1  N.  Janney,  Zeitschr.  f.  physiol.  Chem.,  76,  which  also  contains  the  literature. 

2  Pfliiger's  Arch.,  43,  32. 

3  Zeitschr.  f.  physiol.  Chem.,  39;  Schaffer,  Amer.  Journ.  of  Physiol.,  8,  which  contains 
the  literature.     Henriques  and  S6rensen,  Zeitschr.  f.  physiol.  Chem.,  64. 

•  Folin,  Zeitschr.  f.  physiol.  Chem.,  37,  and  Journ.  of  biol.  Chem.,  8;    Steel,  ibid.,  8. 

6  Ronchese,  see  Maly's  Jahresber.,  38,  321;  Malfatti,  Zeitschr.  f.  anal.  Chem.,  47; 
il.  BjOrn-Anderseii  and  M.  Lauritzen,  Zeitschr.  f.  physiol.  Chem.,  64;  L.  de  Jager, 
ibid.,  62;  Folin  and  Maccallum,  Journ.  of  biol.  Chem.,  11. 


CALCIUM  AND  MAGNESIUM.  7GD 

than  1  gram,  and  of  this  amount  §  la  magnesium  and  ^  calcium  phos- 
phate. This  statement,  as  found  by  Renwall  and  Gross,  is  not  correct, 
or  at  least  is  not  true  in  general,  as  they  found  more  calcium  than  mag- 
nesium in  the  urine.  Long  and  Gephart  j  obtained  similar  results. 
In  acid  urines  the  mono-  as  well  as  the  dihydrogen  earthy  phosphates 
are  found,  and  the  solubility  of  the  first,  among  which  the  calcium  salt 
CaHP04  is  especially  insoluble,  is  particularly  augmented  by  the  presence 
in  the  urine  of  dihydrogen  alkali  phosphates  and  sodium  chloride  (Ott  2) . 
The  quantity  of  alkaline  earths  in  the  urine  depends  on  the  composi- 
tion of  the  food.  The  lime-salts  absorbed  are  in  great  part  excreted 
again  into  the  intestine,  and  the  quantity  of  lime-salts  in  the  urine  is 
therefore  no  measure  of  their  absorption.  The  introduction  of  readily 
soluble  lime-salts  or  the  addition  of  hydrochloric  acid  to  the  food  may 
therefore  cause  an  increase  in  the  quantity  of  lime  in  the  urine,  while 
the  reverse  takes  place  on  adding  alkali  phosphate  to  the  food.  Accord- 
ing to  Granstrom  starvation  in  rabbits  or  the  introduction  of  food  which 
yields  an  acid  ash  and  causes  an  acid  urine  produces  the  same  effect  as 
the  introduction  of  acid.  The  observation  of  de  Jager3  is  significant, 
namely,  he  found  that  the  partaking  of  CaS04  and  to  a  higher  degree 
of  MgSCU  causes  an  increase  in  the  urine  ammonia  and  of  acid.  Noth- 
ing is  known  with  certainty  in  regard  to  the  constant  and  regular  change 
in  the  elimination  of  calcium  and  magnesium  salts  in  disease,  and  in  these 
conditions  the  excretion  is  chiefly  dependent  upon  the  diet  and  the  forma- 
tion and  introduction  of  acid.4 

The  quantity  of  calcium  and  magnesium  is  determined  according  to 
the  ordinary  well-known  methods. 

Iron  occurs  in  the  urine  only  in  small  quantities,  and  it  does  not  exist  as  a 
salt,  but  as  an  organic  combination  of  a  colloidal  nature.  The  earlier  reports  in 
regard  to  the  iron  present  seem  to  show  that  the  quantity  ranges  from  1  to  11 
milligrams  per  liter  of  urine.  Hoffmann,  Neumann  and  Mayer  found  lower 
results — an  average  of  1.09  and  0.983  milligrams  and  according  to  recent  determi- 
nations of  Wolter  and  Reich  8  the  quantity  is  about  1  milligram.  The  quantity 
of  silicic  acid  is  ordinarily  stated  to  amount  to  about  0.3  p.  m.     H.  Schulz  6  found 

1  Renwall,  Skand.  Arch.  f.  Physiol.,  16;  Gross,  Biochem.  Centralbl.,  4,  189;  Long 
and  Gephart,  Journ.  Amer.  Chem.  Soc,  34. 

2  Zeitschr.  f.  physiol.  Chem.,  10. 

s  Granstrom,  Zeitschr.  f.  physiol.  Chem.,  58;  de  Jager,  Bioch.  Zeitschr.,  38. 

4  See  page  758,  Albu  and  Neuberg,  1.  c,  and  E.  Zak,  Ueber  Harn  bei  Lungenent- 
ziindung,  Wien.  klin.  Wochenschr.,  21. 

6  Kunkei,  cited  from  Maly's  Jahresber.,  11;  Giacosa,  ibid.,  16;  Kobert,  Arbeiten 
des  Pharm.  Inst,  zu  Dorpat,  7;  Magnier,  Ber.  d.  deutsch.  chem.  Gesellsch.,  7;  Gott- 
lieb, Arch.  f.  exp.  Path.  u.  Pharm.,  26;  Jolles,  Zeitschr.  f.  anal.  Chem.,  36;  Hoff- 
mann, Zeitschr.  f.  anal.  Chem.,  40;  Neumann  and  Mayer,  Zeitschr.  f.  physiol. 
Chem.,  37;  Wolter,  Bioch.  Zeitschr.,  24;  Reich,  ibid.,  36. 

6  Pfluger's  Arch,,  144. 


770  URINE. 

0.1046  to  0.2594  grains  per  day  on  a  mixed  diet.    Traces  of  hydrogen  peroxide 
also  occur  in  the  urine. 

The  gases  of  the  urine  are  carbon  dioxide,  nitrogen,  and  traces  of 
oxygen.  The  quantity  of  nitrogen  is  not  quite  1  vol.  per  cent.  The 
carbon  dioxide  varies  considerably.  In  acid  urines  it  is  hardly  one-half 
as  great  as  in  neutral  or  alkaline  urines. 

IV.     THE   QUANTITY  AND   QUANTITATIVE   COMPOSITION   OF  URINE. 

The  quantity  and  composition  of  urine  are  liable  to  great  variation. 
The  circumstances  which  under  physiological  conditions  exercise  a  great 
influence  are  the  following:  the  blood-pressure,  and  the  rapidity  of  the 
blood-current  in  the  glomeruli.  The  quantity  of  urinary  constituents, 
especially  water  in  the  blood;  and,  lastly,  the  condition  of  the  secretory 
glandular  elements.  Above  all,  the  quantity  and  concentration  of  the 
urine  depend  on  the  quantity  of  water  which  is  introduced  into  the  blood 
or  which  leaves  the  body  in  other  ways.  The  excretion  of  urine  is  increased 
by  drinking  freely  or  by  reducing  the  quantity  of  water  otherwise  removed; 
and  it  is  decreased  by  a  diminished  ingestion  of  water  or  by  a  greater  loss 
of  water  in  other  ways.  Ordinarily  in  man  just  as  much  water  is  elimi- 
nated by  the  kidneys  as  by  the  skin,  lungs,  and  intestine  together.  At 
lower  temperatures  and  in  moist  air,  since  under  these  conditions  the 
elimination  of  water  by  the  skin  is  diminished,  the  excretion  of  urine 
may  be  considerably  increased.  Diminished  introduction  of  water  or 
increased  elimination  of  water  by  other  means — as  in  violent  diarrhoea  or 
vomiting,  or  in  profuse  perspiration — greatly  diminishes  the  amount  of 
urine  excreted.  For  example,  the  urine  may  sink  as  low  as  500-400  cc. 
per  day  in  intense  summer  heat,  while  after  copious  draughts  of  water 
the  elimination  of  3000  cc.  of  urine  has  been  observed  during  the  same 
time.  The  quantity  of  urine  voided  in  the  course  of  twenty-four  hours 
varies  considerably  from  day  to  day,  the  average  being  ordinarly  cal- 
culated as  1500  cc.  for  healthy  adult  men  and  1200  cc.  for  women.  The 
minimum  elimination  occurs  during  the  early  morning  between  2  and  4 
o'clock;  the  maximum,  in  the  first  hours  after  waking  and  from  1-2 
hours  after  a  meal. 

The  quantity  of  solids  excreted  per  day  is  nearly  constant,  even  though  the 
quantity  of  urine  may  vary,  and  it  is  quite  constant  when  the  manner  of  living 
is  regular.  Therefore  the  percentage  of  solids  in  the  urine  is  naturally  in  inverse 
proportion  to  the  quantity  of  urine.  The  average  amount  of  solids  per  twenty- 
four  hours  is  calculated  as  60  grams.  The  quantity  may  be  calculated  with  approx- 
imate accuracy  from  the  specific  gravity  if  the  second  and  third  decimals  of  this 
factor  be  multiplied  by  Haser's  coefficient,  2.33.  The  product  gives  the  amount 
of  solids  in  1000  cc.  of  urine,  and  if  the  quantity  of  urine  eliminated  in  twenty- 
four  hours  be  measured,  the  quantity  of  solids  in  twenty-four  hours  may  be 
easily  calculated.     For  example,  1050  cc.  of  urine  of  a  specific  gravity  1.021  was 


QUANTITATIVE  COMPOSITION.  771 

eliminated  in  twenty-four  hours;  therefore  the  quantity  of  solids  excreted  was 
21  X 2.33  =48.9  and  iqqq  =  51.35  grams.  Long  >  has  made  a  new  determina- 
tion of  the  coefficient  for  the  specific  gravity  taken  at  25°  C.  and  finds  that  it  is 
equal  to  2.<'>,  which  almost  corresponds  to  Haser's  coefficient  at  15°  C. 

Those  hodics  which,  under  physiological  conditions,  affect  the  density 
of  the  urine  are  common  salt  and  urea.  The  specific  gravity  of  the  first 
is  2.15  and  the  last  only  1.32,  so  it  is  easy  to  understand,  when  the  relative 
proportion  of  these  two  bodies  essentially  deviates  from  the  normal, 
why  the  above  calculation  from  the  specific  gravity  is  not  exact.  The 
same  is  true  when  a  urine  poor  in  normal  constituents  contains  large 
amounts  of  foreign  bodies,  such  as  albumin  or  sugar. 

As  above  stated,  the  percentage  of  solids  in  the  urine  generally  decreases 
with  a  greater  elimination,  and  a  very  considerable  excretion  of  urine 
(polyuria)  has  therefore,  as  a  rule,  a  lower  specific  gravity.  An  important 
exception  to  this  rule  is  observed  in  urine  containing  sugar  (diabetes 
mellitus),  in  which  there  is  a  copious  excretion  with  a  very  high  specific 
gravity  due  to  the  sugar.  In  cases  where  very  little  urine  is  excreted 
(oliguria),  e.g.,  during  profuse  perspiration,  in  diarrhoea,  and  in  fevers, 
the  specific  gravity  of  the  urine  is  as  a  rule  very  high;  the  percentage  of 
solids  is  also  high  and  the  urine  has  a  dark  color.  Sometimes,  as  for 
example,  in  certain  cases  of  albuminuria,  the  urine  may  have  a  lew  specific 
gravity  notwithstanding  the  oliguria,  and  be  poor  in  solids  and  light  in 
color. 

In  certain  cases  it  is  interesting  to  know  the  relation  between  the 
earlwn  and  the  nitrogen,  or  the  quotient  C/N.  This  factor  may  vary 
between  0.6  and  1 ;  as  a  rule,  it  amounts  on  an  average  to  0.87,  but  changes 
according  to  the  nature  of  the  food  and  is  higher  after  a  diet  rich  in  carbo- 
hydrates than  after  food  rich  in  fat  (Pregl,  Tangl,  Langstein  and 
Steinitz).  According  to  Magnus-Alsleben  it  rises  after  body  exer- 
tion, but  in  healthy  individuals  the  variation  is  independent  of  the 
kind  of  food.  In  the  urine  analyses  of  Bouchez  2  a  variation  between 
0.62  and  0.90  was  observed  which  showed  no  regular  relation  to  the  food. 
On  account  of  the  great  variations  which  the  composition  of  the  urine 
shows  it  is  difficult  and  of  little  value  to  give  a  tabular  review  of  the 
composition  of  the  urine.  The  following  table  contains  only  approximate 
values  and  it  must  not  be  overlooked  that  the  results  are  not  given  for 
1000   parts  of  urine,   but  only  approximate  figures  for  the  quantities 


1  Journ.  Amer.  Chem.  Soc,  25. 

2  Pregl,  Pfliiger's  Arch.,  75,  which  contains  the  earlier  literature.  Tangl,  Arch.  f. 
(Anat.  u.)  Physiol.,  1899,  Suppl.;  Langstein  and  Steinitz,  Centralbl.  f.  Physiol.,  19; 
Magnus-Alsleben,  Zeitschr.  f.  klin.  Med.,  68,  Bouchez,  footnote  1,  page  767. 


772  URINE. 

of  the  most  important  constituents  which  are  eliminated  during  the 
course  of  twenty-four  hours  in  a  volume  of  1500  cc.  of  urine.  These 
figures  apply  only  to  a  diet  which  corresponds  to  Voit's  standard  figures, 
namely  118  grams  protein,  56  grams  fat,  and  500  grams  carbohydrate 
per  day,  and  to  a  man  of  average  weight. 

Daily  quantity  of  solids  =  55-70  grams. 

Organic  constituents               35-45  grams.         Inorganic  constituents  20-25  grams. 

Urea 25-35.0  grams.         Sodium  chloride  (NaCl) .  10-15.0  grams. 

Uric  acid 0.7      "              Sulphuric  acid  (H2S04). .  2.5      " 

Creatinine 1.5      "              Phosphoric  acid  (P205) . .  2.5      " 

Hippuric  acid 0.7     "             Potash  (K20) 3.3     " 

Ammonia  (NH3) 0.7      " 


Magnesia  (MgO)    \  n  fi 

Lime  (CaO)  /■•••■  u,a 


Urine  contains  on  an  average  40  p.  m.  solids.  The  quantity  of  urea  is 
about  20  p.  m.,  and  common  salt  about  10  p.  m. 

The  physico-chemical  methods  are  being  used  in  urinary  analysis  even  to  a 
greater  extent  than  in  the  analysis  of  other  animal  fluids.  A  great  number 
of  eryoscopie  determinations,  but  fewer  conduct ivitjr  determinations,  have  been 
made.  A  constant  relation  between  the  values  found  by  physico-chemical  methods 
and  the  analytical  methods  has  been  sought,  for  example,  between  the  freezing- 
point  depression  and  the  specific  gravity  or  the  common  salt  content  and  others;  or 
have  been  made  to  find  certain  constants  in  the  composition  of  the  urine  based 
upon  the  results  of  various  methods,  and  in  this  way  to  obtain  an  explanation 
as  to  the  mechanism  of  the  excretion  of  urine  in  order  to  apply  them  for  diag- 
nostic purposes.  The  results  obtained  are,  as  is  to  be  expected,  so  variable  and 
dependent  upon  so  many  conditions  which  cannot  be  controlled  that  definite 
conclusions  must  be  drawn  with  the  greatest  caution.  In  regard  to  the  value  and 
usefulness  of  the  various  constants  and  relations  which  are  based  upon  theoretical 
considerations,  opinions  are  unfortunately  still  too  divergent  and  as  the  plan  and 
scope  of  this  book  do  not  allow  of  more  detailed  description  of  these  facts  we 
must  refer  to  larger  works  on  the  urine  and  diseases  of  the  kidneys. 

V.  CASUAL  URINARY   CONSTITUENTS. 

The  casual  appearance,  in  the  urine,  of  medicinal  agents  or  of  urinary 
constituents  resulting  from  the  introduction  of  foreign  substances  into 
the  organism  is  of  practical  importance,  because  such  compounds  may 
interfere  in  certain  urinary  investigations;  they  also  afford  a  good  means 
of  determining  whether  certain  substances  have  been  introduced  into  the 
organism  or  not.  From  this  point  of  view  a  few  of  these  bodies  will  be 
spoken  of  in  a  following  section  (on  the  pathological  urinary  constituents) . 
The  presence  of  these  foreign  bodies,  in  the  urine,  is  of  special  interest 
in  those  cases  in  which  they  serve  to  elucidate  the  chemical  transformations 
which  certain  substances  undergo  within  the  organism.  As  inorganic 
substances  generally  leave  the  body  unchanged,1  they  are  of  very  little 


1  In  regard  to  the  behavior  of  certain  of  these  bodies,  see  Heffter,  Die  Ausscheidung 
korperfremden  Substanzen  im  Ham,  Ergebnisse  d.  Physiol.,  2,  Abt.  1. 


CASUAL    URINARY   CONSTITUENTS.  773 

interest  from  this  standpoint;  but  the  changes  which  certain  organic 
substances  undergo  when  introduced  into  the  animal  body  may  be  studied 
by  the  transformation  products  as  found  in  the  urine. 

The  bodies  belonging  to  the  fatty  series  undergo,  though  not  without 
exceptions,  a  combustion  leading  toward  the  final  products  of  metab- 
olism; still,  often  a  greater  or  smaller  part  of  the  bodies  in  question 
escape  oxidation  and  appear  unchanged  in  the  urine.  A  part  of  the  acids 
belonging  to  this  series,  which  are  otherwise  decomposed  into  water  and 
carbonates,  and  render  the  urine  neutral  or  alkaline,  may  act  in  this  manner. 
The  volatile  fatty  acids  poor  in  carbon  are  less  easily  oxidized  than  those 
rich  in  carbon,  and  they  therefore  pass  unchanged  into  the  urine  in 
large  amounts.  This  is  especially  true  of  formic  and  acetic  acids 
(Schotten,  Gr6hant  and  Quinquaud  r).  In  birds,  according  to  Gaglio 
and  Giunti,  oxalic  acid  is  not  oxidized.  Opinions  on  the  behavior  of 
oxalic  acid  in  mammalia  and  man,  are  conflicting;  the  investigations 
of  Salkowski  and  especially  of  Hildebrandt  and  Dakin  2  show 
that  oxalic  acid,  when  introduced  in  medium  amounts,  is  in  part 
oxidized  in  the  animal  body.  Racemic  acid,  d-l  tartaric  acid,  passes 
(in  dogs)  in  part  into  the  urine,  and  this  unburned  part  is  optically  inactive 
according  to  Neuberg  and  Saneyoshi.  The  statement  of  Biron3 
that  Z-tartaric  acid  is  more  readily  burned  than  e?-tartaric  acid  is  accord- 
ingly incorrect,  and  the  cW-tartaric  acid  therefore  does  not  belong  to 
those  substances  which  are  asymmetrically  attacked  in  the  animal 
body.  Malic  acid  and  citric  acid  belong  to  those  acids  which  are  in  great 
part  burned  in  the  body.4 

The  destruction  of  normal  fatty  acids  with  several  membered  chains 
takes  place,  our  belief  being  based  upon  the  work  of  Knoop  and  Dakin  5 
especially,  in  an  oxidation  in  the  0-position,  i.e.,  in  the  group  which  is  in 
the  /3-position  to  the  carboxyl  group  at  the  end.     The  conversion  into  an 


1  Schotten,  Zeitschr.  f.  physiol.  Chem.,  7;  Grehant  and  Quinquaud,  Compt.  Rend., 
104. 

2  Gaglio,  Arch.  f.  exp.  Path.  u.  Pharm.,  22;  Giunti,  Chem.  Centralbl.,  1897,  2; 
Marfori,  Maly's  Jahresber.,  20  and  27;  Pohl,  Arch.  f.  exp.  Path.  u.  Pharm.,  37;  Sal- 
kowski, Berl.  klin.  Wochenschr.,  1900;  Pierallini,  Virchow's  Arch.,  160;  Stradomsky, 
i&td.,163;  Klemperer  and  Tritschler,  Zeitschr.  f.  klin.  Med.,  44;  Hildebrandt,  Zeitschr. 
f.  physiol.  Chem.,  35;  Dakin,  Journ.  of  biol.  Chem.,  3. 

3  Biron,  Zeitachr.  f.  physiol.  Chem.,  25;  Neuberg  and  Saneyoshi,  Bioch.  Zeitschr., 
36.  O 

4  Pohl,  Arch.  f.  exp.  Path.  u.  Pharm.,  37,  which  also  contains  reports  on  the  inter- 
mediary products  formed  in  the  oxidation  of  the  fatty  bodies;  K.  Ohta,  Bioch. 
Zeitschr.,  44. 

5  F.  Knoop,  Hofmeister's  Beitrage,  6  and  Habilit.-Schrift,  Freiburg,  1904;  Dakin, 
Journ.  of  biol.  Chem.,  4,  5,  6  and  9. 


774  URINE. 

acid  having  two  carbon  atoms  less  takes  place  according  to  this  assump- 
tion according  to  the  formula: 

R.CH2.CH2.COOH-*R.CH(OH).CH2.COOHR.CO.CH2.COO±^H-> 

R.COOH. 

The  animal  body  has  therefore  the  ability  to  transform  oxyacids  (alcohol 
acids)  into  keto-acids  by  oxidation  as  well  as  the  reverse,  the  conversion 
of  keto-acids  into  oxyacids,  and  this  behavior,  which  is  indicated  by  the 
above  formula,  makes  it  difficult  to  state  which  products  are  primary 
and  which  are  secondary.  As  example  of  such  a  reversible  process  we 
will  mention  the  following;  the  0-oxybutyric  acid  CH3.CH(OH).CH2COOH 
is  transformed  by  oxidation  into  the  keto-acid,  acetoacetic  acid, 
CH3.CO.CH2COOH,  and  this  latter  by  reduction  is  changed  into  /3- 
oxybutyric  acid.  Both  processes  may  take  place,  as  Friedmann  and 
Maase,  Dakin  and  Wakeman  *  have  shown,  in  the  liver,  and  as  these 
two  so-called  acetone  bodies  have  great  importance  in  diabetes,  they 
may  serve  also  as  an  example  of  the  first  stages  of  a  ^-oxidation  (of 
n.  butyric  acid). 

Most  of  the  investigations  on  the  demolition  of  fatty  acids  have  been 
carried  out  by  Knoop,  Dakin,  Friedmann  and  others  upon  substituted, 
especially  phenyl-substituted  fatty  acids,  and  in  speaking  of  the  behavior 
of  the  cyclic  compounds  we  will  discuss  the  behavior  of  these. 

The  amino-acids  are,  when  large  amounts  are  introduced  into  the 
animal  body,  eliminated  unchanged,  and  even  under  physiological  con- 
ditions traces  of  the  amino-acids  formed  in  the  animal  body  can  pass 
into  the  excretions — glycocoll  in  the  urine  and  serine  in  the  perspiration. 
Otherwise  they  are  as  a  rule  decomposed  and  a  deamidation  takes  place, 
the  ammonia  split  off  serving  for  material  for  the  formation  of  urea. 
The  two  components  of  a  racemic  a-amino-acid  behave  differently  in  that 
the  alien  component  is  burned  with  greater  difficulty  and  less  completely 
than  the  component  occurring  in  the  body  protein,  which  is  burned 
more  readily  and  more  completely. 

In  the  demolition  of  the  a-amino-acids,  fatty  acids,  poorer  in  carbon, 
are  formed;  the  detailed  manner  of  this  demolition  has  been  explained 
in  various  ways. 

According  to  a  long-accepted  view  it  was  believed  that  a  hydrolytic 
splitting  off  of  ammonia  with  the  formation  of  the  corresponding  oxyacid 
(alcohol  acid)  took  place,  according  to  the  formula  R.CHNH2.COOH-f- 
H20  =  R.CH(OH).COOH+NH3,    and    then    a    further    demolition    to 


1  Friedmann  and  Maase,  Bioch.  Zeitschr.,  27;  Dakin  and  Wakeman,  Journ.  of  biol. 
Chem.,  8. 


CASUAL  URINARY   CONSTITUENTS.  775 

R.COOH.  The  appearance  of  lactic  acid  in  the  urine  of  rabbits  after 
feeding  alanine  is  an  example  of  such  deamidation.1  The  possibility 
is  not  excluded  that  in  the  first  place  the  keto-acid,  pyruvic  acid, 
CH3.CO.COOH,  is  formed  from  the  alanine  and  then  the  lactic  acid, 
CH3.CHOH.COOH,  formed  from  this  as  a  secondary  reduction  product. 

In  agreement  with  the  views  of  Xeubauer  2  it  is  now  rather 
generally  conceded  that  the  hydrolytic  deamidation  is  not  as  important 
as  the  oxidative  deamidation,  with  the  formation  of  keto-acids 
R.CH(NH2).COOH+0  =  R.CO.COOH+NH3,  although  this  is  not  the 
only  possibility.  The  proofs  for  the  correctness  of  this  view  have  been 
obtained  essentially  by  experiments  with  aromatic  amino-acids  and 
will  be  given  as  examples  of  such  deamidation. 

Dakin  and  Dundley  3  have  shown  that  all  a-amino-acids  investi- 
gated by  them  can  be  decomposed  under  special  conditions  so  that  they 
to  a  certain  degree  yield  ammonia  and  an  a-keto-aldehyde. 

R.CH.NH2.COOH->R.CO.CHO+NH3. 

Thus,  with  alanine,  and  as  the  reaction  to  all  appearances  is  reversible, 
they  consider  the  relationship  between  alanine  and  lactic  acid  is  as  follows- 

CH3.CH.NH2.COOE^CH3.CO.CHO^CH3CHOH.COOH. 

They  also  found  it  probable,  that  the  a-keto-aldehydes  represent  the 
first  step  in  the  demolition  of  the  a-amino-acids  whereby  the  regular 
demolition  of  these  acids  takes  place  over  the  a-keto-acids  and  not  over 
oxyacids,  which  explains  also  the  formation  of  sugar  from  certain  amino- 
acids  (over  methylglyoxal  as  intermediary  step). 

The  deamidation  after  previous  oxidation  with  the  formation  of 
keto-acids  has  awakened  special  interest  because  recently  in  perfusion 
experiments  on  dog-livers  the  reverse  process,  namely  a  synthesis  of  amino- 
acids  from  keto-acids  (in  part  also  from  oxyacids)  and  ammonia  has  been 
performed  (Knoop,  Embden  and  Schmitz,  Kondo4).  Among  such 
syntheses  we  can  here  call  attention  to  the  synthesis  in  the  dog-liver 
of  alanine,  phenylalanine  and  tyrosine  from  pyruvic  acid  (also  lactic  acid), 
phenylpyruvic  acid  and  p-oxyphenyl  pyruvic  acid,  or  of  a-amino-n-butyric 
acid  from  a-keto-butyrie  acid  (all  as  ammonium  salts). 


1  See  Langstein  and  Neuberg,  Arch.  f.  (Anat.  u.)  Physiol.,  1903.     Suppl.  Bd. 
1  Deutsch.  Arch.  f.  klin.  Med.,  95,  and  Habilit.  Schrift.,  Leipzig,   1908.     See  also 
further  on  in  regard  to  the  literature  on  the  demolition  of  the  aromatic  amino-acids. 

3  Journ.  of  biol.  Chem.,  14. 

4  Knoop,  Zeitschr.   f.   physiol.  Chem.,  67  and  71;    Embden  and  Schmitz,   Bioch. 
Zeitschr.,  29  and  38;  Kondo,  ibid.,  38. 


77G  URINE. 

The  residue  of  the  amino-acids  remaining  after  deamidation  can 
naturally,  according  to  the  rule  governing  the  fatty  acids,  be  burned 
and  in  certain  cases  this  combustion  takes  place  with  the  formation  of 
acetone  bodies  (which  see).  The  fatty  acid  residue  can  also  be  used,  be- 
sides in  the  synthesis  of  amino-acids,  also  in  the  synthesis  of  other 
substances,  and  in  Chapter  VII  the  formation  of  carbohydrates  from 
amino-acids  has  been  mentioned. 

Among  the  amino-acids  the  cystine,  or  better  the  cysteine, 

CH2.  (SH)  .CH  (NH2)  .COOH, 

show  a  special  behavior.  On  oxidation  in  the  SH  group  and  splitting 
cff  of  CO2  (see  page  149)  it  is  transformed  into  a  new  amino-acid,  taurine 
(H2N)CH2.CH2(S020H).  Taurine,. which  when  conjugated  with  cholic  acid 
forms  taurocholic  acid,  occurring  in  the  bile  and  which  is  habitually  decom- 
posed in  the  intestine  or  other  parts  of  the  animal  body,  can  when  intro- 
duced as  such  into  the  human  body,  at  least  in  part,  be  eliminated  in 
the  urine  as  such  or  as  tauro-carbamic  acid,  H2N.CO.NH.C2H4.SO2OH 
(Salkowski  l).  Otherwise  as  end-products  of  the  demolition  of  cystine 
and  taurine  an  increased  elimination  of  urinary  sulphur,  sulphuric  acid  and 
thiosulphate,  have  been  observed  (Blum,  Abderhalden  and  Samuely2). 
The  sulphydryl  group  of  cysteine  also  serves  in  the  formation  of  sulpho- 
cyanide,  which  is  formed  from  the  nitriles,  introduced  into  the  animal 
body,  by  the  HCN  (Lang).  The  loosely  combined  sulphur  of  the  pro- 
teins, according  to  the  observations  of  Pascheles,  in  alkaline  reaction  and 
body  temperature,  can  be  readily  transformed,  with  the  cyan  alkali  into 
sulphocyanide  alkali.  The  alkali  sulphocyanides  when  ingested  are 
almost  quantitatively  eliminated  in  the  urine,  according  to  Pollak.3 

By  substituting  one  of  the  hydrogen  atoms  in  the  NH2  group  of 
normal  a-amino-acids  by  an  alkyl  radical  (methyl)  the  combustion  of  the 
acids  of  the  series  C2  and  C4  is  considerably  retarded  and  almost  entirely 
prevented  in  the  members  of  the  C5  and  C6  series  (Friedmann).4  Sar- 
cosine  (methyl  glycocoll),  (CH3)NH.CH2.COOH,  is  not  readily  burnt,  and 
therefore  passes  in  great  part  unchanged  into  the  urine,  but  perhaps  also 
passes  in  small  part  into  the  corresponding  uramino-acid,  methylhydan- 
toic  acid,   NH2.CO.N(CH3).CH2.COOH    (Schultzen  5),   is  an    example 

1  Ber.  d.  d.  Chem.  Gesellsch.,  6,  and  Virchow's  Arch.,  58. 

'  Bhjm,  Hofmeister's  Beitrage,  5;  Abderhalden  and  Samuely,  Zeitschr.  f.  physiol. 
Chem.,  46. 

'  Lang,  Arch.  f.  exp.  Path.  u.  Pharm.,  34;  Pascheles,  ibid.;  Pollak,  Hofmeister's 
Beitrage,  2. 

4  Hofmeister's  Beitrage,  11. 

6  Ber.  d.  d.  Chem.  Gesellsch.,  5.  See  also  Baumann  and  v.  Mering,  ibid.,  8,  and 
E.  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  4. 


CONJUGATION   WITH  SUL1T1UKIC  AND  GLUCURONIC  ACIDS.     777 

of  this  kind.  Substitution  of  both  hydrogen  atoms  of  th<'  ami  no-group 
by  methyl  groups  seems  to  make  the  demolition  of  the  amino-acids  still 
more  difficult  (Friedmann).  Ordinary  betaine  (trimethyl  glycocoll) 
passes,  according  to  Kohlhauch,1  in  part  unburned  into  the  urine  in 
camivora  as  well  as  herbivora. 

The  combustion  of  the  aliphatic  bodies  can  be  retarded  or  prevented 
also  by  substitutions  of  other  kinds  and  by  combining  with  other  sub- 
stances. 

By  substitution  with  halogens,  bodies  otherwise  readily  oxidizable  are 
converted  into  difficultly  oxidizable  ones.  While  the  aldehydes  are  readily 
and  completely  burnt  like  the  primary  and  secondary  alcohols  of  the 
fatty  series,  the  halogen-substituted  aldehydes  and  alcohols,  are,  on  the 
contrary,  difficultly  oxidizable.  The  halogen-substitution  products  of 
methane  (chloroform,  iodoform,  and  bromoform)  are  at  least  in  part 
destroyed,  and  the  corresponding  alkali  compounds  of  the  halogen  pass 
into  the  urine.2 

By  conjugation  with  sulphuric  acid,  the  alcohols  which  are  otherwise 
readily  oxidizable  may  be  protected  against  combustion,  and  conse- 
quently the  alkali  salt  of  ethyl-sulphuric  acid  is  not  burnt  in  the  body 
(Salkowski3). 

Conjugation  with  other  substances  can  prevent  the  combustion  of 
the  aliphatic  bodies  as  shown  in  the  conjugation  of  glycocoll  with  benzoic 
acid  into  hippuric  acid.  A  conjugation  can  also  be  a  mutual  protection 
against  the  combustion  of  two  bodies  as  in  the  case  of  glucuronic  acid 
and  certain  substances. 

Conjugation  with  glucuronic  acid  occurs,  according  to  the  investiga- 
tions of  Sundvik  and  especially  of  O.  Neubauer,  in  many  substituted 
as  well  as  non-substitued  alcohols,  aldehydes,  and  ketones.  Chloral 
hydrate,  CCl3CH(OH)2,  passes,  after  it  has  been  converted  into  tri- 
chlorethyl-alcohol  by  a  reduction,  into  a  levogyrate  reducing  acid,  uro- 
chloralic  acid  or  trichlorethylglucuronic  acid,  CCI3.CH2.CGH9O7  (Musculus 
and  v.  Mering).  Of  the  primary  alcohols  investigated  by  Neubauer* 
(upon  rabbits  and  dogs)  methyl  alcohol  gave  no  conjugated  glucuronic 
acid,  and  ethyl  alcohol  only  a  small  amount.     Isobutyl  alcohol  and  active 


1  Zeitschr.  f.  Biol.,  57. 

2  See  Harnack  and  Griindler,  Berlin,  klin.  Wochenschr.,  1883;  Zeller,  Zeitschr.  f. 
physiol.  Chem.,  8;  Kast,  ibid.,  11;  Binz,  Arch.  f.  exp.  Path.  u.  Pharm.,  28;  Zeehuisen, 
Maly's  Jahresber.,  23. 

3  Pfliiger's  Arch.,  4. 

4  Sundvik,  Maly's  Jahresber.,  16;  Musculus  and  v.  Mering,  Ber.  d.  deutsch.  chem. 
Gesellsch.,  8;  also  v.  Mering,  ibid.,  15;  Zeitschr.  f.  physiol.  Chem.,  6;  Kulz,  Pfliiger's 
Arch.,  28  and  33;  O.  Neubauer,  Arch.  f.  exp.  Path.  u.  Pharm.,  46;  Saneyoshi,  Bioch. 
Zeitschr.,  36. 


778  UEINE. 

amyl  alcohol  yielded  relatively  large  quantities.  Secondary  alcohols 
produced  conjugated  glucuronic  acids,  and  indeed  to  a  greater  .extent  than 
the  primary  alcohols,  especially  in  rabbits.  The  ketones  are  reduced  in 
part  into  secondary  alcohols  and  are  partly  excreted  as  the  conjugated 
acid.     This  could  be  shown  for  acetone  with  rabbits  but  not  with  dogs. 

The  homo-  and  heterocyclic  compounds  pass,  as  far  as  is  known, 
into  the  urine  as  such,  or  after  a  previous  partial  oxidation  or  synthesis 
with  other  bodies,  and  appear  as  so-called  aromatic  compounds.  This 
applies  at  least  to  foreign  substances  that  are  introduced  into  the  body. 

The  fact  that  benzene  may  be  oxidized  outside  of  the  body  into  carbon 
dioxide,  oxalic  acid,  and  volatile  fatty  acids  has  been  known  for  a  long 
time;  and  as  in  these  cases  a  rupture  of  the  benzene  ring  must  take  place, 
so  also,  it  must  be  admitted,  when  aromatic  substances  undergo  a  com- 
bustion in  the  animal  body,  a  splitting  of  the  benzene  nucleus  with  the 
formation  of  fatty  bodies  must  be  the  result.  If  this  does  not  occur,  then 
the  benzene  nucleus  is  eliminated  with  the  urine  as  an  aromatic  compound 
of  one  kind  or  another.  The  manner  in  which  this  benzene  ring  is  opened 
is  not  known.  Still  Jaff£  l  has  detected  muconic  acid  in  the  urine  of 
dogs  and  rabbits  which  had  been  fed  for  a  long  time  with  benzene,  and 
suggest  one  way  in  which  the  benzene  can  be  split  in  the  animal  body. 

CH  CH 

•\       y\ 

HC     CH    HC      COOH 

— *  .     That  the  demolition  of  the  benzene  nucleus 

HC     CH     HC      COOH 

CH  CH 

occurs  in  certain  cases,  as  in  tyrosine  and  phenylalanine  according  to 
the  present  view,  over  homogentisic  acid,  has  already  been  mentioned. 
The  most  striking  example  of  a  complete  combustion  of  the  benzene 
nucleus  is  given  by  tyrosine,  which  as  previously  mentioned  (page  737) 
can  be  absorbed  even  in  large  quantities  and  decomposed  without  the 
observer  being  able  to  detect  any  of  the  cleavage  products  of  it  in  the  urine. 
Other  examples  of  readily  and  at  least  in  greatest  part  combustible  aroma- 
tic substances  are  phenyl-a-lactic  acid,  p-oxyphenylpyruvic  acid  and  a-amino 
cinnamic  acid.  According  to  Juvalta  and  Porcher  phthalic  acid  is 
also  burnt  in  the  animal  body.  The  last  investigator  found  that  the  three 
phthalic  acids  have  varying  effects,  as  the  o-acid  is  almost  completely 
burnt  in  dogs,  while  about  75  per  cent  of  the  m-  and  p-acids  are  excreted 
unconsumed.  This  corresponds  with  the  rule  found  by  R.  Cohn,2  that 
among    the    di-derivatives   of   benzene   the   ortho-compounds  are   more 


1  Zeitschr.  f.  physiol.  Chem.,  62. 
1  Ibid.,  17. 


OXIDATION  IN  THE  NUCLEUS  AND  SIDE  CHAIN.  779 

readily  destroyed  than  the  corresponding  meta-  and  para-compounds. 
The  claims  of  Juvalta  and  Porcher  are  unfortunately  disputed  by 
Pribram  and  Pohl.1 

An  oxidation  in  the  side  chain  of  aromatic  compounds  is  often  found, 
and  may  also  occur  in  the  nucleus  itself.  As  an  example,  benzene  is  first 
oxidized  to  oxybenzene  (Schultzen  and  Naunyn),  and  this  is  then 
further  in  part  oxidized  into  dioxybenzenes  (Baumann  and  Preusse). 
Naphthalene  appears  to  be  converted  into  oxy naphthalene,  and  probably 
a  part  also  into  dioxynaphthalene  (Lesnik  and  M.  Nencki).  The  hydro- 
carbon with  an  amino-  or  imino-group  may  also  be  oxidized  by  a  sub- 
stitution of  hydroxy!  for  hydrogen,  especially  when  the  formation  of  a 
derivative  in  the  para-position  is  possible  (Klingenberg).  For  example, 
aniline,  C6H5.NH2,  passes  into  paraminophenol,  which  latter  passes  into 
the  urine  as  its  ethereal-sulphuric  acid,  H2N.C6H4.O.SO2.OH  (F.  Muller). 
Acetanilid  is  in  part  converted  into  acetyl  paraminophenol  (Jaffe  and 
Hilbert,  K.  Morner),  and  carbazol  into  oxycarbazol  (Klingenberg).2 

An  oxidation  of  the  side  chain  may  occur  by  the  hydrogen  atoms  being 
replaced  by  hydroxy), or  may  also  take  place  with  the  formation  of  carboxyl; 
thus,  for  example,  toluene,  C6H5.CH3  (Schultzen  and  Naunyn),  ethyl- 
benzene,  CGH5.C2H5,  and  propylbenzene,  C6H5.C3H7  (Nencki  and  Giacosa)3 
besides  many  other  bodies,  are  oxidized  into  benzoic  acid.  Cymene  is 
oxidized  to  cumic  acid,  xylene  to  toluic  acid,  methylpyridine  to  pyridine- 
carboxylic  acid  in  the  same  way. 

If  several  side  chains  are  present  in  the  benzene  nucleus,  then  only  one 
is  always  oxidized  into  carboxyl.  Thus  xylene,  CeH^CHV^,  is  oxidized 
into  toluic  acid,  CeH^CH^OOOH  (Schultzen  and  Naunyn);  mesitylene, 
C6H3(CH3)3,  into  mesitylenic  acid,  C6H3(CH3)2.COOH  (L.  Nencki); 
cymene,  (CH3)2CH.C6H4.CH3,  into  cumic  acid,  (CH3)CH.C6H4.COOH 
(M.  Nencki  and  Ziegler  4). 

If  the  side-chain  has  several  members,  then  the  behavior  is  dif- 
ferent and  in  these  cases  the  demolition  of  aromatic  amino-acids  and 
fatty  acids  is  especially  to  be  considered. 


1  Juvalta,  Zeitschr.  f.  physiol.  Chem.,  13;  Pribram,  Arch.  f.  exp.  Path.  u.  Pharm., 
51;  Porcher,  Bioch.  Zeitschr.,  14;  Pohl,  ibid.,  16. 

2  Schultzen  and  Naunyn,  Arch.  f.  (Anat.  u.)  Physiol.,  1867;  Baumann  and  Preusse, 
Zeitschr.  f.  physiol.  Chem.,  3,  156.  See  also  Nencki  and  Giacosa,  ibid.,  4;  Lesnik  and 
Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  24;  F.  Muller,  Deutsch.  med.  Wochenschr., 
1887;  Jaffe  and  Hilbert,  Zeitschr.  f.  physiol.  Chem.,  12;  Morner,  ibid.,  13;  Klingen- 
berg, "  Studien  liber  die  Oxydation  aromatischer  Substanzen,"  etc.,  Inaug.-Diss., 
Rostock,  1891. 

3  Zeitschr.  f.  physiol.  Chem.,  4. 

4  Nencki,  Arch.  f.  exp.  Path.  u.  Pharm.,  1;  Nencki  and  Ziegler,  Ber.  d.  d.  Chem. 
Gesellsch..  5;  see  also  O.  Jacobsen,  ibid.,  12. 


780  UKINE. 

The  aromatic  amino-acids  are,  like  the  amino-acids  in  general,  decom- 
posed to  fatty  acids  and  have  one  carbon  atom  less.  For  example  phenyl- 
amino-acetic  acid  is  in  part  converted  into  benzoic  acid  (O.  Neubauer)  ; 
o-  and  ra-tyrosine  yield  o-  and  m-oxyphenylacetic  acid  respectively 
(Blum,  Flatow);  p-chlorphenylalanine  passes  according  to  Friedmann 
and  Maase  into  p-chlorphenylacetic  acid,  and  phenyl-a-aminobutyric 
acid  is  changed,  as  Knoop  x  showed,  into  phenylpropionic  acid.  As 
intermediary  steps  in  this  demolition  we  have,  as  in  the  other  amino-acids, 
part  the  hydrolytic  cleavage  of  NH2  groups  and  part  the  demolition  by 
way  of  the  corresponding  keto-acid. 

As  an  example  of  a  demolition  of  the  first  kind  we  have  for  a  long 
time  considered  the  finding  by  Schotten,  of  mandelic  acid 

C6H5.CH(OH).COOH 

in  the  urine  after  feeding  phenylaminoacetic  acid,  C6H5.CH(NH2).COOH. 
According  to  O.  Neubauer  2  the  process  is  nevertheless  of  another  kind, 
namely,  mandelic  acid  is  produced  secondarily  by  reduction  from  the 
intermediarily  formed  keto-acid,  phenylglyoxylic  acid,  C6H5.CO.COOH. 
As  example  of  a  hydrolytic  deamidation  we  will  use  the  production,  as 
first  observed  by  Blendermann,  of  p-oxyphenyl-lactic  acid, 

HO.C6H4.CH2.CH(OH).COOH 

from  tyrosine  (in  rabbits).  This  acid  has  also  been  found  in  the  urine 
by  Schultzen  and  Riess  in  acute  yellow  atrophy  of  the  liver,  and  by 
Baumann  in  phosphorus  poisoning,  although  the  earlier  investigators 
incorrectly  considered  the  acid  as  oxymandelic  acid.  That  this  acid, 
which  was  considered  as  oxymandelic  acid,  is  Z-p-oxyphenyl-lactic  acid 
has  been  proved  by  Ellinger  and  Kotake  and  Fromherz.3 

As  shown  especially  by  the  investigations  of  O.  Neubauer  the  demoli- 
tion of  the  aromatic  amino-acids  passes  ordinarily  by  way  of  the  cor- 
responding keto-acid.  As  stated  above  (page  737)  in  regard  to  the  forma- 
tion of  homogentisic  acid,  the  demolition  of  tyrosine,  according  to  Neu- 
bauer, passes  over  the  p-oxyphenylpyruvic  acid,  HO.CeH4.CH2.CO.COOH. 
According  to  him  phenylamino-acetic  acid  also  yields  phenylglyoxylic  acid; 


1  Neubauer,  Deutsch.  Arch.  f.  klin.  Med.,  95;  L.  Blum,  Arch.f.  exp.  Path.  u.  Pharm., 
59;  Flatow,  Zeitschr.  f.  physiol.  Chem.,  64;  F.  Knoop,  ibid.,  67;  Friedmann  and 
Maase,  Bioch.1,  Zeitschr.,  27. 

2  Schotten,  Zeitschr.  f.  physiol.  Chem.,  8;  O.  Neubauer,  1.  c. 

3  Blendemann,  Zeitschr.  f.  physiol.  Chem.,  6;  Schultzen  and  Riess,  Chem.  Ctotralbl., 
1869;  Baumann,  Zeitschr.  f.  physiol.  Chem.,  6;  Ellinger  and  Kotake,  ibid.,  65;  From- 
herz, ibid.,  70. 


DEMOLITION  OF  AROMATIC  FATTY  ACIDS.  781 

the  m-tyrosine  passes  according  to  Flatow  l  in  part  as  ra-oxyphenyl 
pyruvic  acid  in  the  urine.  The  keto-acids  give  also  the  same  end  products 
as  the  corresponding  amino-acids.  Thus  o-tyrosine,  like  o-oxyphenyl- 
pyruvic  acid,  yields  o-oxyphenylacetic  acid  (Flatow)  as  end  product; 
the  i>-chlnrph<  ni/lalanine  and  the  p-chlorphcnylpijrui>ic  acid  pass  into  the 
p-chlorphenylacetic  acid,  which  is  not  the  case  with  the  oxyacid,  the  p-chlor- 
phenyl-lactic  acid  (Friedmann  and  Maase  2) .  This  last-mentioned  case  is 
an  example  of  the  more  ready  combustibility  of  the  keto-acids  as  com- 
pared to  the  oxyacids.  Another  such  example  is  the  p-oxyphenyl  pyruvic 
acid,  which  is  in  great  part  burned,  while  the  p-oxyphenyl-lactic  acid  is 
hardly  burned  at  all  (Kotake,  Suwa).  A  correspondingly  different 
behavior  is  shown  by  these  two  acids  in  perfusion  experiments  with 
the  excised  liver  of  the  dog.  The  oxyphenylpyruvic  acid,  like  tyrosine, 
shows  itself  to  be  an  acetone  former  while  oxyphenyl-lactic  acid,  on 
the  contrary,  does  not  (Neubauer  and  Gross,  E.  Schmitz3).  The 
ready  combustibility  of  the  keto-acids  indicate  that  these  acids  and 
not  the  oxyacids  are  the  important  intermediary  cleavage  products. 

In  regard  to  the  demolition  of  aromatic  fatty  acids,  Knoop  4  has  found 
that  the  acids  with  even  carbon  chains,  such  as  phenyl  butyric  acid  and 
phenyl  caproic  acid,  are  converted  into  phenylacetic  acid,  which  con- 
jugates with  glycocoll  to  form  phenaceturic  acid,  while  the  acids  with 
uneven  carbon '  chains,  like  phenylpropionic  and  phenylvaleric  acid, 
yield  benzoic  acid,  which  then  is  eliminated  as  hippuric  acid.  This 
behavior  is  in  close  agreement  with  the  generally  accepted  oxidation  of 
fatty  at  the  /3-group,  for  which  Dakin  has  also  given  important  support. 
Thus  Dakin  found  after  feeding  phenylpropionic  acid  to  cats,  that 
phenyl-&-cx])propionic  add,  benzoylacetic  acid  and  acetophenone,  the  latter 
passing  into  benzoic  acid  or  hippuric  acid,  wTere  formed,  which  pre- 
supposes an  oxidation  in  the  /3-position.  According  to  the  investigations 
of  Dakin  and  Friedmann  5  the  conditions  are  still  very  complicated. 
Certain  of  the  processes  are  reversible,  oxidations  as  well  as  reductions 
occur,  and  a-/3-unsaturated  acids  may  also  be  formed  as  intermediary 
products.  Dakin  as  well  as  Friedmann  have  obtained  cinnamic  acid 
as  intermediary  product  in  the  demolition  of  phenylpropionic  acid,  and 


1  Neubauer,  Deutsch.  Arch.  f.  klin.  Med.,  95;  Flatow,  Zeitschr.f.  physio! .  Chem.,  6-4. 
1  Flatow,  1.  c;  Friedmann  and  Masse,  Bioch.  Zeitschr.,  27. 

5  Kotake,  Zeitschr.  f.  physiol.  Chem.,  69;    Suwa,  ibid.,  72;    Neubauer  and  Gross, 
ibid.,  67;  Schmitz.  Bioch.  Zeitschr.,  28. 

4  Hofmeister's  Beitrage,  6,  and  Habilit.-Schrift,  Freiburg,  1904. 

6  Dakin,  Journ.  of  biol.  Chem.,  4,  5,  6,  8,  and  9;    Friedmann,  6ee  Med.  Klinik,. 
No.  28,  1911,  and  Bioch.  Zeitschr.,  35. 


782 


URINE. 


this  is  probably  formed  from  the  phenyl-/3-oxypropionic  acid  by  the  with- 
drawal of  water: 

C6H5.CH  (OH)  .CH2.COOH  -  H20  =  C6H5.CH  :CH.COOH. 

Friedmann  has  also  (in  part  with  Sasaki)1  studied  the  decomposition,  of 
fur  fur  propionic  acid  and  found  that  pyromucic  acid  with  furfuracrylic 
acid  as  intermediary  step,  was  formed: 

C4H3O.CH2CH2COOH-»C4H3O.CH:CH.COOH^C4H30.COOH. 

The  above-mentioned  investigators  are  therefore  of  the  opinion  that  the 
demolition  takes  place  in  part  over  the  a-/3-unsaturated  acids  and  in  part 
over  the  /3-keto-acids  or  /3-alcohol  acids. 

According  to  the  investigations  of  Dakin  and  Friedmann  and  to  the  schematic 
illustration  which  they  give,  we  can  consider  the  demolition  of  phenylpropionic 
acid  as  follows: 


C«H=.CH.,.CH,COOH  (Phenylpropionic  acid) 


(Cinnamicacid)    C,H,..CH:  CH.COOH 


O.HB.CO.    CH0.COOH  (Benzoylaceticacid) 


C6H5.CO  .CH3 

( Acetophenone) 


C6H5.CH  (OH).  CH2.COOH 

•  (Phenyl-£-oxypropionic  acid) 


C6H5.COOH 

(Benzoic  add) 


■C6H6.COOH 

(Benzoic  acid) 


C0H5.CO.  NH.  CH2.COOH 

(Hippuric  acid) 

Reductions  may  also  occur  and  besides  the  examples  of  the  reduction 
of  keto-acids  to  alcohol-acids,  we  will  mention  as  further  examples  the 
conversion,  as  observed  by  E.  Meyer,2  of  nitrobenzene,  C6H5NO2,  or  of. 
nitrophenol,  HO.CfjH4.XO2  into  aminophenol,  HO.C0H4.NH2,  and  also  the 
behavior  of  ?n-nitro-benzaldehyde  in  the  animal  body  as  mentioned  below. 

Syntheses  of  aromatic  substances  with  other  atomic  groups  occur 
frequently.  To  these  syntheses  belongs,  in  the  first  place,  the  conjugation 
of  benzoic  acid  with  glycocoll  to  form  hippuric  acid,  the  discovery  of  which 
is  generally  ascribed  to  Wohler,  but  according  to  Heffter3  more  cor- 


1  Sasaki,  Bioch.  Zeitschr.,  25;  Friedmann,  ibid.,  35. 
*  Zeitschr.  f.  physiol.  Chem.,  40. 

'  Die  Ausscheidung  korperfremder  Substanzen  im  Harn,  Ergebnisse  der  Physiol., 
4,  252. 


SYNTHESES  OF  AROMATIC  SUBSTANCES.  783 

rectly  to  Keller  and  Ure.  All  the  numerous  aromatic  substances  which 
are  converted  into  benzoic  acid  in  the  body  are  voided  partly  as  hippuric 
acid.  This  statement  is  not  true  for  all  species  of  animals.  According 
to  the  observations  of  Jaffe,1  benzoic  acid  does  not  pass  into  hippuric 
acid  in  birds,  but  after  conjugation  with  ornithin,  into  the  correspond- 
ing acid,  ornithuric  acid,  (a-5-dibenzoyldiamino  valeric  acid).  Not  only 
are  the  oxybcnzoic  acids  and  the  substituted  benzoic  acids  conjugated  with 
glycocoll,  forming  corresponding  hippuric  acids,  but  also  the  above- 
mentioned  acids,  toluic,  mesitylenic,  cumic,  and  phenylacetic  acids.  These 
acids  are  voided  as  toluric,  mcsitylcnuric,  cuminuric,  and  phenaceturic 
acids. 

It  must  be  remarked  in  regard  to  the  oxybcnzoic  acids  that  a  conjugation  with 
glycocoll  has  been  shown  only  with  salicylic  and  p-oxybenzoic  acid  (Bertagnim, 
and  others),  while  Baumann  and  Herter  2  find  it  only  very  probable  for  m- 
oxyhenzoic  acid.  According  to  Baldoni,3  in  dogs,  the  salicylic  acid  docs  not  pass 
into  salicyluric  acid,  and  he  indeed  found  two  acids  which  he  calls  ursalicylic 
acid,  CuHuOs  and  uramin-salicylic  acid,  CieHieNOs.  The  oxybcnzoic  acids  are 
also  in  part  eliminated  as  conjugated  sulphuric  acids,  which  is  especially  true  for 
?/;-oxybenzoic  acid.  The  three  aminobenzoic  acids,  according  to  the  experiments 
of  Hildebrandt,  on  rabbits,  appeared  at  least  in  part  unchanged  in  the  urine. 
Salkowski  found,  as  was  later  confirmed  by  R.  Cohn,4  that  in  rabbits  ///-amino- 
benzoic  acid  passes  in  part  into  vmviiiiobenzoic  acid,  H2N.CO.HX.C6H.i.COOH. 
It  is  also  in  part  eliminated  as  aminohippuric  acid. 

The  behavior  of  the  halogen-substituted  compounds  of  toluene  varies  in 
different  animals  according  to  Hildebrandt's  experiments.  In  dogs  they  are 
converted  into  the  corresponding  substituted  hippuric  acid.  In  rabbits  o-brom- 
toluene  is  completely  changed  to  hippuric  acid,  the  m-  and  p-bromtoluene  only 
partly.  The  three  chlortoluenes  are  converted  in  rabbits  into  the  corresponding 
benzoic  acid  and  are  eliminated  as  such  and  not  as  hippuric  acid. 

The  substituted  aldehydes  are  of  special  interest  as  substances  which 
may  undergo  conjugation  with  glycocoll.  According  to  the  investiga- 
tions of  R.  Cohn  5  on  this  subject,  o-nitrobcnzaldeh]/de  when  introduced 
into  a  rabbit  is  only  in  a  very  small  part  converted  into  nitrobenzoic 
acid,  and  the  chief  mass,  about  90  per  cent,  is  destroyed  in  the  body. 
According  to  Sieber  and  Smirnowt  6  m-nitrobenzaldchyde  passes  in  dogs 
into  w-nitrohippuric  acid,  and  according  to  Cohn  into  urea-m-nitro- 
hippurate,  but  in  rabbits  a  different  action  results.  In  this  case  not  only 
does  an  oxidation  of  the  aldehyde  into  benzoic  acid  take  place,  but  the 

1  Ber.  d.  d.  chem.  Gesellsch.,  10  and  11. 

2  Zeitschr.  f.  physiol.  Chem.,  1,  where  Bertagnini's  work  is  also  cited.  See  also- 
Dautzenberg,  Maly's  Jahresber.,  11,  231. 

3  Arch.  f.  exp.  Path.  u.  Pharm.,  1908,  Suppl.  Bd.  (Schmiedeberg's  Festschrift). 

4  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  7;  Cohn,  ibid.,  17;  Hildebrandt,  Hof- 
meister's  Beit  rage,  3. 

6  Zeitschr.  f.  physiol.  Chem.,  17. 
6  Monatshefte  f.  Chem.,  8. 


784  URINE. 

nitro-group  is  also  reduced  to  an  amino-group,  and  finally  acetic  acid 
attaches  itself  to  this  with  the  expulsion  of  water,  so  that  the  final  product 
is  m-acetylaminobenzoic  acid,  (CH3.CO).NH.C6H4COOH.  The  p-nitro- 
benzaldehyde  acts  in  rabbits  in  part  like  the  m-aldehyde  and  passes  in 
part  into  p-acetylaminobenzoic  acid.  Another  part  is  converted  into 
p-nitrobenzoic  acid,  and  the  urine  contains  a  chemical  combination  of 
equal  parts  of  these  two  acids.  According  to  Sieber  and  Smirnow  p- 
nitrobenzaldehyde  yields  only  urea  p-nitrohippurate  in  dogs.  The  above- 
mentioned  pyridine-carboxylic  acid,  formed  from  methylpyridine 
(a-picoline)  passes  into  the  urine  after  conjugation  with  glycocoll  as 
a-pyridinuric  acid.1 

To  those  substances  which  undergo  a  conjugation  with  glycocoll 
belongs  also  furfur ol  (the  aldehyde  of  pyromucic  acid,  C4H3O.CHO), 
which,  when  introduced  into  rabbits  and  dogs,  as  shown  first  by  Jaffe 
and  Cohn,2  and  then  further  shown  by  Sasaki  and  Friedmann,  is  elimi- 
nated in  two  ways  from  the  body.  The  furfurol  can,  by  a  similar  synthesis 
to  Perkin's  reaction,  be  transformed  into  the  unsaturated  Sicidfurfuracrylic 
acid  C4H3O.CH:CH.COOH,  and  also  into  pyromucic  acid  C4H3O.COOH. 
These  two  acids  pass,  after  conjugation  with  glycocoll,  into  the  urine  as 
furfuracryluric  acid  and  pyromucuric  acid.  In  birds  the  pyromucic  acid 
is  conjugated  with  ornithine  and  is  eliminated  as  pyromucinornithuric  acid. 

It  has  not  been  proved  how  thiophene,  C4H4S,  behaves  in  the  animal 
body.  Of  methylthiophene  (thiotolene),  C4H3S.CH3,  a  very  small  part  is 
oxidized  to  thiophenic  acid,  C4H3S.COOH  (Levy).  This  acid,  as  shown 
by  Jaffe  and  Levy,3  is  conjugated  with  glycocoll  in  the  body  (rabbits) 
and  eliminated  as  thiophenuric  acid. 

Another  very  important  synthesis  of  aromatic  substances  is  that  of 
the  ethereal-sulphuric  acids.  Phenols,  and  in  particular  the  hydroxylated 
aromatic  hydrocarbons  and  their  derivatives  are  voided  as  ethereal-sul- 
phuric acids,  according  to  Baumann,  Herter  and  others.4 

A  conjugation  of  aromatic  acids  with  sulphuric  acid  occurs  less  often. 
The  two  previously  mentioned  aromatic  acids,  p-oxyphenylacetic  and 
p-oxyphenylpropionic  acid,  are  in  part  eliminated  in  this  form.  Gentisic 
acid  (hydroquinone-carboxylic  acid)  also  increases,  according  to  Lik- 
hatscheff,5  the  quantity  of  ethereal-sulphuric  acid  in  the  urine,  while 

1  In  regard  to  the  extensive  literature  on  glycocoll  conjugations  we  refer  the  reader 
to  O.  Kuhling,  Ueber  Stoffwechselprodukte  aromatischer  Korper.  Inaug.-Diss., 
Berlin,  1887. 

2  Ber.  d.d.  Chem.  Gesellsch.,  20  and  21;  Sasaki  and  Friedmann,  footnote  1,  page  782. 

3  Levy,  Ueber  das  Verhalten  einiger  Thiophenderivate,  etc.,  Inaug.-Diss.,  Konigs- 
berg,  1889;  Jaffe  and  Levy,  Ber.  d.  d.  chem.  Gesellsch.,  21. 

*  In  regard  to  the  literature,  see  O.  Kuhling,  1.  c. 
6  Zeitschr.  f.  physiol.  Chem.,  21. 


CONJUGATION  OF  AROMATIC  SUBSTANCES.  785 

Rost  asserts,  contrary  to  earlier  claims,  that  the  same  occurs  with  gallic 
acid  (trioxybenzoic  acid)  and  tannic  acid.1 

Although  Nencki  and  Rekowski  2  have  shown  that  acetophenone  (phen- 
ylmethylketone),  CeH5.CO.CH3,  is  oxidized  to  benzoic  acid  and  eliminated 
as  hippuric  acid,  the  aromatic  oxyketones  with  hydroxyl  groups,  such  as 
resacetophenone,  2,  4  dioxacetophenone  (HO)2.CcH3.CO.CH3,  pass  into 
the  urine  as  ethereal-sulphuric  acids  and  in  part  after  conjugation  with 
glucuronic  acid.     Euxanthon,  which  is  also  an  aromatic  ketone,  namely 

CO 
dioxyxanthon,  HO.CoH3<  >CeH3.0H,  passes  into  the  urine  as  eux- 

xcr 

anthic  acid  after  conjugation  with  glucuronic  acid. 

A  conjugation  of  other  aromatic  substances  with  glucuronic  acid, 
which  last  is  protected  from  combustion,  occurs  rather  often.  The  phenols, 
as  above  stated  (page  725),  pass  in  part  as  conjugated  glucuronic  acids 
into  the  urine.  The  same  is  true  for  the  homologues  of  the  phenols,  for 
certain  substituted  phenols,  and  for  many  aromatic  substances,  also 
hydrocarbons  after  previous  oxidation  and  hydration.  Thus  Hilde- 
brandt  and  Fromm  and  Clemens  3  have  shown  that  the  ter penes  and  cam- 
phors, by  oxidation  or  hydration,  or  in  certain  cases  by  both,  are  converted 
into  hydroxyl  derivatives  when  the  body  in  question  is  not  previously 
hydroxylized,  and  that  these  hydroxyl  derivatives  are  eliminated  as  con- 
jugated glucuronic  acids.  Conjugated  glucuronic  acids  are  detected  in 
the  urine  after  the  introduction  of  various  substances  into  the  organsim, 
e.g.,  therapeutic  agents,  such  as  ter penes,  borneol,  menthol,  camphor  (cam- 
phoglucuronic  acid  was  first  observed  by  Schmiedeberg),  naphthalene, 
oil  of  turpentine,  oxyquinolines,  antipyrine,  and  many  other  bodies.4 
Orthonitrotoluene  in  dogs  passes  first  into  o-nitrobenzyl  alcohol  and  then 


1  In  regard  to  the  behavior  of  gallic  and  tannic  acids  in  the  animal  body,  see  C. 
Morner,  Zeitschr.  f.  physiol.  Chem.,  16,  which  also  contains  the  earlier  literature;  also 
Harnack,  ibid.,  24,  and  Rost,  Arch.  f.  exp.  Path.  u.  Pharm.,  38,  and  Sitzungsber.  d. 
Gesellsch.  zur  Beford.  d.  ges.  Naturwiss.  zu  Marburg,  1898. 

2  Arch.  d.  scienc.  biol.  de  St.  Petersbourg,  3,  and  Ber.  d.  deutsch.  chem. 
Gesellsch.,  27. 

3  Hildebrandt,  Arch.  f.  exp.  Path.  u.  Pharm.,  45,  46;  Zeitschr.  f.  physiol.  Chem., 
36;  with  Fromm,  ibid.,  33;  and  with  Clemens,  ibid.,  37;  Fromm  and  Clemens,  ibid., 
34.  Extensive  investigations  on  the  behavior  of  alicylic  compounds  with  the  glucuronic 
acid  conjugation  in  the  organism  have  been  carried  out  by  Hiimalainen,  Skand.  Arch, 
f.  Physiol.,  27. 

4  See  O.  Kiihling,  1.  c,  which  gives  the  literature  up  to  1887;  also  E.  Kiilz,  Zeitschr. 
f.  Biologie,  27;  the  works  of  Hildebrandt,  Fromm  and  Clemens,  see  footnote  3; 
Brahm,  Zeitschr.  f.  physiol.  Chem.,  28;  Fenyvessy,  ibid.,  30;  Bonanni,  Hofmeister's 
Beitrage,  1;  Lawrow,  Ber.  d.  d.  chem.  Gesellsch.,  33. 


786  URINE. 

Into  a  conjugated  glucuronic  acid,  uronitrotoluolic  acid  (JaffIs1).  The 
glucuronic  acid  split  off  from  this  conjugated  acid  is  levogyrate  and  hence 
is  not  identical,  but  only  isomeric,  with  the  ordinary  glucuronic  acid. 
Diindhylaminobcnzaldehyde,  according  to  Jaff£,  is  converted  in  part 
into  dimethylaminobenzoglucuronic  acid  in  rabbits.  The  same  conjugated 
glucuronic  acid  is  also  produced,  according  to  Hildebrandt,2  from  p- 
dimcthijltoluidine,  which  is  first  changed  into  p-dimethylaminobenzoic 
acid.  Indol  and  skatol  seem,  as  above  stated  (page  731),  to  be  eliminated 
in  the  urine  partly  as  conjugated  glucuronic  acids.  The  mercapturic 
acids,  which  will  be  mentioned  below,  also  belong  to  those  substances 
which  are  conjugated  with  glucuronic  acid,  and  after  this  conjugation 
appear  in  the  urine. 

A  conjugation  of  carbamic  acid,  NH2COOH,  with  amino-acids  to  form 
wamino-acids,  R.CH.NH.(CONH2)COOH,  or  their  anhydrides,  the 
hydantoins,  have  also  been  observed  in  several  cases,  as  after  feeding 
sarcosin,  amino-benzoic  acid,  phenylalanine,  taurine,  tyrosine.  It  must 
be  remarked  that  according  to  Lippich  and  Dakin,3  the  uramino-acids 
can  be  easily  produced  as  transformation  products  from  the  urea  in  the 
concentration  of  the  urine  by  the  aid  of  heat. 

Syntheses  with  a  simultaneous  acetylation  are  of  great  interest. 
Such  a  synthesis  is  the  formation  of  the  mercapturic  acids.  These  acids, 
which  are  produced  in  the  body  of  dogs  after  the  introduction  of  brom- 
and  chlorbenzene  (Baumann  and  Preusse,  Jaff£,  Friedmann  4)  are 
acetylated  derivatives  of  the  protein  cystine,  and  the  acetylated  brom- 
phenylcysteine  is  CH2.S(C6H4Br).CH.NH(COCH3).COOH.  Another 
example  of  a  synthesis  with  acetylation  is  the  phenylaminoacetic  acid, 
which,  as  Neubauer  and  Warburg  5  have  shown,  in  perfusion  exper- 
iments with  dog's  livers,  gives  among  other  products  also  acetylated 
phenylaminoacetic  acid,  C6H5.CHNH(COCH3).COOH. 

The  synthesis  of  amino-acids,  with  simultaneous  acetylation,  as 
recently  observed  by  Knoop,  are  of  specially  great  interest.  After  the 
introduction  of  7-phenyl-a-keto  butyric  acid  into  the  body  of  a  dog, 
the  formation  of  the  corresponding  acetylated  amino-acid, 

C6H5.CH2.CH2.CHNH(COCH3).COOH 


1  Zeitsehr.  f.  physiol.  Chem.,  2. 

2  JafiY',  Zeitsehr.  f.  physiol.  Chem.,  43;  Hildebrandt,  Hofmeister's  Beitrage,  7. 

3  Lippich,  Ber.  d.  d.  chem.  Gesellsch.,  41;  Dakin,  Journ.  of  biol.  Chem.,  8;  Weiland, 
Bioch.  Zeitsehr.,  38. 

4  Baumann  and  Preusse,  Zeitsehr.  f.  physiol.  Chem.,  5;    Jaff6,  Ber.  d.  deutsch. 
chem.  Gesellsch.,  12;  Friedmann,  Hofmeister's  Beitrage,  4. 

6  Neubauer  and  0.  Warburg,  Zeitsehr.  f.  physiol.  Chem.,  70. 


METHYLATION.  787 

was  observed.  In  perfusion  experiments  with  dog  livers,  Embden  and 
Schmitz  l  have  shown  the  formation  of  phenylalanine,  tyrosine  and  other 
amino-aeids,  as  has  already  been  mentioned  (pages  530  and  775)  by 
synthesis  from  the  ammonium  salts  of  the  corresponding  keto-arids  and 
also  in  part  from  the  oxy-acids. 

Mdhylation  also  often  occurs,  and  as  an  example  we  will  mention  that 
His  has  shown  that  'pyridine,  C5H5N,  is  transformed  in  dogs  into  methyl- 
pyridine,  and  then  passes  into  the  urine  as  methyl pyridylammonium 
hydroxide.  Pyridine  behaves  similarly  in  hens  (Hoshiai),  pigs  and  goats, 
(Totani  and  Hoshiai)  while  according  to  Abderhalden  2  and  collabora- 
tors, in  rabbits  it  passes  unchanged  into  the  urine.  Further  examples 
of  methylation,  although  not  aromatic  substances,  are  the  conversion 
of  guanidine  acetic  acid  into  creatine  (Jaffe)  and  the  observation  of 
Takeda  3  of  the  appearance  of  aminobutyrobetaine  in  the  urine  of  dogs 
with  phosphorus  poisoning. 

Several  alkaloids,  such  as  quinine,  morphine,  and  strychnine,  may  pass  into 
the  urine.  After  the  ingestion  of  turpentine,  balsam  of  copaiba,  and  resins,  these 
may  appear  in  the  urine  as  resin  acids.  Different  kinds  of  coloring-matters,  such 
as  alizarin,  crysophanic  acid,  after  rhubarb  or  senna,  and  the  coloring-matter  of 
the  blueberry,  etc.,  may  also  pass  into  the  urine.  After  rhubarb,  senna,  or  santonin 
the  urine  assumes  a  yellow  or  greenish-yellow  color,  which  is  transformed  into 
a  beautiful  red  by  the  addition  of  alkali.  Phenol  produces,  as  above  mentioned, 
a  dark-brown  or  dark-green  color  which  depends  mainly  on  the  decomposition 
products  of  hydroquinone  and  humin  substances.  After  naphthalene  the  urine 
has  a  dark  color,  and  several  other  medicinal  agents  produce  a  special  coloration. 
Thus  after  antipyrine  it  becomes  yellow  or  blood-red.  After  balsam  of  copaiba 
the  urine  becomes,  when  strongly  acidified  with  hydrochloric  acid,  gradually 
rose-  and  purple-red.  After  naphthalene  or  naphthol  the  urine  gives  with  con- 
centrated sulphuric  acid  (1  cc.  of  concentrated  acid  and  a  few  drops  of  urine) 
a  beautiful  emerald-green  color,  which  is  probably  due  to  naphthol-glucuronic 
acid.  Odoriferous  bodies  also  pass  into  the  urine.  After  asparagus  the  urine 
acquires  a  digusting  odor  which  is  probably  due  to  methylmercaptan.  After 
turpentine  the  urine  may  have  a  peculiar  odor  similar  to  that  of  violets. 

VI.     PATHOLOGICAL   CONSTITUENTS    OF   URINE. 

Proteid.  The  appearance  of  slight  traces  of  proteid  in  normal  urine 
has  been  observed  by  many  investigators,  such  as  Posner  Pl6sz,  v. 
Noorden,  Leube,  and  others.  According  to  K.  Morner  4  proteid 
regularly  occurs  as  a  normal  urinary  constituent  to  the  extent  of  22-78 

1  Knoop,  Zeitschr.  f.  physiol.  Chem.,  67;  Embden  and  Schmitz,  Bioch.  Zeitschr.,  29 
and  38. 

8  His,  Arch.  f.  exp.  Path.  u.  Pharm.,  22;  Cohn,  Zeitschr.  f.  physiol.  Chem.,  18; 
Hoshiai,  ibid.,  62;  with  Totani,  ibid.,  68;  Abderhalden  and  collaborators,  ibil..  .V.» 
and  62. 

5  Jaff<5,  Zeitschr.  f.  physiol.  Chem.,  48;  Takeda,  Pfluger's  Arch.,  133. 

*  Skand.  Arch.  f.  Physiol.,  6  (literature). 


788  URINE. 

milligrams  per  liter.  Frequently  traces  of  a  substance  similar  to  a 
nucleoalbumin,  which  is  easily  mistaken  for  mucin,  and  whose  nature 
will  be  treated  of  later,  appears  in  the  urine.  In  diseased  conditions 
proteid  occurs  in  the  urine  in  a  variety  of  cases.  The  albuminous  bodies 
which  most  often  occur  are  serglobulin  and  seralbumin.  Proteoses  (or 
peptones)  are  also  sometimes  present.  The  quantity  of  proteid  in  the 
urine  is  in  most  cases  less  than  5  p.m.,  rarely  10  p.  m.,  and  only  very  rarely 
does  it  amount  to  50  p.  m.  or  over.  Cases  are  known,  however,  where  it 
was  even  more  than  80  p.  m. 

Among  the  many  reactions  proposed  for  the  detection  of  proteid  in 
urine,  the  following  are  to  be  recommended: 

The  Heat  Test.  Filter  the  urine  and  test  its  reaction.  An  acid 
urine  may,  as  a  rule,  be  boiled  without  further  treatment,  and  only  in 
especially  acid  urines  is  it  necessary  to  first  treat  with  a  little  alkali. 
An  alkaline  urine  is  made  neutral  or  faintly  acid  before  heating.  If  the 
urine  is  poor  in  salts,  add  1/10  vol.  of  a  saturated  common-salt  solution 
before  boiling;  then  heat  to  the  boiling-point,  and  if  no  precipitation, 
cloudiness,  or  opalescence  appears,  the  urine  in  question  contains  no 
coagulable  proteid,  but  it  may  contain  proteoses  or  peptones.  If  a  pre- 
cipitate is  produced  on  boiling,  this  may  consist  of  proteid,  or  of  earthy 
phosphates,1  or  of  both.  The  monohydrogen  calcium  phosphate  decom- 
poses on  boiling,  and  the  normal  phosphate  may  separate  out.  The 
proper  amount  of  acid  is  now  added  to  the  urine,  so  as  to  prevent  any 
mistake  caused  by  the  presence  of  earthy  phosphates,  and  to  give  a  better 
and  more  flocculent  precipitate  of  the  proteid.  If  acetic  acid  is  used 
for  this,  then  add  1-3  drops  of  a  25  per  cent  acid  to  each  10  cc.  of  the 
urine  and  boil  after  the  addition  of  each  drop.  On  using  nitric  acid, 
add  1-2  drops  of  the  25  per  cent  acid  to  each  cubic  centimeter  of  the 
boiling-hot  urine. 

On  using  acetic  acid,  when  the  quantity  of  proteid  is  very  small, 
and  especially  when  the  urine  was  originally  alkaline,  the  proteid  may 
sometimes  remain  in  solution  on  the  addition  of  the  above  quantity  of 
acid.  If,  on  the  contrary,  less  acid  is  added,  the  precipitate  of  calcium 
phosphate,  which  forms  in  amphoteric  or  faintly  acid  urines,  is  liable 
not  to  dissolve  completely,  and  this  may  cause  it  to  be  mistaken  for  a 
proteid  precipitate.  If  nitric  acid  is  used  for  the  heat  test,  the  fact  must 
not  be  overlooked  that  after  the  addition  of  only  a  little  acid  a  combina- 
tion between  it  and  the  proteid  is  formed  which  is  soluble  on  boiling  and 
which  is  only  precipitated  by  an  excess  of  the  acid.  On  this  account  the 
large  quantity  of  nitric  acid,  as  suggested  above,  must  be  added,  but  in 
this  case  a  small  part  of  the  proteid  is  liable  to  be  dissolved  by  the  excess 
of  the  nitric  acid.  When  the  acid  is  added  after  boiling,  which  is  absolutely 
necessary,  the  liability  of  a  mistake  is  not  so  great.  It  is  on  these  grounds 
that  the  heat  test,  although  it  gives  very  good  results  in  the  hands  of 
experts,  is  not  recommended  to  physicians  as  a  positive  test  for  proteid. 

1  In  regard  to  the  cause  of  the  phosphate  precipitation  on  boiling  the  urine,  see 
Malfatti,  Hofrneister's  Beitriige,  8. 


PROTEIDS   IN  THE  URINE.  789 

A  confounding  with  mucin,  when  this  body  occurs  in  the  urine,  is 
easily  prevented  in  the  heat  test  with  acetic  acid  by  acidifying  another 
portion  with  acetic  acid  at  the  ordinary  temperature.  Mucin  and 
nucleoalbumin  substances  similar  to  mucin  arc  hereby  precipitated.  If 
in  the  performance  of  the  heat  and  nitric-acid  test,  a  precipitate  first 
appears  on  cooling  or  is  strikingly  increased,  then  this  -hows  the  presence 
of  proteoses  in  the  urine,  either  alone  or  mixed  with  coagulable  proteid. 
In  this  case  a  further  investigation  is  necessary  (see  below).  In  a  urine 
rich  in  urates  a  precipitate  consisting  of  uric  acid  separates  on  cooling. 
This  precipitate  is  colored  and  granular,  and  is  hardly  to  be  mistaken 
for  a  proteose  or  proteid  precipitate. 

Heller's  test  is  performed  as  follows  (see  page  99):  The  urine  is 
very  carefully  floated  on  the  surface  of  nitric  acid  in  a  test-tube,  or  the 
urine  is  placed  in  a  test-tube  and  then  the  acid  is  slowly  added  by  means 
of  a  funnel,  drawn  out  to  a  point,  and  extending  to  the  bottom.  In  the 
presence  of  albumin  a  white  disk,  or  as  we  ordinarily  say  a  white  ring  or 
at  least  a  sharply  defined  cloudiness,  appears  at  the  point  of  contact  of 
the  two  fluids.  With  this  test  a  red  or  reddish-violet  transparent  ring 
is  always  obtained  with  normal  urine;  it  depends  upon  the  indigo  color- 
ing-matters and  can  hardly  be  mistaken  for  the  white  or  whitish  proteid 
ring.  In  a  urine  rich  in  urates,  another  complication  may  occur,  due 
to  the  formation  of  a  ring  produced  by  the  precipitation  of  uric  acid. 
The  uric-acid  ring  does  not  lie,  like  the  proteid  ring,  between  the  two 
liquids,  but  somewhat  higher.  For  this  reason  two  simultaneous  rings 
may  exist  in  urines  which  are  rich  in  urates  and  do  not  contain  very  much 
proteid.  The  disturbance  caused  by  uric  acid  is  easily  prevented  by 
diluting  the  urine  with  1-2  vols,  of  water  before  performing  the  test. 
The  uric  acid  now  remains  in  solution,  and  the  delicacy  of  Heller's 
test  is  so  great  that  after  dilution  only  in  the  presence  of  insignificant 
traces  of  proteid  does  this  test  give  negative  results.  In  a  urine  very 
rich  in  urea  a  ring-like  separation  of  urea  nitrate  may  also  appear.  This 
ring  consists  of  shining  crystals,  and  it  does  not  appear  in  urine  previously 
diluted.  A  confusion  with  resinous  acids,  which  also  give  a  whitish 
ring  with  this  test,  is  easily  prevented,  since  these  acids  are  soluble  on  the 
addition  of  ether.  Stir,  add  ether,  and  carefully  shake  the  contents  of 
the  test-tube.  If  the  cloudiness  is  due  to  resinous  acids,  the  urine  gradually 
becomes  clear,  and  on  evaporating  the  ether  a  sticky  residue  of  resinous 
acids  is  obtained.  A  liquid  which  contains  true  mucin  does  not  give 
a  precipitate  with  this  test,  but  it  gives  a  more  or  less  strongly  opalescent 
ring,  which  disappears  on  stirring.  The  liquid  does  not  contain  any 
precipitate  after  stirring,  but  is  somewhat  opalescent.  If  a  faint,  not 
wholly  typical  reaction  is  obtained  with  Heller's  test  after  some  time 
with  undiluted  urine,  while  the  diluted  urine  gives  a  pronounced  reaction, 
the  presence  is  shown  of  the  substance  which  used  to  be  called  mucin 
or  nucloealbumin.  In  this  case  proceed  as  described  below  for  the  detec- 
tion of  nucleoalbumin. 

If  the  above-mentioned  possible  errors  and  the  means  by  which  they 
may  be  prevented  are  borne  in  mind,  there  is  hardly  another  test  for 
proteid  in  the  urine  which  is  at  the  same  time  so  easily  performed,  so 
delicate,  and  so  positive  as  Heller's.  With  this  test  even  0.002  per 
cent  of  albumin  may  be  detected  without  difficulty.  »>till  the  student 
must  not  be  satisfied  with  this  test  alone,  but  should  apply  at  least  a 


790  URINE. 

second  one,  such  as  the  heat  test.  In  performing  this  test  the  (primary) 
proteoses  are  also  precipitated. 

The  reaction  with  meta phosphoric  acid  is  very  convenient  and  easily 
performed.  It  is  not  quite  so  delicate  and  positive  as  Heller's  test. 
The  proteoses  are  also  precipitated  by  this  reagent. 

Reaction  with  Acetic  Acid  and  Potassium  Ferrocyanide.  Treat  the 
urine  first  with  acetic  acid  until  it  contains  about  2  per  cent,  and  then 
add  drop  by  drop  a  potassium-ferrocyanide  solution  (1:20),  carefully 
avoiding  an  excess.  This  test  is  very  good,  and  in  the  hands  of  experts 
it  is  even  more  delicate  than  Heller's.  In  the  presence  of  a  very  small 
quantity  of  proteid  it  requires  more  practice  and  dexterity  than  Hel- 
ler's, as  the  relative  quantities  of  reagent,  proteid,  and  acetic  acid  influence 
the  result  of  the  test.  The  quantity  of  salts  in  the  urine  likewise  seems 
to  have  an  influence.     This  reagent  also  precipitates  proteoses. 

Spiegler's  Test.  Spiegler  recommends  a  solution  of  8  parts  mercuric 
chloride,  4  parts  tartaric  acid,  20  parts  glycerin,  and  200  parts  water  as  a  very 
delicate  reagent  for  proteid  in  the  urine.  A  test-tube  is  half  filled  with  this 
reagent  and  the  urine  is  allowed  to  flow  upon  its  surface  drop  by  drop  from  a 
pipette  along  the  wall  of  the  test-tube.  In  the  presence  of  proteid  a  white  ring  is 
obtained  at  the  point  of  contact  between  the  two  liquids.  The  delicacy  of  this 
test  is  1 :350,000.  Jolles  *  does  not  consider  this  reagent  suited  for  urines  very 
poor  in  chlorine,  and  for  this  reason  he  has  changed  it  as  follows:  10  grams  mer- 
curic chloride,  20  grams  succinic  acid,  10  grams  NaCl,  and  500  cc.  water. 

Reaction  with  sulphosalicylic  acid.  Treat  the  urine  either  with  a  20  per  cent 
watery  solution  of  sulphosalicylic  acid  or  a  few  crystals  of  the  acid.  This  reagent 
does  not  precipitate  the  uric  acid  or  the  resin  acids.     (Roch's2  test.) 

As  every  normal  urine  contains  traces  of  proteid,  it  is  apparent  that 
very  delicate  reagents  are  to  be  used  only  with  the  greatest  caution.  For 
ordinary  cases  Heller's  test  is  sufficiently  delicate.  If  no  reaction  is 
obtained  with  this  test  within  2\  to  3  minutes,  the  .urine  tested  contains 
less  than  0.003  per  cent  of  proteid,  and  is  to  be  considered  free  from  pro- 
teid in  the  ordinary  sense. 

The  use  of  precipitating  reagents  presumes  that  the  urine  to  be  investi- 
gated is  perfectly  clear,  especially  in  the  presence  of  only  very  little 
proteid.  The  urine  must  first  be  filtered.  This  is  not  easily  done  with 
urine  containing  bacteria,  but  a  clear  urine  may  be  obtained,  as  suggested 
by  A.  Jolles,  by  shaking  the  urine  with  infusorial  earth.  Although 
a  little  proteid  is  retained  in  this  procedure  and  lost,  it  does  not  seem  to 
be  of  any  importance  (Grutzner,  Schweissinger3). 

The  different  color  reactions  cannot  be  directly  used,  esspecially  in  deep-colored 
urines  which  contain  only  little  proteid.  The  common  salt  of  the  urine  has  a 
disturbing  action  on  Millon's  reagent.     To  prove  mora  positively  the  presence 

1  Spiegler,  Wien.  klin.  Wochenschr.,  1892,  and  Centralbl.  f.  d.  klin.  Med.,  1893; 
Jolles,  Zeitschr.  f.  physiol.  Chem.,  21. 

2  Pharmaceut.  Centralbl.,  1889,  and  Zeitschr.  f.  anal.  Chem.,  29. 

*  Jolles,  Zeitschr.  f.  anai.  Chem.,  29;  Grutzner,  Chem.  Centralbl.,  1901,  1; 
Schweissinger,  ibid. 


PROTEIDS  IN  THE   URINE.  791 

of  protein,  the  precipitate1  obtained  in  the  boiling  test  may  be  filtered,  washed, 
and  then  tested  with  MlLLON's  reaction.  The  precipitate  may  also  be  dissolved 
in  dilute  alkali  and  the  biurel  teal  applied  to  the  solution.  The  presence  of  pro- 
teoses or  peptones  in  the  urine  is  directly  tested  for  by  this  last-mentioned  test. 

In  testing  the  urine  for  proteid  one  should  never  be  satisfied  with  one 
reaction  alone,  but  must  apply  the  heat  test  and  Heller's,  or  the  pctas- 
sium-ferrocyanide  test.  In  using  the  heat  test  alone  the  proteoses  may 
be  easily  overlooked,  but  these  are  detected,  on  the  contrary,  by  Heller's 
or  the  potassium-ferrocyanide  test.  If  only  one  of  these  tests  is  employed, 
no  sufficient  intimation  of  the  kind  of  proteid  present  can  be  obtained, 
whether  it  consists  of  proteoses  or  coagulable  proteid. 

For  practical  purposes  several  dry  reagents  for  proteid  have  been  recommended. 
Besides  the  metaphosphoric  acid  may  be  mentioned  Stutz's  or  Furbringer's 
gelatin  capsules,  which  contain  mercuric  chloride,  sodium  chloride,  and  citric 
acid;  and  Geissler's  albumin-test  papers,  which  consist  of  strips  of  filter-paper 
some  of  which  have  been  dipped  in  a  solution  of  citric  acid,  and  some  into  a 
solution  of  mercuric-cliloride  and  potassium-iodide  solution,  and  then  dried. 

If  the  presence  of  proteid  has  been  positively  proved  in  the  urine  by 
the  above  tests,  it  then  remains  necessary  to  determine  its  character. 

The  Detection  of  Globulin  and  Albumin.  In  detecting  serglobulin 
the  urine  is  exactly  neutralized,  filtered,  and  treated  with  magnesium 
sulphate  in  substance  until  it  is  completely  saturated  at  the  ordinary 
temperature,  or  with  an  equal  volume  of  a  saturated  neutral  solution  of 
ammonium  sulphate.  In  both  cases  a  white,  flocculent  precipitate  is 
formed  in  the  presence  of  globulin.  In  using  ammonium  sulphate  with 
a  urine  rich  in  urates,  a  precipitate  consisting  of  ammonium  urate  may 
separate.  This  precipitate  does  not  appear  immediately,  but  only  after 
a  certain  time,  and  it  must  not  be  mistaken  for  the  globulin  precipitate. 
In  detecting  seralbumin  heat  the  filtrate  from  the  globulin  precipitate 
to  boiling-point,  or  add  about  1  per  cent  acetic  acid  to  it  at  the  ordinary 
temperature. 

For  the  detection  and  also  for  the  quantitative  estimation  of  the  various 
globulins  (fibringlobulin,  euglobulin,  and  pseudoglobulin)  Oswald  1  has  pro- 
posed the  fractional  precipitation  with  ammonium  sulphate. 

Proteoses  and  peptones  have  been  repeatedly  found  in  the  urine  in 
different  diseases.  Reliable  reports  are  at  hand  on  the  occurrence  of 
proteoses  in  the  urine.  The  statements  in  regard  to  the  occurrence  of 
peptones  date  from  a  time  when  the  conception  of  proteoses  and  pep- 
tones was  different  from  that  of  the  present  day,  and  in  part  they  are 
based  upon  investigations  using  untrustworthy  methods.  According 
to  Ito  2  true  peptones  are  sometimes  found  in  the  urine  in  cases  of  pneu- 


1  Munch,  med.  Wochenschr.,  1904.  See  also  Zak  and  Necker,  Deutsch.  Arch.  f. 
klin.  Med.,  88. 

2  In  regard  to  the  literature  on  proteoses  and  peptones  in  urine,  see  Huppert- 
Neubauer,  Ham- Analyse,  10.  Aufl.,  466  to  492;  also  A.  Stoffregen,  Ueber  das  Vorkom- 
men  von  Pepton  im  Harn,  Sputum,  und  Eiter  (Inaug.-Diss.,  Dorpat,  1891);  E.  Hirsch- 
feldt,  Ein  Beitrage  «ur  Frage  der  Peptonurie  (Inaug.-Diss.,  Dorpat,  1892);  and  espe- 


792  URINE. 

monia;  what  has  been  designated  as  urine  peptones  seems  to  have  been 
chiefly  deuteroproteoses. 

In  detecting  the  proteoses,  the  proteid-free  urine,  or  urine  boiled  with  addi- 
tion of  acetic  acid,  is  saturated  with  ammonium  sulphate,  which  precipitates  the  pro- 
teoses. Several  errors  are  here  possible.  The  urobilin,which  may  give  a  reaction 
similar  to  the  biuret  reaction,  is  also  precipitated  and  may  lead  to  mistakes  (Sal- 
kowski,  Stokvis  l).  The  following  modification  by  Bang  and  Devoto's2  method 
can  be  used  to  advantage:  The  urine  is  heated  to  boiling  with  ammonium  sul- 
phate (8  parts  to  10  parts  urine)  and  boiled  for  a  few  seconds.  The  hot  liquid 
is  centrifuged  for  \  to  1  minute  and  separated  from  the  sediment.  The  urobilin 
is  removed  from  this  by  extraction  with  alcohol.  The  residue  is  suspended  in 
a  little  water,  heated  to  boiling,  filtered,  whereby  the  coagulable  proteid  is  retained 
on  the  filter,  and  any  urobilin  still  present  in  the  filtrate  is  shaken  out  with  chloro- 
form. The  watery  solution,  after  removal  of  the  chloroform,  is  used  for  the  biuret 
test.     For  clinical  purposes  this  method  is  very  serviceable. 

According  to  Salkowski  the  urine  treated  with  10-per  cent  hydrochloric 
acid  is  precipitated  with  phosphotungstic  acid,  then  warmed,  the  liquid  decanted 
from  the  resin-like  precipitate,  this  washed  with  water,  and  then  dissolved  in  a 
little  water  with  the  aid  of  some  caustic  soda,  warmed  again  until  the  blue  color 
disappears,  cooled,  and  finally  tested  with  copper  sulphate.  This  method  has 
been  somewhat  modified  by  v.  Aldor  and  Cerny.3  In  regard  to  other  more 
complicated  methods  we  refer  to  Huppert-Neubauer. 

Morawitz  and  Dietschy  4  first  remove  the  proteid  from  the  urine  made 
faintly  acid  with  acid  potassium  phosphate  by  the  addition  of  double  the  volume 
of  96-per  cent  alcohol  and  warming  on  the  water-bath  for  several  hours.  \  From 
the  concentrated  filtrate  acidified  with  a  little  sulphuric  acid  the  proteoses  can 
be  precipitated  by  saturating  with  zinc  sulphate.  After  the  removal  of  the  urobilin 
by  alcohol  and  extracting  with  water,  the  biuret  test  may  be  applied. 

If  the  proteoses  have  been  precipitated  from  a  larger  portion  of  urine  by 
ammonium  sulphate,  this  precipitate  is  tested  for  the  presence  of  different  pro- 
teoses for  the  reasons  given  in  Chapter  II.  The  following  serves  as  a  preliminary 
determination  of  the  character  of  the  proteoses  present  in  the  urine.  If  the  urine 
contains  only  deuteroproteose  it  does  not  become  cloudy  on  boiling,  does  not  give 
Heller's  test,  does  not  become  cloudy  on  saturating  with  NaCl.in  neutral  reaction, 
but  does  become  turbid  on  adding  acetic  acid  saturated  with  this  salt.  In  the 
presence  of  only  protoproteose  the  urine  gives  Heller's  test,  is  precipitated 
even  in  neutral  solution  on  saturating  with  NaCl,  but  does  not  coagulate  on  boil- 
ing. The  presence  of  heteroproteose  is  shown  by  the  urine  behaving  like  the 
above  with  NaCl  and  nitric  acid,  but  shows  a  difference  on  heating.  It  gradually 
becomes  cloudy  on  warming  and  separates  at  about  60°  C.  a  sticky  precipitate 
which  attaches  itself  to  the  sides  of  the  vessel  and  which  dissolves  at  boiling  tem- 
perature on  acidifying  the  urine ;  the  precipitate  reappears  on  cooling. 

In  close  relation  to  the  proteoses  stands  the  so-called  Bence-Jones 
proteid,  which  occurs  in  the  urine  in  rare  cases  in  diseases  with  changes 

daily  Stadelmann,  Untersuchungen  liber  die  Peptonurie,  Wiesbaden,  1894;  Ehrstrom, 
Bidrag  till  kannedomen  om  Albumosurien,  Helsingfors,  1900;  Ito,  Deutsch.  Arch, 
f.  klin.  Med.,  71. 

1  Salkowski,  Berlin,  klin.  Wochenschr.,  1897;  Stokvis,  Zeitschr.  f.  Biologie,  34. 

1  Devoto,  Zeitschr.  f.  physiol.  Chem.,  15;  Bang,  Deutsch.  med.  Wochenschr.,  1898. 

3  Salkowski,  Centralbl.  f.  d.  med.  Wissensch.,  1894;  v.  Aldor,  Berl.  klin.  Wochenschr., 
36;  Cerny,  Zeitschr.  f.  analyt.  Chem.,  40. 

*  Arch.  f.  exp.  Path.  u.  Pharrn.,  54. 


QUANTITATIVE  ESTIMATION  OF  PROTEID  IN   URINE.         793 

in  the  spinal  marrow.  It  gives  a  precipitate  on  heating  to  40-60°  C, 
which  on  further  heating  to  boiling  dissolves  again  more  or  less  completely, 
depending  upon  the  reaction  and  upon  the  amount  of  salt  present.  In 
salt-free  solution  the  precipitate  is  not  dissolved,  on  heating  to  boiling, 
at  least  not  always.  It  does  not  separate  on  dialysis,  but  can  be  pre- 
cipitated from  the  urine  by  double  the  volume  of  a  saturated  ammonium- 
sulphate  solution  or  by  alcohol.  It  has  also  been  obtained  as  crystals 
(Grutterink  and  de  Graaff,  Magnus-Levy  2).  This  body  shows  a 
varying  behavior  in  the  different  cases  in  which  it  has  been  found  and  its 
nature  has  not  been  explained.  From  the  investigations  of  the  above- 
mentioned  and  other  experimenters  (Moitessier,  Abderhalden  and 
Rostoski)  we  can  draw  the  conclusion  that  this  proteid  is  similar  to  the 
proteoses  in  several  reactions,  but  that  nevertheless  it  stands  close  to  the 
genuine  protein  bodies.  It  also  yields  primary  as  well  as  secondary 
proteoses  on  peptic  digestion  (Grutterink  and  de  Graaff),  and  yields 
the  same  hydrolytic  cleavage  products  as  the  other  proteins  (Abderhalden 
and  Rostoski). 

Quantitative  Estimation  of  Proteid  in  Urine.  Of  all  the  methods  pro- 
posed thus  far,  the  coagulation  method  (boiling  with  the  addition  of 
acetic  acid)  when  performed  with  sufficient  care  gives  the  best  results. 
The  average  error  need  never  amount  to  more  than  0.01  per  cent,  and  it 
is  generallv  smaller.  With  this  method  it  is  best  to  first  find  how  much 
acetic  acid  must  be  added  to  a  small  portion  of  the  urine,  which  has  been 
previously  heated  on  the  water-bath,  to  completely  separate  the  pro- 
teid so  that  the  filtrate  will  not  respond  to  Heller's  test.  Then  coagulate 
20-50-100  cc.  of  the  urine.  Pour  the  urine  into  a  beaker  and  heat  on 
the  water-bath,  add  the  required  quantity  of  acetic  acid  slowly,  stirring 
constantly,  and  heat  at  the  same  time,  Filter  while  warm,  wash  first 
with  water,  then  with  alcohol  and  ether,  dry  and  weigh,  incinerate  and 
weigh  again.  In  exact  determinations  the  filtrate  must  not  give  Hel- 
ler's test. 

The  separate  estimation  of  globulins  and  albumins  is  done  by  carefully 
neutralizing  the  urine  and  precipitating  with  MgS04  added  to  saturation  (Hammar- 
stex),  or  simply  by  adding  an  equal  volume  of  a  saturated  neutral  solution  of 
ammonium  sulphate  (HoVmeister  and  Pohl2).  The  precipitate  consisting  of 
globulin  is  thoroughly  washed  with  a  saturated  magnesium-sulphate  or  half- 
saturated  ammonium-sulphate  solution,  dried  continuously  at  110°  C,  boiled 
with  water,  extracted  with  alcohol  and  ether,  then  dried,  weighed,  incinerated, 
and  weighed  again.  The  quantity  of  albumin  is  calculated  as  the  difference 
between  the  quantity  of  globulin  and  the  total  proteids. 

Approximate  Estimation  of  Proteid  in  Urine.  Of  the  methods  suggested  for 
this  purpose  none  has  been  more  extensively  employed  than  Esbach's. 


'Magnus-Levy,  Zeitschr.  f.  physiol.  Chem.,  30  (literature);  Grutterink  and  de 
Graaff,  ibid.,  34  and  36;  Moitessier,  Compt.  rend.  soc.  biolog.,  57;  Ville  and  Derrien, 
ibid.,  62;  Abderhalden  and  Rostoski,  Zeitschr.  f.  physiol  Chem.,  46;  see  also  Hopkins 
and  Savory,  Journ.  of  Physiol.,  42. 

2  Hammarsten,  Pfliiger's  Arch.,  17;  Hofmeister  and  Pohl,  Arch.  f.  exp.  Path.  u. 
Pharm.,  20. 


794  URINE. 

Esbach's  l  Method.  The  acidified  urine  (with  acetic  acid)  is  poured  into  a 
specially  graduated  tube  to  a  certain  mark,  and  then  the  reagent  (a  2-per  cent 
citric-acid  and  1  per  cent  picric-acid  solution  in  water)  is  added  to  a  second  mark, 
the  tube  closed  with  a  rubber  stopper  and  carefully  shaken,  avoiding  the  pro- 
duction of  froth.  The  tube  is  allowed  to  stand  twenty-four  hours,  and  then  the 
height  of  the  precipitate  on  the  graduation  is  read  off.  The  reading  gives  directly 
the  quantity  of  proteid  in  1000  parts  of  the  urine.  Urines  rich  in  proteid  must 
first  be  diluted  with  water.  The  results  obtained  by  this  method,  are,  however, 
dependent  upon  the  temperature;  and  a  difference  in  temperature  of  5°  to  6.5° 
C.  may  cause  an  error  of  0.2-0.3  per  cent  deficiency  or  excess  in  urines  containing 
a  medium  quantity  of  proteid  (Christensen  and  Mygge).  The  method  sug- 
gested by  Tsuschija  2  seems  to  be  more  reliable,  and  consists  in  precipitating  the 
proteid  by  an  alcoholic  solution  of  phosphotungstic  acid  containing  hydrochloric 
acid. 

Other  methods  for  the  approximate  estimation  of  proteid  are  the  optical 
methods  of  Christensen  and  Myg'ge,  and  of  Walbum,3  of  Roberts  and  Stollni- 
kow  as  modified  by  Brandberg,  with  Heller's  test,  which  has  been  simplified 
for  practical  purposes  by  Mittelbach.  The  density  methods  of  Lang,  Huppert 
and  Zahor  are  also  very  good.  In  regard  to  these  and  other  methods  we  refer  to 
Huppert-Xeubauer's  Harn-Analyse,  10.  Aufl. 

There  is  at  present  no  trustworthy  method  for  the  quantitative  estimation 
of  proteoses  and  peptone  in  the  urine. 

Nucleoalbumin  and  Mucin.  According  to  K.  Morner  traces  of  urinary 
mucoids  may  pass  into  solution  in  the  urine;  otherwise  normal  urine  con- 
tains no  mucin.  There  is  no  doubt  that  there  may  be  cases  where  true 
mucin  appears  in  the  urine;  in  most  cases  mucin  has  probably  been  mis- 
taken for  so-called  nucleoalbumin.  The  occurrence,  under  some  circum- 
stances, of  nucleoalbumin  in  the  urine  is  not  to  be  denied,  as  such  sub- 
stances occur  in  the  renal  and  urinary  passages;  still  in  most  cases  this 
nucleoalbumin,  as  shown  by  K.  Morner,4  is  of  an  entirely  different  kind. 

All  urine,  according  to  Morner,  contains  a  little  proteid  and  in 
addition  substances  which  precipitate  proteid.  If  the  urine  freed  from 
salts  by  dialysis  is  shaken  with  chloroform  after  the  addition  of  1-2  p.  m. 
acetic  acid,  a  precipitate  is  obtained  which  acts  like  a  nucleoalbumin. 
If  the  acid  filtrate  is  treated  with  seralbumin,  a  new  and  similar  precipitate 
is  obtained,  due  to  the  presence  of  a  residue  of  the  substance  which  pre- 
cipitates proteids.  The  most  important  of  these  proteid-precipitating 
substances  is  chondroitin-sulphuric  acid  and  nucleic  acid,  although  the 
latter  appears  to  a  much  smaller  extent.  Taurocholic  acid  may  in  a  few 
instances,  especially  in  icteric  urines,  be  precipitated.  The  substances 
isolated  by  different  investigators  from  urine  by  the  addition  of  acetic 
acid  and  called  "  dissolved  mucin  "  or  "  nucleoalbumin  "  are  considered 

1  In  regard  to  the  literature  on  this  method  and  the  numerous  experiments  to 
determine  its  value,  see  Huppert-Neubauer,  10  Aufl.,  853  and  Neuberg,  Der  Ham, 
g.  765. 

2  Christensen,  Virchow's  Arch.,  115;  Tsuschija,  Centralbl.  f.  Med.,  1908. 
'  Deutsch.  med.  Wochenschrift,  1908. 

« Skand.  Arch,  f .  Physiol.,  6. 


DETECTION  OF  NUCLEOALBUMINS.  795 

by  Morner  to  be  a  combination  of  proteid  chiefly  with  chondroitin- 
sulphuric  acid,  and  to  a  less  extent  with  nucleic  acid,  and  also  perhaps 
with  tauroeholic  acid. 

As  normal  urine  habitually  contains  an  excess  of  substances  capable 
of  precipitating  proteids,  it  is  apparent  that  an  increased  elimination  of 
so-called  nucleoalbumin  may  be  caused  simply  by  an  augmented  excretion 
of  proteid.  This  happens  to  a  still  greater  extent  in  cases  where  the 
proteid  as  well  as  the  proteid-precipitating  substance  is  eliminated  to  an 
increased  extent. 

Detection  of  so-called  Nucleoalbumins.  When  a  urine  becomes  cloudy 
or  precipitates  on  the  addition  of  acetic  acid,  and  when  it  gives  a  more 
typical  reaction  with  Heller's  test  after  the  dilution  of  the  urine  than 
before,  one  is  justified  in  making  tests  for  mucin  and  nucleoalbumin. 
As  the  salts  of  the  urine  interfere  considerably  with  the  precipitation 
of  these  substances  by  acetic  acid,  they  must  first  be  removed  by  dialysis. 
As  large  a  quantity  of  urine  as  possible  is  dialyzed  (with  the  addition  of 
chloroform)  until  the  salts  are  removed.  The  acetic  acid  is  added  until 
it  contains  2  p.  m.,  and  the  mixture  allowed  to  stand.  The  precipitate 
is  dissolved  in  water  by  the  aid  of  the  smallest  possible  quantity  of  alkali 
and  precipitated  again.  In  testing  for  chrondroitin-sulphuric  acid  a 
part  is  warmed  on  the  water-bath  with  about  5  per  cent  hydrochloric 
acid.  If  positive  results  are  obtained  on  testing  for  sulphuric  acid  and 
reducing  substance,  then  chondroproteid  was  present.  If  a  reducing 
substance  can  be  detected  but  no  sulphuric  acid,  then  mucin  is  probably 
there.  If  it  does  not  contain  any  sulphuric  acid  or  reducing  substance, 
a  part  of  the  precipitate  is  exposed  to  pepsin  digestion  and  another  part 
used  for  the  determination  of  any  organic  phosphorus.  If  positive  results 
are  obtained  from  these  tests,  then  nucleoalbumin  and  nucleoproteid 
must  be  differentiated  by  special  tests  for  nuclein  bases.  No  positive 
conclusion  can  be  drawn  except  by  using  very  large  quantities  of  urine. 
The  filtrate  from  the  nucleoalbumin  can  be  used  for  the  ordinary  proteid 
tests. 

Nucleohistone.  In  a  case  of  pseudoleucacmia  A.  Jolles  found  a  phos- 
phorized  protein  substance  which  he  considers  as  identical  with  nucleohistone. 
Histone  is  claimed  to  have  been  found  in  some  cases  by  Krehl  and  AIatthes, 
and  by  Kolisch  and  Buuian.1 

The  nitrogen  contained  in  the  substances  precipitated  by  alcohol,  called  the 
"colloidal  nitrogen  "  by  Salkowski  and  whose  quantity  is  doubled  in  carcinoma 
as  compared  to  the  normal,  consists  in  great  part  of  oxyproteic  acids.  Accord- 
ing to  Salkowski  and  Kojo  2  this  can  be  precipitated  by  basic  lead  acetate  and 
the  nitrogen  determined  therein. 

Blood  and  Blood-coloring  Matters.  The  urine  may  contain  blood  from 
hemorrhage  in  the  kidneys  or  other  parts  of  the  urinary  passages  (ilema- 

1  Jolles,  Ber.  d.  deutsch.  chem.  Gesellsch.,  30;  Krehl  and  Matthes,  Deutsch.  Arch, 
f.  klin.  Med.,  54;  Kolisch  and  Burian,  Zeitschr.  f.  klin.  Med.,  29. 

2  Salkowski,  Berl.  klin.  Wochenschr.,  1905  and  1910;  Kojo,  Zeitschr.  f.  physiol. 
Chem.,  73. 


796  URINE. 

turia).  In  these  cases,  when  the  quantity  of  blood  is  not  very  small, 
the  urine  is  more  or  less  cloudy  and  colored  reddish,  yellowish  red,  dirty 
red,  brownish  red,  or  dark  brown.  In  recent  hemorrhages  in  which  the 
blood  has  not  decomposed  the  color  is  nearer  blood-red.  Blood-corpuscles 
may  be  found  in  the  sediment,  sometimes  also  blood-casts  and  smaller 
or  larger  blood-clots. 

In  certain  cases  the  urine  contains  no  blood-corpuscles,  but  only  dis- 
solved blood-coloring  matters,  haemoglobin,  or,  and  indeed  quite  often, 
methsemoglobin  (ilemoglobinuria)  .  The  blood-pigments  appear  in  the 
urine  under  different  conditions,  as  in  dissolution  of  blood  in  poisoning 
with  arseniuretted  hydrogen,  chlorates,  etc.,  after  serious  burns,  after 
transfusion  of  blood,  and  also  in  the  periodic  appearance  of  haemoglo- 
binuria  with  fever.  In  hemoglobinuria  the  urine  may  also  have  an  abun- 
dant grayish-brown  sediment  rich  in  proteid  which  contains  the  remains 
of  the  stromata  of  the  red  blood-corpuscles.  In  animals,  hsemoglobinuria 
may  be  produced  by  many  causes  which  force  free  haemoglobin  into  the 
plasma. 

To  detect  blood  in  the  urine,  we  make  use  of  the  microscope,  the  spec- 
troscope, the  guaiac  test,  and  Heller's  or  Heller-Teichmann's  test. 

Microscopic  Investigation.  The  blood-corpuscles  may  remain  undis- 
solved for  a  long  time  in  acid  urine;  in  alkaline  urine,  on  the  contrary, 
they  are  easily  changed  and  dissolved.  They  often  appear  entirely 
unchanged  in  the  sediment;  in  some  cases  they  are  distended  and  in 
others  unequally  pointed  or  jagged  like  a  thorn-apple.  In  hemorrhage  of 
the  kidneys  a  cylindrical  clot  is  sometimes  found  in  the  sediment  which  is 
covered  with  numerous  red  blood-corpuscles,  forming  casts  of  the  urinary 
passages.     These  formations  are  called  blood-casts. 

The  spectroscopic  investigation  is  naturally  of  very  great  value;  and  if 
it  be  necessary  to  determine  not  only  the  presence  but  also  the  kind  of 
coloring-matter,  this  method  is  indispensable.  In  regard  to  the  optical 
behavior  of  the  various  blood-pigments  we  must  refer  to  Chapter  V. 

Guaiac  Test.  Mix  in  a  test-tube  equal  volumes  of  tincture  of 
guaiac  and  old  turpentine  which  has  become  strongly  ozonized  by  the 
action  of  air  under  the  influence  of  light.  To  this  mixture,  which  must 
not  have  the  slightest  blue  color,  add  the  urine  to  be  tested.  In  the 
presence  of  blood  or  blood-pigments,  first  a  bluish-green  and  then  a  beau- 
tiful blue  ring  appears  where  the  two  liquids  meet.  On  shaking  the  mixture 
it  becomes  more  or  less  blue.  Normal  urine  or  one  containing  proteid 
does  not  give  this  reaction.  According  to  Liebermann  l  this  reaction 
is  brought  about  by  the  blood  pigments  acting  as  catalyst  upon  the 
organic  peroxides  existing  in  the  turpentine,  accelerating  the  decomposi- 
tion of  these  and  the  active  oxygen  taken  up  by  the  guaiaconic  acid 
which  is  oxidized  to  guaiac  blue  (guaiaconic  acid  ozonide).     Urine  con- 

1  Pfluger's  Arch.,  104. 


BLOOD   PIGMENTS.     HiEMATOPORPHYRIN.  797 

I 

taining  pus,  even  when  no  blood  is  present,  gives  a  blue  color  with  these 
reagents;  but  in  this  case  the  tincture  of  guaiac  alone,  without  tur- 
pentine, is  colored  blue  by  the  urine  (Vitali  ').  This  is  at  least  true 
for  a  tincture  that  has  been  exposed  for  some  time  to  the  action  of  air 
and  sunlight.  The  blue  color  produced  by  pus  differs  from  that  pro- 
duced by  blood-coloring  matters  by  disappearing  on  heating  the  urine 
to  boiling.  A  urine  alkaline  by  decomposition  must  first  be  made  faintly 
acid  before  performing  the  reaction.  The  turpentine  should  be  kept 
exposed  to  sunlight,  while  the  tincture  of  guaiac  must  be  kept  in  a 
dark  glass  bottle.  These  reagents  to  be  of  use  must  be  controlled  by  a 
liquid  containing  blood.  With  positive  results,  however,  this  test  is 
not  absolutely  decisive,  because  other  bodies  may  give  a  similar  reaction, 
but  when  properly  performed  it  is  so  extremely  delicate  that  when  it 
gives  negative  results  any  other  test  for  blood  is  superfluous.2 

As  the  delicacy  of  the  above-mentioned  tests  is  sufficient  for  ordinary 
purposes  it  is  not  necessary  to  give  the  new  blood-tests  suggested  recently. 

Heller-Teichmann's  Test.  If  a  neutral  or  faintly  acid  urine  containing 
blood  is  heated  to  boiling,  one  always  obtains  a  mottled  precipitate  consist- 
ing of  proteid  and  haematin.  If  caustic  soda  is  added  to  the  boiling-hot  test, 
the  liquid  becomes  clear  and  turns  green  when  examined  in  thin  layers  (due  to 
haematin  alkali),  and  a  red  precipitate,  appearing  green  by  reflected  light,  re-forms, 
consisting  of  earthy  phosphates  and  hsematin.  This  reaction  is  called  Heller's 
blood-test.  If  this  precipitate  is  after  a  time  collected  on  a  small  filter,  it  may  be 
used  for  the  haemin  test  (see  page  293).  If  the  precipitate  contains  only  a  little 
blood-coloring  matter  with  a  larger  quantity  of  earthy  phosphates,  then  wash 
it  with  dilute  acetic  acid,  which  dissolves  the  earthy  phosphates,  and  use  the 
residue  for  the  preparation  of  Teichmann's  haemin  crystals.  If,  on  the  contrary, 
the  amount  of  phosphates  is  very  small,  then  first  add  a  little  MgCb  solution 
to  the  urine,  heat  to  boiling,  and  add  simultaneously  with  the  caustic  potash 
some  sodium-phosphate  solution.  In  the  presence  of  only  very  small  quantities 
of  blood,  first  make  the  urine  very  faintly  alkaline  with  ammonia,  add  tannic 
acid,  acidify  with  acetic  acid,  and  use  this  precipitate  in  the  preparation  of  the 
haemin  crystals  (Sturve  3). 

0.  and  R.  Adler  4  have  recommended  leucomalachite  green  or  benzidine  in 
the  presence  of  peroxide  and  acetic  acid  as  especially  sensitive  reagents  for  blood. 

Haematoporphyrin.  Since  the  occurrence  of  haematoporphyrin  in  the 
urine  in  various  diseases  has  been  made  very  probable  by  several  investi- 
gators, such  as  Neusser,  Stokvis,  MacMunn,  Le  Nobel,  Copeman,  and 
others,5  Salkow'ski  has  positively  shown  the  presence  of  this  pigment 
in  the  urine  after  sulfonal  intoxication.     It  was  first  isolated  in  a  pure 


1  See  Maly's  Jahresber.,  18. 

2  For  more  details  in  regard  to  the  preparation  of  the  reagents  and  the  performance 
of  the  reaction  see  O.  Schumm.,  Zeitschr.  f.  physiol.  Chem.,  50. 

3  Zeitschr.  f.  anal.  Chem.,  11. 

4  Zeitschr.  f.  physiol.  Chem.,  41. 

s  A  very  complete  index  of  the  literature  on  haematoporphyrin  in  the  urine  may  be 
found  in  R.  Zoja,  Su  qualche  pigmento  di  alcune  urine,  etc.,  in  Arch.  Ital.  di.  clin. 
Med.,  1893. 


798  URINE. 

crystalline  state  by  Hammarsten  *  from  the  urine  of  insane  women  after 
sulfonal  intoxication.  According  to  Garrod  and  Saillet2  traces  of 
hsematoporphyrin  (Saillet's  urospectrin)  regularly  occur  in  normal 
urines.  It  is  also  found  in  the  urine  during  different  diseases.  It  was 
found  in  great  abundance  in  a  case  of  typhoid  fever  (Arnold3)  but 
otherwise  it  generally  occurs  only  in  small  amounts.  It  has  been  found  in 
considerable  quantities  in  the  urine  after  the  lengthy  use  of  sulfonal. 

Urine  containing  hsematoporphyrin  is  sometimes  only  slightly  colored, 
while  in  other  cases,  as  for  example,  after  the  use  of  sulfonal,  it  is  more 
or  less  deep  red.  In  these  last-mentioned  cases  the  color  depends,  in 
greatest  part,  not  upon  the  hsematoporphyrin,  but  upon  other  red  or 
reddish-brown  pigments  which  have  not  been  sufficiently  studied. 

In  the  detection  of  small  quantities  of  hsematoporphyrin  proceed  as 
suggested  by  Garrod.  Precipitate  the  urine  with  a  10-per  cent  caustic- 
soda  solution  (20  cc.  for  every  100  cc.  of  urine).  The  phosphate  pre- 
cipitate containing  the  pigment  is  dissolved  in  alcohol-hydrochloric  acid 
(15-20  cc.)  and  the  solution  investigated  with  the  spectroscope.  In  more 
exact  investigations  make  the  solution  alkaline  with  ammonia,  add  enough 
acetic  acid  to  dissolve  the  phosphate  precipitate,  shake  with  chloroform, 
which  takes  up  the  pigment,  and  test  this  solution  with  the  spectroscope. 

In  the  presence  of  larger  quantities  of  hsematoporphyrin  the  urine 
is  first  precipitated,  according  to  Salkowski,  with  an  alkaline  barium- 
chloride  solution  (a  mixture  of  equal  volumes  of  barium-hydroxide  solu- 
tion, saturated  in  the  cold,  and  a  10-per  cent  barium-chloride  solution), 
or,  according  to  Hammarsten,4  with  a  barium-acetate  solution.  The 
washed  precipitate,  which  contains  the  hsematoporphyrin,  is  allowed 
to  stand  some  time  at  the  temperature  of  the  room,  with  alcohol  contain- 
ing hydrochloric  or  sulphuric  acid,  and  then  filtered.  The  nitrate  shows 
the  characteristic  spectrum  of  hsematoporphyrin  in  acid  solution  and  gives 
the  spectrum  of  alkaline  hsematoporphyrin  after  saturation  with  ammonia. 
If  the  alcoholic  solution  is  mixed  with  chloroform  and  a  large  quantity 
of  water  added  and  carefully  shaken,  sometimes  a  lower  layer  of  chloro- 
form is  obtained  which  contains  very  pure  hsematoporphyrin,  while  the 
upper  layer  of  alcohol  and  water  contains  the  other  pigments  besides 
some  hsematoporphyrin. 

Other  methods  which  have  no  advantage  over  this  one  of  Garrod  have  been 
suggested  by  Riva  and  Zoja  as  well  as  Saillet.5 

Baumstark  6  found  in  a  case  of  leprosy  two  characteristic  coloring-matters 
in   the   urine,    "  urorubrohsematin "    and    "  urofuscohsematin,"    which,    as   their 

1  Salkowski,  Zeitschr.  f.  physiol.  Chem.,  15;  Hammarsten,  Skand.  Arch.  f.  Physiol.,  3. 

2  Garrod,  Journ.  of  Physiol.,  13  (contains  review  of  literature)  and  17;  Saillet,. 
Revue  de  Mi'decine,  16. 

3  Zeitschr.  f.  physiol.  Chem.,  82. 

*  Salkowski,  1.  c;  Hammarsten,  1.  c. 

6  Riva  and  Zoja,  Maly's  Jahresber.,  24;  Saillet,  1.  c.     See  also  Nebelthau,  Zeitschr. 
f.  physiol.  Chem.,  27. 
6  Pfliiger's  Arch.,  9. 


PUS.     BILE  ACIDS.  799 

names  indicate,  seem  to  stand  in  close  relation  to  the  blood-coloring  matters. 
UroTubrohccmatin,  CosHniNgFeiOj*,  contains  iron  and  shows  in  acid  solution  an 
absorption-hand  in  front  of  D  and  a  broader  one  back  of  D.  In  alkaline  solution 
it  shows  four  bands— behind  D,  at  E,  beyond  F,  and  behind  G.  It  is  not  soluble 
either  in  water,  alcohol,  ether,  or  chloroform.  It  gives  a  beautiful  brownish-red 
non-dichroic  liquid  with  alkalies.  Urofuscohoematin,  CssHiobNsC^,  which  is  free 
from  iron,  shows  no  characteristic  spectrum;  it  dissolves  in  alkalies,  producing 
a  brown  color.  It  remains  to  be  proven  whether  these  two  pigments  are  related 
to  (impure)  lueinatoporphyrin. 

Melanin.  In  the  presence  of  melanotic  cancers  dark  pigments  are  some- 
times eliminated  with  the  urine.  K.  Morner  has  isolated  two  pigments  from 
such  a  urine,  of  which  one  was  soluble  in  warm  50-75  per  cent  acetic  acid,  while 
the  other,  on  the  contrary,  was  insoluble.  The  one  seemed  to  be  phymatorhusin 
(see  Chapter  XV).  Usually  the  urine  does  not  contain  any  melanin,  but  a 
chromogen  of  melanin,  a  meianogen.  In  such  cases  the  urine  gives  Eislet's 
reaction,  becoming  dark-colored  with  oxidizing  agents,  such  as  concentrated 
nitric  acid,  potassium  bichromate,  and  sulphuric  acid,  as  well  as  with  free  sulphuric 
acid.  They  also  give  Thormahlen's  reaction  namely  a  beautiful  blue  coloration 
with  sodium  nitroprusside  and  then  acetic  acid.  Urine  containing  melanin  or 
meianogen  is  colored  black  by  a  ferric-chloride  solution  (v.  Jaksch  *)■ 

In  a  case  of  melanotic  sarcoma  H.  Eppinger  2  has  isolated  from  the  urine 
a  crystalline  meianogen  of  the  composition  C9Hi2N2S04,  and  which  was  insoluble 
in  ether.  It  gave  the  ordinary  meianogen  reactions  and,  according  to  him  is 
probably  an  amidated  ethereal  sulphuric  acid  of  methylpyrrolidinoxycarboxylic 
acid,  which  is  derived  from  tryptophane. 

Pus  occurs  in  the  urine  in  various  inflammatory  affections,  especially 
in  catarrh  of  the  bladder  and  in  inflammation  of  the  pelvis  of  the 
kidneys,  or  of  the  urethra. 

Pus  is  best  detected  by  means  of  the  miscroscope.  The  pus-cells  are 
rather  easily  destroyed  in  alkaline  urines.  In  detecting  pus  we  make 
use  of  Donne's  pus  test,  which  is  performed  in  the  following  way:  Pour 
off  the  urine  from  the  sediment  as  carefully  as  possible,  place  a  small 
piece  of  caustic  alkali  on  the  sediment,  and  stir.  If  the  pus-cells  have 
not  been  previously  changed,  the  sediment  is  converted  by  this  means 
into  a  slimy  tough  mass. 

The  pus-corpuscles  swell  up  in  alkaline  urines,  and  dissolve,  or  at  least 
are  so  changed  that  they  cannot  be  recognized  under  the  microscope. 
The  urine  in  these  cases  is  more  or  less  slimy  or  fibrous,  and  the  proteid 
can  be  precipitated  in  large  flakes  by  acetic  acid,  so  that  it  might  possibly 
be  mistaken  for  mucin.  The  closer  investigation  of  the  precipitate 
produced  by  acetic  acid,  and  especially  the  appearance  or  non-appearance 
of  a  reducing  substance  after  boiling  it  with  a  mineral  acid,  demonstrates 
the  nature  of  the  precipitated  substance.  Urine  containing  pus  always 
contains  proteid. 

Bile-acids.  The  reports  in  regard  to  the  occurrence  of  bile-acids  in  the 
urine  under  physiological  conditions  do  not  agree.  According  to  Dragen- 
dorff  and  Hone  traces  of  bile-acids  occur  in  the  urine;  according  to  Mao 

^hormahlen,  Virchow's  Arch.,  108;  v.  Jaksch,  Zeitschr.  f.  physiol.  Chem.,  13. 
1  Bioch.  Zeitschr.,  28. 


800  URINE. 

kay  and  v.  Udranszky  and  K.  Morner1  they  do  not.  Pathologically 
they  are  present  in  the  urine  in  hepatogenic  icterus,  although  not  invar- 
iably. 

Detection  of  Bile-acids  in  the  Urine.  Pettenkofer's  test  gives  the  most 
decisive  reaction ;  but  as  it  gives  similar  color  reactions  with  other  bodies,  it  must 
be  supplemented  by  the  spectroscopic  investigation.  The  direct  test  for  bile- 
acids  is  easily  performed  after  the  addition  of  traces  of  bile  to  a  normal  urine. 
But  the  direct  detection  in  a  colored  icteric  urine  is  more  difficult  and  gives  very 
misleading  results;  the  bile-acid  must  therefore  always  be  isolated  from  the  urine. 
This  may  be  done  by  the  following  method  of  Hoppe-Seyler,  which  is  slightly 
modified  in  non-essential  points. 

Hoppe-Seyler's  Method.  Concentrate  the  urine  and  extract  the  residue 
with  strong  alcohol.  The  filtrate  is  freed  from  alcohol  by  evaporation  and  then 
precipitated  by  basic  lead  acetate  and  ammonia.  The  washed  precipitate  is 
treated  with  boiling  alcohol,  filtered  hot,  the  filtrate  treated  with  a  few  drops 
of  soda  solution,  and  evaporated  to  dryness.  The  dry  residue  is  extracted  with 
absolute  alcohol,  filtered,  and  an  excess  of  ether  added.  The  amorphous  or, 
after  a  longer  time,  crystalline,  precipitate  consisting  of  the  alkali  salts  of  the 
biliary  acids  is  used  in  performing  Pettenkofer's  test. 

Bile-pigments  occur  in  the  urine  in  different  forms  of  icterus.  A 
urine  containing  bile-pigments  is  always  abnormally  colored — yellow, 
yellowish  brown,  deep  brown,  greenish  yellow,  greenish  brown,  or  nearly 
pure  green.  On  shaking  it  froths,  and  the  bubbles  are  yellow  or  yellowish 
green  in  color.  As  a  rule  icteric  urine  is  somewhat  cloudy,  and  the  sedi- 
ment is  frequently,  especially  when  it  contains  epithelium-cells,  rather 
strongly  colored  by  the  bile-pigments. 

Detection  of  Bile-coloring  Matters  in  Urine.  Many  tests  have  been 
proposed  for  the  detection  of  these  substances.  Ordinarily  we  obtain 
the  best  results  with  the  following  three  tests: 

•  Gmelin's  test  may  be  applied  directly  to  the  urine;  but  it  is  better  to 
use  Rosenbach's  modification.  Filter  the  urine  through  a  very  small 
filter,  which  becomes  deeply  colored  from  the  retained  epithelium-cells 
and  bodies  of  that  nature.  After  the  liquid  has  entirely  passed  through 
apply  to  the  inside  of  the  filter  a  drop  of  nitric  acid  which  contains  only 
very  little  nitrous  acid.  A  pale-yellow  spot  will  be  formed  which  is  sur- 
rounded by  colored  rings  which  appear  yellowish  red,  violet,  blue,  and 
green  from  within  outward.  This  modification  is  very  delicate,  and  it 
is  hardly  possible  to  mistake  indican  and  other  coloring-matters  for  the 
bile-pigments.  Several  other  modifications  of  Gmelin's  direct  test,  e.g., 
with  concentrated  sulphuric  acid  and  nitrate,  etc.,  have  been  proposed, 
but  they  are  neither  simpler  nor  more  delicate  than  Rosenbach's  modifica- 
tion. 

Huppert's  Reaction.  In  a  dark-colored  urine  or  one  rich  in  indican 
good  results  are  not  always  obtained  with  Gmelin's  test.  In  such  cases, 
as  also  in  urines  containing  blood-coloring  matters  at  the  same  time, 
the  urine  is  treated  with  lime-water,  or  first  with  some  CaCb  solution, 

'Cited  from  Huppert-Neubauer,  Harn-Analyse,  10.  Aufl.,  229. 


BILE  PIGMENTS.  801 

and  then  with  a  solution  of  sodium  or  ammonium  carbonate.  The  pre- 
cipitate which  contains  the  bile-coloring  matter  is  filtered,  washed,  dis- 
solved in  alcohol  which  contains  5  cc.  of  concentrated  hydrochloric  acid 
in  100  cc.  (I.  Munk),  and  heated  to  boiling,  when  the  solution  becomes 
green  or  bluish  green.  According  to  Xakayama  i  this  reaction  is  more 
delicate  on  using  a  mixture  of  ferric  chloride,  acid,  and  alcohol. 

Hammarsten's  Reaction.  For  ordinary  cases  it  is  sufficient  to  add 
a  few  drops  of  urine  to  about  2-3  cc.  of  the  reagent  (see  page  432),  when 
the  mixture  immediately  after  shaking  turns  a  beautiful  green  or  bluish 
green,  which  color  remains  for  several  days.  In  the  presence  of  only 
very  small  quantities  of  bile-pigments,  especially  when  blood  or  other 
pigments  are  simultaneously  present,  pour  about  10  cc.  of  the  acid  or 
nearly  neutral  (not  alkaline)  urine  into  the  tube  of  a  small  centrifugal 
machine  and  add  BaCb  solution  and  centrifuge  for  about  one  minute. 
The  liquid  is  decanted  and  the  sediment  stirred  with  about  1  cc.  of  the 
reagent  and  centrifuged  again.  A  beautiful  green  solution  is  obtained 
which  may  be  changed,  by  the  addition  of  increased  quantities  of  the  acid 
mixture,  to  blue,  violet,  red,  and  reddish  yellow.  The  green  color  may 
be  obtained  in  the  presence  of  1  part  bile-pigment  in  500,000-1,000,000 
parts  urine.  In  the  presence  of  large  amounts  of  other  pigments  calcium 
chloride  is  better  suited  than  barium  chloride. 

Bouma2  has  suggested  the  use  of  alcohol  containing  ferric  chloride 
and  hydrochloric  acid  instead  of  the  above-mentioned  acid  mixture.  He 
has  also  wrorked  out  a  colorimetric  method  of  quantitative  estimation 
of  bilirubin  in  urine  by  means  of  this  reagent. 

As  above  indicated,  we  have  a  great  many  other  tests  besides  these 
given  above.  A  very  complete  summary  of  these  tests  and  the  literature 
thereof  can  be  found  in  the  work  of  Obermayer  and  Popper. 

For  ordinary  purposes  the  above-mentioned  tests  are  sufficiently 
delicate,  and  according  to  Hammarsten  it  is  not  advisable,  as  also  in  the 
case  of  the  detection  of  proteid,  sugar,  etc.,  to  increase  the  delicacy  of 
a  test  so  that  it  shows  the  presence  of  the  traces  of  the  questionable 
substance  in  normal  urine.  If  in  certain  cases  a  greater  delicacy  is 
required  than  is  obtained  with  the  above  tests,  then  we  must  recommend 
the  flotation  test  of  Obermeyer  and  Popper  3  with  iodine  and  salt. 

Medicinal  coloring-matters  produced  from  santonin,  rhubarb,  senna,  etc., 
may  give  an  abnormal  color  to  the  urine  and  may  be  mistaken  for  bile-pigments, 
or,  in  alkaline  urines,  perhaps  for  blood-eoloring  matters.  If  hydrochloric  acid 
is  added  to  such  a  urine,  it  becomes  yellow  or  pale  yellow,  while  on  the  addition 
of  an  excess  of  alkali  it  takes  on  a  more  or  less  beautiful  red  color. 


1  Munk,  Arch.  f.  (Anat.  u.)  Physiol.,  1898;   Nakayama,  Zeitschr.  f.  physiol.  Chem., 
36. 

*  Deutsch.  med.  Wochenschr.,  1902  and  1904 

*  Wien.  med.  Wochenschr.,  21. 


802  URINE. 

Sugar  in  Urine. 

The  occurrence  of  traces  of  glucose  in  the  urine  of  perfectly  healthy 
persons  has  been,  as  above  stated  (page  749),  quite  positively  proven.  If 
sugar  appears  in  the  urine  in  constant  and  especially  in  large  quantities, 
it  must  be  considered  as  an  abnormal  constituent.  In  a  previous  chapter 
several  of  the  principal  causes  of  glycosuria  in  man  and  animals  were  men- 
tioned, and  the  reader  is  referred  to  Chapters  VII  and  VIII  for  the  essen- 
tial facts  in  regard  to  the  appearance  of  sugar  in  the  urine. 

In  man  the  appearance  of  glucose  in  the  urine  has  been  observed 
under  various  pathological  conditions,  such  as  lesions  of  the  brain  and 
especially  of  the  medulla  oblongata,  abnormal  circulation  in  the  abdomen, 
diseases  of  the  heart,  lungs  and  liver,  cholera,  and  many  other  diseases. 
The  continued  presence  of  sugar  in  human  urine,  sometimes  in  very  con- 
siderable quantities,  occurs  in  diabetes  mellitus.  In  this  disease  there 
may  be  elimination  of  1  kilogram  or  even  more  of  glucose  per  day. 
In  the  beginning  of  the  disease,  when  the  quantity  of  sugar  is  still  very 
small,  the  urine  often  does  not  appear  abnormal.  In  the  more  developed, 
typical  cases  the  quantity  of  urine  voided  increases  considerably,  to 
3-6-10  liters  per  day.  The  percentage  of  the  physiological  constituents 
is  as  a  rule  very  low,  while  their  absolute  daily  quantity  is  increased. 
The  urine  is  pale,  but  of  a  high  specific  gravity,  1.030-1.040  or  even  higher. 
The  high  specific  gravity  depends  upon  the  quantity  of  sugar  present, 
which  varies  in  different  cases,  but  may  reach  10  per  cent.  The  urine  is 
therefore  characterized  in  typical  cases  of  diabetes  by  the  very  large 
quantity  voided,  by  the  pale  color  and  high  specific  gravity,  and  by  its 
containing  sugar. 

That  the  urine  after  the  introduction  into  the  system  of  certain  medici- 
nal agents  or  poisonous  bodies  contains  reducing  substances,  conjugated 
glucuronic  acids,  which  may  be  mistaken  for  sugar,  has  already  been  men- 
tioned. 

Glucose  in  urine.  The  properties  and  reactions  of  this  sugar  have  been 
considered  in  a  previous  chapter,  and  it  remains  but  to  mention  the  methods 
for  the  detection  and  quantitative  determination  of  glucose  in  the  urine. 

The  detection  of  sugar  in  the  urine  is  ordinarily,  in  the  presence  of  not 
too  small  quantities,  a  very  simple  task.  The  presence  of  only  very  small 
quantities  may  make  its  detection  sometimes  very  difficult  and  laborious. 
A  urine  containing  proteid  must  first  have  the  proteid  removed  by  coagu- 
lation with  acetic  acid  and  heat  before  it  can  be  tested  for  sugar.  ■ 

The  tests  which  are  most  frequently  employed  and  are  especially 
recommended  are  as  follows: 

Trommer's  Test.  In  a  typical  diabetic  urine  or  one  rich  in  sugar  this 
test  succeeds  well,  and  it  may  be  performed  in  the  manner  suggested  on 


SUGAR  IN   URINE.  803 

page  214.  This  test  may  lead  to  very  great  mistakes  in  urine  poor  in 
sugar,  especially  when  they  have  at  the  same  time  normal  or  increased 
amounts  of  physiological  constituents,  and  therefore  it  cannot  be  recom- 
mended to  physicians  or  to  persons  inexperienced  in  such  work.  Normal 
urine  contains  reducing  substances,  such  as  uric  acid,  creatinine,  and  others, 
and  therefore  a  reduction  takes  place  in  all  urines  on  using  this  test.  A 
separation  of  copper  suboxide  does  not  generally  occur,  but  still  if  one 
varies  the  proportion  of  the  alkali  to  the  copper  sulphate  and  boils,  there 
takes  place  an  actual  separation  of  suboxide  in  normal  urines,  or  a  peculiar 
yellowish  red  liquid  due  to  finely  divided  cuprous  hydroxide.  This  occurs 
especially  on  the  addition  of  much  alkali  or  too  much  copper  sulphate, 
and  by  careless  manipulation  the  inexperienced  worker  may  therefore 
sometimes  obtain  apparently  positive  results  in  a  normal  urine.  On  the 
other  hand,  as  the  urine  contains  substances  such  as  creatinine  and 
ammonia  (from  the  urea),  which  in  the  presence  of  only  a  little  sugar 
may  keep  the  copper  suboxide  in  solution,  the  investigator  may  easily 
overlook  small  quantities  of  sugar  that  may  be  present. 

The  delicacy  of  Trommer's  test  can  be  increased  by  the  suggestion  made  by 
Worm-Muller.1  As  "by  this  rather  complicated  and  tedious  method  small 
amounts  of  sugar  cannot  be  detected  in  certain  urines,  and  also  as  special  urines 
from  healthy  persons  readily  give  inconclusive  results,  and  finally  as  Schondorff 
has  shown  in  numerous  cases  that  the  physiological  sugar  content  of  the  urine 
responds  to  this  test  in  perfectly  healthy  persons  because  of  its  extreme  delicacy, 
it  does  not  seem  advisable  in  Hammarsten's  opinion  to  recommend  this  test  to 
the  physician.  Bang  and  Bohmansson  2  have  recently  also  shown  its  unre- 
liability. 

Almen's  bismuth  test,  which  has  been  incorrectly  called  Nylander's 
test,  is  performed  with  the  alkaline-bismuth  solution  prepared  as  described 
on  page  214.  For  each  test  10  cc.  of  urine  are  taken  and  treated  with 
1  cc.  of  the  bismuth  solution  and  boiled  for  a  few  minutes.  In  the 
presence  of  sugar  the  urine  becomes  dark  yellow  or  yellowish  brown; 
then  it  grows  darker,  cloudy,  dark  brown,  or  nearly  black,  and  non- 
transparent.  After  a  longer  or  shorter  time  a  black  deposit  appears, 
the  supernatant  liquid  gradually  clears,  but  still  remains  colored.  In 
the  presence  of  only  very  little  sugar  the  test  does  not  become  black  or 
dark  brown,  but  simply  deeper  colored,  and  not  until  after  some  time 
is  there  seen  on  the  upper  layer  of  the  phosphate  precipitate  a  dark  or 
black  layer  (of  bismuth?).  In  the  presence  of  much  sugar  a  larger 
amount  of  the  reagent  may  be  used  without  disadvantage.  In  a  urine 
poor  in  sugar  only  1  cc.  of  the  reagent  for  every  10  cc.  of  the  urine  must 
be  employed. 


1  In  regard  to  this  test  see  Pfluger,  Pfluger's  Arch.,  105  and  106;    Hammarsten, 
ibid.,  116,  and  Zeitschr.  f.  physiol.  Chem.,  50. 

2  Schondorff,  Pfluger's  Arch.,  121;  Bohmansson,  Bioch.  Zeitschr.,  19. 


804  URINE. 

Small  amounts  of  proteid  may  retard  this  reaction  and  reduce  the 
delicacy  of  the  test.  Large  quantities  of  proteid  may,  however,  give 
rise  to  an  error  by  forming  bismuth  sulphide,  and  therefore  it  must 
always  be  first  removed.  The  assertion  of  Bechhold  that  mercury 
compounds  in  the  urine  disturb  the  test  has  not  been  substantiated  by 
Zeidlitz  on  properly  performing  the  test,  and  recently  Rehfuss  and 
Hawk  x  came  to  the  same  conclusion.  Those  sources  of  error  which 
in  Trommer's  test  are  caused  by  the  presence  of  uric  acid  and  creatinine 
are  removed  by  using  this  test.  The  bismuth  test  is,  moreover,  readily 
performed,  and  on  this  account  is  to  be  recommended  to  the  physician. 

The  bumping  and  ejection  of  the  fluid  can  be  readily  prevented  by  heating 
over  a  very  small  flame  after  the  test  has  been  brought  to  a  boil,  and  by  gently 
shaking  the  contents  of  the  not  too  narrow  test-tube.  The  recommendation 
of  heating  for  a  longer  time  in  the  water-bath,  fifteen  minutes  or  more,  is  to  be 
discarded,  as  the  delicacy  of  the  test  is  thereby  so  much  increased  that  it  gives 
a  reaction  with  a  physiological  sugar  content  of  0.02  per  cent. 

When  the  amount  of  sugar  in  the  urine  is  not  less  than  0.1  per  cent 
a  positive  reaction  is  obtained  if  the  test  is  boiled  for  2-3  minutes  and 
then  allowed  to  stand  quietly  for  5  minutes.  The  phosphate  precipitate 
is  then  black  or  nearly  black.  In  detecting  smaller  quantities  of  sugar 
— 0.05  per  cent,  the  test  as  a  rule  must  be  boiled  longer — about  5  minutes. 

The  value  of  this  test  lies  in  the  fact  that  it  positively  detects  small 
quantities  of  sugar — 0.1  per  cent  or  somewhat  less,  and  that  when  the 
urine  gives  negative  results  we  can  consider  it  free  from  sugar  in  a  clinical 
sense.  Like  Trommer's  test  it  is  a  reduction  test,  and  shows  also  certain 
other  reducing  bodies  besides  the  sugar.  These  bodies  are  certain  con- 
jugated glucuronic  acids  which  may  appear  in  the  urine.  After  the  use 
of  certain  therapeutic  agents,  such  as  rhubarb,  senna,  antipyrine,  salol, 
turpentine  and  others,  the  bismuth  test  gives  .positive  results.  From 
this  it  follows  that  we  should  never  be  satisfied  with  this  test  alone,  espe- 
cially when  the  reduction  is  not  very  great. 

According  to  Bohmansson  and  Bang  this  test  is  perfectly  reliable 
if  about  20  cc.  of  the  urine  is  treated  with  5  cc.  of  25  per  cent  HC1  and 
2  grams  blood-charcoal  (a  teaspoonful)  added  and  shaken  every  once  in 
a  while  during  five  minutes  and  then  filtered.  The  filtrate  on  neutraliza- 
tion with  caustic  soda  is  used  for  the  Almen  test.  The  disturbing  reduc 
ing  substances  are  removed  by  the  animal  charcoal,  but  the  sugar  is  not. 

According  to  Andersen  2  this  procedure  cannot  be  used  in  the  quan- 


1  Bechhold,  Zeitschr.  f.  physiol.  Chem.,  46;  Zeidlitz,  Upsala  Lakaref.  Forh.  (N.  F.), 
11  (Hammarsten  Festschr.);  Rehfuss  and  Hawk,  Journ.  of  biol.  Chem.,  7. 

2  Bohmansson   and    Bang,  Bioch.   Zeitschr.,    19,   and   Zeitschr.   f.  physiol.   Chem., 
63;  Andersen,  Bioch.  Zeitschr.,  37. 


SUGAR  IN   URINE.  805 

titative  estimation  of  sugar  as  a  part  of  the  sugar  is  retained  by  the 
use  of  hydrochloric  acid  and  blood-charcoal.  According  to  Andersen 
the  pigments  and  the  disturbing  substances  can  be  removed  by  per- 
cipitation  with  mercuric  nitrate.  It  can  be  more  simply  done  by  treating 
40  cc.  of  the  urine  with  10  cc.  acetic  acid  of  50  per  cent  strength  and  4 
grams  blood-charcoal,  shaking  as  above  described  and  filtering.  In  the 
presence  of  acetic  acid  no  sugar  is  taken  up  by  the  charcoal  and  as  this 
simple  method  can  be  used  for  the  quantitative  estimation  it  can  therefore 
be  used  in  the  qualitative  tests  for  sugar. 

Fermentation  Test.  On  using  this  test  the  process  must  vary  accord- 
ing as  the  bismuth  test  shows  small  or  large  quantities  of  sugar.  If  a 
rather  strong  reduction  is  obtained,  the  urine  may  be  treated  with  yeast 
and  the  presence  of  sugar  determined  by  the  generation  of  carbon 
dioxide.  In  this  case  the  acid  urine,  or  that  faintly  acidified  with  a  little 
tartaric  acid  is  treated  with  compressed  yeast,  or  yeast  which  has  pre- 
viously been  washed  by  decantation  with  water.  Pour  this  urine 
to  which  the  yeast  has  been  added  into  a  Schrotter's  gas  burette  or  a 
Lohnstein's  saccharimeter  (see  below).  As  the  fermentation  proceeds, 
the  carbon  dioxide  collects  in  the  upper  part  of  the  tube,  while  a  correspond- 
ing quantity  of  liquid  is  expelled  below.  As  a  control  in  this  case  two 
similar  tests  must  be  made,  one  with  normal  urine  and  yeast  to  learn 
the  quantity  of  gas  usually  developed,  and  the  other  with  a  sugar  solu- 
tion and  yeast  to  determine  the  activity  of  the  yeast.  According  to 
Victorow  l  the  fermentation  is  complete  after  six  hours  at  a  tem- 
perature of  34-36°  C. 

If,  on  the  contrary,  only  a  faint  reduction  with  the  bismuth  test 
is  found,  no  positive  conclusion  can  be  drawn  from  the  absence  of  any 
carbon  dioxide  or  the  appearance  of  a  very  insignificant  quantity.  The 
urine  absorbs  considerable  amounts  of  carbon  dioxide,  and  in  the  presence 
of  only  small  amounts  of  sugar  the  fermentation  test  as  above  performed 
may  lead  to  negative  or  inaccurate  results.  In  this  case  proceed  in  the 
following  way:  Treat  the  acid  urine,  or  urine  which  has  been  faintly 
acidified  with  tartaric  acid,  with  yeast  whose  activity  has  been  tested 
by  a  special  test  on  a  sugar  solution,  and  allow  it  to  stand  six  to  twelve 
hours  at  about  34-36°  C.  Then  test  again  with  the  bismuth  test,  and 
if  the  reaction  now  gives  negative  results,  then  sugar  was  previously 
present.  But  if  the  reaction  continues  to  give  positive  results,  then  it 
shows,  if  the  yeast  is  active,  the  presence  of  other  reducing,  unfer- 
mentable  substances. 

In  performing  the  fermentation  test  care  should  be  taken  that  the  urine 
be   acid   before   as  well  as  after  fermentation.     If  the   reaction  becomes 


1  Pfliiger's  Arch.,  118. 


806  URINE. 

alkaline  during  fermentation  (alkaline  fermentation),  then  the  test 
must  be  discarded.  The  vessel  must  be  perfectly  clean  and  strongly 
heated  before  use.  To  make  sure  the  urine  may  be  boiled  before  fer- 
mentation.1 

If  a  good  polariscope  is  at  hand  it  must  not  be  forgotten  to  control 
the  results  of  the  fermentation  by  determining  the  rotation  before  and 
after  fermentation.  The  phenylhydrazine  test  also,  in  many  otherwise 
doubtful  cases,  gives  good  service  in  testing  urines  for  sugar. 

Phenylhydrazine  Test.  Can  be  performed  in  the  following  manner: 
20-25  cc.  urine  in  a  test-tube  or  in  a  beaker  covered  with  a  watch-glass 
are  treated  with  1  gram  phenylhydrazine  hydrochloride  and  2  grams 
sodium  acetate,  and  after  solution  of  the  salts  it  is  warmed  on  the  water- 
bath  for  three-quarters  of  an  hour.  In  the  presence  of  sugar  even  dur- 
ing the  warming,  a  precipitate  occurs,  or  in  the  presence  of  only  a  little 
sugar,  at  least  after  the  gradual  cooling,  a  yellow,  crystalline  precipitate 
forms.  If  the  precipitate  is  very  slight,  it  can  be  collected  to  advantage 
by  means  of  a  centrifuge  and  investigated  by  aid  of  the  microscope. 
One  finds  at  least  a  few  phenylglucosazone  crystals  in  the  sediment 
while  the  appearance  of  smaller  or  larger  yellow  platelets  or  strongly 
refractive,  brown  globules  is  not  indicative  of  sugar.  In  the  presence 
of  large  amounts  of  sugar  in  the  urine  a  large  quantity  of  the  yellow 
needles  of  phenylglucosazone  or  a  mass  of  them  are  obtained. 

This  reaction  is  very  reliable,  and  by  it  the  presence  of  0.03  per  cent 
sugar  can  be  detected  (Rosenfeld,  Geyer2).  In  doubtful  cases  it  is 
necessary  to  investigate  the  nature  of  the  precipitate.  For  this  purpose 
dissolve  a  large  quantity  of  the  crystals  in  hot  alcohol,  treat  the  filtrate 
with  water,  and  boil  off  the  alcohol.  Still  better,  the  precipitate  is 
dissolved,  according  to  Neuberg,  in  some  pyridine,  and  again  precipi- 
tated as  crystals  by  the  addition  of  benzene,  ligroin,  or  ether.  If  the 
characteristic  yellow  crystalline  needles,  whose  melting-point  (204- 
205°  C.)  may  also  be  determined,  are  now  obtained,  then  this  test  is 
decisive  for  the  presence  of  sugar.  It  must  not  be  forgotten  that  fructose 
gives  the  same  osazone  as  glucose,  and  that  a  further  investigation  is 
necessary  in  certain  cases,  and  also  that  the  impure  crystals  of  phen- 
ylducosazone  have  a  much  lower  melting-point  than  the  pure  ones. 

The  following  modification  by  A.  Neumann  is  simple,  practical,  and  at  the 
same  time  sufficiently  delicate.  5  cc.  of  the  urine  are  treated  with  2  cc.  of  acetic 
acid  (30-per  cent)  saturated  with  sodium  acetate,  2  drops  of  pure  phenylhydrazine 

1  On  the  performance  of  the  fermentation  test  and  certain  sources  of  error,  see 
Salkow.sk i,  Berlin,  klin.  Wochenschr.,  1905  (Ewald-Festnummer),  and  Pfliiger,  Pfliiger's 
Arch.,  105  and  111. 

2  Rosenfeld,  Deutsch.  med.  Wochenschr.,  1888;  Geyer,  cited  from  Roos,  Zeitschr.. 
f.  physiol.  Chem.,  15. 


SUGAR  IN   URINE.  807 

added,  and  the  mixture  boiled  in  a  test-tube  until  it  measures  3  cc.  After  quickly 
cooling  warm  again  and  then  allow  it  to  cool  slowly.  After  5-10  minutes  beautifully 
formed  crystals  are  obtained  even  in  the  presence  of  only  0.02  per  cent  sugar. 
According  to  the  experience  of  ElAMMABSTEN  this  modification,  even  in  the  presence 
of  0.1  per  cent  sugar  in  concentrated  urines,  does  not  always  give  a  positive  reac- 
tion.    Salkowski  '  has  suggested  an  even  more  simple  method. 

The  value  of  the  phem  ihydrazine  test  lias  been  considerably  debated, 
and  the  objection  has  been  made  that  glucuronic  acids  also  give  a  similar 
precipitate.  A  confounding  with  glucuronic  acid  is,  according  to  Hirschl, 
not  to  be  apprehended  when  the  test  is  heated  in  the  water-bath  for  a 
long  time  (one  hour).  Kistermann  found  this  precaution  insufficient, 
and  Roos  states  that  the  phenylhydrazine  test  always  gives  a  positive 
result  with  human  urine,  which  coincides  with  E.  Holmgren's  2  and 
Hammersten's  experience.  This  test  only  show's  a  non-physiological 
quantity  of  sugar  when  a  rather  abundant  crystallization  is  obtained  from 
a  small  quantity  of  urine  (about  5-10  cc.)  Too  great  a  delicacy  of  this 
test  is  not  to  be  recommended. 

Rubxer's  test  is  performed  as  follows:  The  urine  is  precipitated  with  an  excess 
of  a  concentrated  lead-acetate  solution  and  the  nitrate  carefully  treated  with 
enough  ammonia  to  produce  a  flocculent  precipitate.  It  is  then  heated  to  boiling, 
when  the  precipitate  becomes  flesh-colored  or  pink  in  the  presence  of  sugar. 

Polarization.  This  test  is  of  great  value,  especially  as  in  many  cases 
it  quickly  differentiates  between  glucose  and  other  reducing,  sometimes 
levogyrate,  substances,  such  as  the  conjugated  glucuronic  acids.  In 
the  presence  of  only  very  little  sugar  the  value  of  this  test  depends  on 
the  delicacy  of  the  instrument  and  the  dexterity  of  the  observer.  As  a 
urine  which  showrs  no  rotation  or  is  actually  faintly  levorotatory,  may 
contain  0.2  per  cent  glucose  or  perhaps  even  more,  this  test  must  be 
combined  with  the  fermentation  test  if  we  are  seeking  very  small  amounts 
of  sugar.  The  sugar  in  these  cases  can  be  detected  only  by  the  use  of  a 
very  accurate  and  delicate  instrument.  This  method  is  in  many  cases 
not  serviceable  for  the  physician.  If  the  urine  is  to  be  clarified  and 
partly  decolorized  by  precipitation  with  lead  acetate,  it  must  be  done  in 
acid  solution  with  acetic  acid.3 

In  the  isolation  of  sugar  and  carbohydrates  from  the  urine  the  benzoic-acid 
esters  may  be  prepared  according  to  Baumann's  method.  The  urine  is  made 
alkaline  with  caustic  soda  to  precipitate  the  earthy  phosphates,  the  filtrate  treated 
with  10  cc.  of  benzoyl  chloride  and  120  cc.  of  10  per  cent  caustic  soda  solution 
for  even'  100  cc.  of  the  filtrate  (Reixbold  4),  and  shaken  until  the  odor  of  benzoyl 

1  Neumann,  Arch.  f.  (Anat.  u.)  Physiol.,  1899,  Suppl.  See  also  Margulies,  Berlin. 
klin.  Woehenschr.,  1900;  Salkowski,  Arbeiter  aus  dem  pathol.  Inst.,  Berlin,  1906. 

*  Hirschl,  Zeitschr.  f.  physiol.  Chein.,  14;  Kistermann,  Deutsch.  Arch.  f.  klin. 
Med.,  50;  Roos,  1.  c;  Holmgren,  Maly's  Jahresber.,  27. 

1  See  Grossmann,  Bioch.  Zeitschr.,  1. 

4  Pfluger's  Arch.,  91. 


808  URINE. 

chloride  has  disappeared.  After  standing  sufficiently  long  the  ester  is  collected, 
finely  divided,  and  saponified  with  an  alcoholic  solution  of  sodium  ethylate  in  the 
cold  according  to  Baisch's  method,1  and  the  various  carbohydrates  separated 
according  to  his  suggestion. 

If  small  quantities  of  sugar  are  to  be  isolated  from  the  urine,  precipitate  the 
urine  first  with  sugar  of  lead,  filter,  precipitate  the  filtrate  with  ammoniacal 
basic  lead  acetate,  wash  this  precipitate  with  water,  decompose  it  with  H2S  when 
suspended  in  water  and  use  the  filtrate  for  the  special  tests.  Schondorff  2 
has  suggested  a  method  for  the  detection  and  estimation  of  very  small  amounts 
of  sugar  based  upon  the  work  of  Patein  and  Dufau.  This  method  depends 
upon  precipitating  the  nitrogenous  substances  with  mercuric  nitrate. 

To  the  physician,  who  naturally  wants  simple  and  quick  methods, 
the  bismuth  test  is  especially  to  be  recommended.  If  this  test  gives  neg- 
ative results,  the  urine  is  to  be  considered  as  free  from  sugar  in  a  clinical 
sense.  If  it  gives  positive  results,  the  presence  of  sugar  must  be  con- 
trolled by  other  tests,  especially  by  the  fermentation  test. 

Other  tests  for  sugar,  as,  for  example,  the  reaction  with  orthonitrophenyl- 
propiolic  acid,  picric  acid,  diazobenzene-sulphonic  acid,  are  superfluous.  The 
reaction  with  a-naphthol,  which  is  a  reaction  for  carbohydrates  in  general,  for 
glucuronic  acid  and  mucin,  may,  because  of  its  extreme  delicacy,  give  rise  to 
mistakes,  and  is  therefore  not  to  be  recommenced  to  physicians.  Normal  urines 
give  this  test,  and  if  the  strongly  diluted  urine  gives  the  reaction  the  presence 
of  great  quantities  of  carbohydrates  may  be  suspected.  In  these  cases  more 
positive  results  are  obtained  by  using  other  tests.  This  test  requires  great  clean- 
liness, and  it  has  the  inconvenience  that  sufficiently  pure  sulphuric  acid  is  not 
always  readily  procurable.  Several  investigators,  such  as  v.  Udransky,  Luther, 
Roos  and  Treupel,3  have  investigated  this  test  in  regard  to  its  applicability 
as  an  approximate  test  for  carbohydrates  in  the  urine. 

Quantitative  Determination  of  Sugar  in  the  Urine.  The  quantity  of 
sugar  can  be  determined  by  titration,  by  fermentation  of  the  sugar,  by 
-polarization,  and  also  in  other  ways. 

The  titration  methods  are  based  upon  the  property  of  the  sugar  to 
reduce  metallic  oxides  in  alkaline  solutions.  As  the  titration  liquids 
(cupric  oxide  solution  in  the  Fehling-Soxhlet,  Pavy,  Bang,  Bertrand 
methods  and  mercuric  oxide  in  Knapp's  method)  are  also  reduced  by 
other  urinary  constituents,  these  reduction  methods  always  give  too  high 
results.  When  large  quantities  of  sugar  are  present,  as  in  typical 
diabetic  urine,  which  generally  contains  a  lower  percentage  of  normal 
reducing  constituents,  this  is  indeed  of  little  account;  but  when  small 
quantities  of  sugar  are  present  in  an  otherwise  normal  urine,  the  mistake 
may,  on  the  contrary,  be  important,  as  the  reducing  power  of  normal 
urine  may  correspond  to  5  p.  m.  glucose  (see  page  749).  In  such  cases 
the  titration  procedure  must  be  employed  in  connection  with  the  fer- 
mentation method,  which  will  be  described  later. 


1  Zeitschr.  f.  physiol.  Chem.,  19. 

J  Pfluger's  Anh.,  121,  which  cites  the  work  of  Patein  and  Dufau. 

1  See  Roos  and  Treupel,  Zeitschr.  f.  physiol.  Chem.,  15  and  16. 


ESTIMATION  OF  SUGAR  IN   URINE.  809 

Of  the  titration  methods  with  copper  solutions  the  method  suggested 
by  Bang  is  the  simplest,  and  at  the  same  time  seems  to  be  more  reliable 
than  any  of  the  others.  For  this  reason  we  will  describe  only  this  method 
and  refer  to  the  original  works  and  to  Hoppe-Seyler-Thierfelder, 
Handbuch  der  Chem.  Analyses,  1(.)()'.),  for  description  of  the  titration  of 
Fehling's  solution  according  to  Soxhlet  l  and  to  the  titration  accord- 
ing to  Pavy  and  Kumagawa-Suto.2 

Bang's  First  Method.3  The  principle  of  this  method  is  that  when  urine 
is  boiled  with  an  excess  of  a  solution  of  potassium  carbonate,  potassium 
thiocyanate  and  copper  sulphate,  copper  thiocyanide  is  formed,  and  this 
remains  in  solution  as  a  colorless  compound.  The  excess  of  cupric 
oxide  remaining  is  determined  by  titration  with  a  solution  of  hydroxyl- 
amine  until  the  blue  color  disappears.  The  quantity  of  sugar  is  calculated 
from  the  quantity  of  hydroxylamine  used. 

The  following  solutions  are  necessary:  (a)  A  copper  salt  solution 
containing  25  grams  cupric  sulphate  in  2  liters,  and  (6)  a  solution  con- 
taining 0.55  grams  hydroxylamine  sulphate  in  2  liters. 

The  copper  solution  is  prepared  in  the  following  manner:  Dissolve  100  grams 
potassium  bicarbonate  in  1300  ec.  water  in  a  2-liter  graduated  flask,  and  if  nec- 
essary warm  to  50-60°  C.  After  complete  solution  of  the  bicarbonate,  add 
400  grains  potassium  thiocyanate  and  500  grams  potassium  carbonate.  To 
this  solution,  which  must  have  the  temperature  of  the  room,  add  very  slowly 
150  cc.  of  a  copper  sulphate  solution,  which  contains  166.67  grams  copper  sul- 
phate (CuS04+5H^O)  per  liter,  then  add  water  up  to  2  liters.  This  solution 
unfortunately  does  not  keep  indefinitely,  still,  according  to  Andersen,  it  can  be 
kept  in  the  dark  up  to  3  months  and  its  strength  controlled  by  titration  with  the 
hydroxylamine  solution.  The  hydroxylamine  solution  is  prepared  by  dissolving 
200  grams  potassium  thiocyanate  in  about  1500  cc.  water  in  a  2-liter  graduated 
flask  and  adding  a  solution  of  6.55  grams  hydroxylamine  sulphate  in  water;  then 
add  water  to  the  2-liter  mark.  This  solution,  on  the  contrary  keeps,  but  it  must 
be  kept  in  a  dark-colored  bottle.  Equal  volumes  of  each  of  these  two  solutions 
should  exactly  correspond  to  each  other,  and  this  can  be  determined  by  titrating 
at  ordinary  temperature  50  cc.  of  the  copper  solution  (plus  10  cc.  water)  with  the 
hydroxylamine  solution. 

The  presence  of  proteid  does  not  interfere  with  the  reaction,  and  it 
is  not  necessary  to  remove  the  proteid.  The  urine  for  titration  should 
not  contain  more  than  0.6  per  cent  sugar.  If  the  amount  is  lower,  then 
10  cc.  of  urine  is  used  directly;  if  it  is  higher,  then  the  urine  is  corre- 
spondingly diluted  and  of  this  diluted  urine  we  also  make  use  of  10  cc. 
in  the  titration.  The  quantities  of  sugar  given  in  the  table  below  vary 
between  0.9  and  60  milligrams  in  10  cc. 

Performance  of  the  Determination.  10  cc.  of  the  sugar  fluid  are  placed 
in  a  glass  flask  and  treated  with  50  cc.  of  the  copper  solution.  This  is 
heated  on  a  wire-gauze  to  boiling,  boiled  for  three  minutes,  cooled 
quickly  with  water  to  the  temperature  of  the  room  and  then  the  hydrox- 

1  Journ.  f.  prakt.  Chem.,  (N.  F.),  21. 

2  Pavy,  The  Physiology  of  the  Carbohydrates,  London,  1894;  Kumagawa  and  Suto, 
Salkowski's  Festschr.,  1904;  Sahli,  Deutsch.  med.  Wochenschr.,  1905. 

J  Bang,  Bioch.  Zeitschr.,  2,  11,  32,  and  38.  See  also  Funk,  Zeitschr.  f.  physiol. 
Chem.,  56  and  69;  Jessen-Hansen,  Bioch.  Zeitschr.,  10  and  Andersen,  ibid.,  15  and  26. 


810 


URINE. 


ylamine  solution  allowed  to  flow  in  from  a  burette  until  the  blue  color 
disappears  and  the  solution  is  colorless,  or,  in  urine  poor  in  sugar,  is  yellow. 
The  sugar  in  milligrams  is  directly  obtained  from  the  amount  of  hydroxyl- 
amine  solution  used  by  referring  to  the  following  reduction  table : l 


Hydroxyl- 

amine 

solution 

used. 

Milligrams 
sugar. 

Hydroxyl- 

amine 

solution 

used. 

Milligrams 
sugar. 

Hydroxyl- 

amine 

solution 

used. 

Milligrams 
sugar. 

Hydroxyl- 

amine. 

solution 

used. 

Milligrams 
sugar. 

0.75 

60.0 

13.00 

39.0 

25.50 

23.5 

38.00 

10.4 

1.00 

59.4 

13.50 

38.3 

26.00 

22.9 

38.50 

9.9 

1.50 

58.4 

14.00 

37.7 

26.50 

22.3 

39.00 

9.4 

2.00 

57.3 

14.50 

37.1 

27.00 

21.8 

39.50 

9.0 

2.50 

56.2 

15.00 

36.4 

27.50 

21.2 

40.00 

8.5 

3.00 

55.0 

15.50 

35.8 

28.00 

20.7 

40.50 

8.1 

3.50 

54.3 

16.00 

35.1 

28.50 

20.1 

41.00 

7.6 

4.00 

53.4 

16.50 

34.5 

29.00 

19.6 

41.50 

7.2 

4.50 

52.6 

17.00 

33.9 

29.50 

19.1 

42.00 

6.7 

5.00 

51.6 

17.50 

33.3 

30.00 

18.6 

42.50 

6.3 

5.50 

50.7 

18.00 

32.6 

30.50 

18.0 

43.00 

5.8 

6.00 

49.8 

18.50 

32.0 

31.00 

17.5 

43.50 

5.4 

6.50 

48.9 

19.00 

31.4 

31.50 

17.0 

44.00 

4.9 

7.00 

48.0 

19.50 

30.8 

32.00 

16.5 

44.50 

4.5 

7.50 

47.2 

20.00 

30.2 

32.50 

15.9 

45.00 

4.1 

8.00 

46.3 

20.50 

29.6 

33.00 

15.4 

45.50 

3.7 

8.50 

45.5 

21.00 

29.0 

33.50 

14.9 

46.00 

3.3 

9.00 

44.7 

21.50 

28.3 

34.00 

14.4 

46.50 

2.9 

9.50 

44.0 

22.00 

27.7 

34.50 

13.9 

47.00 

2.5 

10.00 

43.3 

22.50 

27.1 

35.00 

13.4 

47.50 

2.1 

10.50 

42.5 

23.00 

26.5 

35.50 

12.9 

48.00 

1.7 

11.00 

41.8 

23.50 

25.8 

36.00 

12.4 

48.50 

1.3 

11.50 

41.1 

24.00 

25.2 

36.50 

11.9 

49.00 

0.9 

12.00 

40.4 

24.50 

24.6 

37.00 

11.4 

12.50 

39.7 

25.00 

24.1 

37.50 

10.9 

For  every  tV  cc.  hydroxylamine  solution  used  more  than  given  in  the  table 
between  49.00-15.00,  subtract  0.1  milligram  from  the  corresponding  sugar  value 
and  0.2  milligram  between  15.00-1.0. 

The  yellow  color  of  the  urine  may  be  somewhat  disturbing  for  the 
end  reaction  so  that  with  little  experience  an  error  of  0.5  cc.  hydroxylamine 
solution  ( =  about  0.5  milligram  sugar)  may  occur.  In  order  to  decolorize 
the  urine  we  can  precipitate,  according  to  Andersen,  with  mercuric 
nitrate,  when  the  greatest  part  of  the  disturbing  reducing  substances  are 
removed,  and  then  the  excess  of  mercury  removed  by  caustic  soda  and 
shaking  with  zinc.  Still  simpler  is  the  suggestion  mentioned  on  page 
805  with  blood-charcoal  after  acidification  with  acetic  acid. 

Bang  2  decolorizes  by  the  addition  of  2  cc.  alcohol  of  95-97  per  cent 
and  a  teaspoonful  of  blood-charcoal  to  18  cc.  urine,  shaking  and  filtering 
immediately.     By  this  means  50  per  cent  of  the  other  reducing  substances 


1  This  table  is  given  with  the  permission  of  the  publisher,  Julius  Springer,  Berlin, 
where  it  can  be  obtained  at  a  low  cost. 

2  Andersen,  Bioch.  Zeitschr.,  15  and  37;  Bang,  ibid.,  38. 


ESTIMATION   OF   SUGAR   IN    URINE.  811 

are  removed.     If  an  acidified  urine  is  used  for  the  titration  then  the  urine 
is  added  to  the  copper  solution  and  not  the  reverse. 

Bang's  Second  Method.  As  the  reagents  necessary  for  the  preceding  titration 
are  expensive,  and  as  the  copper  solution  only  keeps  for  three  months,  and  the 
preparation  of  the  solutions  requires  great  exactitude  and  is  somewhat  difficult, 
and  as  the  method  gives  somewhat  higher  results  than  other  reduction  methods 
due  to  the  high  alkali  and  salt  content  of  the  solutions,  Bang  «  has  recently 
modified  his  original  method.  Instead  of  potassium  thiocyanate  he  uses  potassium 
chloride,  which  can  also  keep  the  cuprous  oxide  in  solution  as  a  colorless  com- 
pound. Also  the  non-reduced  cupric  oxide  remaining,  as  in  the  early  method,  is  not 
determined,  but  the  cuprous  oxide  formed  in  the  reduction  with  the  sugar  is  directly 
determined  by  titration.  This  is  done  by  means  of  a  N/100  (or  N/10  or  X/25) 
iodine  solution,  which  in  the  alkaline  liquid  acts  oxidizinidy  with  the  formation 
of  cupric  oxide,  according  to  the  formula:  CuCl-f-I+K2C03  =CuC03-(-KC*l-(-KI. 
Starch  solution  is  used  as  indicator.  As  the  potassium  chloride  can  only  hold 
small  amounts  of  cuprous  oxide  in  solution,  and  as  the  end-reaction  with  the  blue 
iodine-starch  cannot  be  determined  with  ease  in  the  presence  of  large  amounts 
of  cupric  oxide  in  solution,  but  can  easily  be  clone  with  the  faintly  blue  coloration 
due  to  cupric  oxide,  by  this  method  a  maximum  of  10  milligrams  sugar  can  only  be 
determined.  On  this  account  a  urine  rich  in  sugar  must  be  diluted  considerably 
before  titration.  It  must  also  be  remarked  that  the  iodine  does  not  only  react 
with  the  cuprous  oxide  but  also  with  other  urinary  constituents,  and  the  importance 
of  this  method  on  titration  with  rich  urines,  poor  in  sugar,  has  not  been  sufficiently 
investigated.  This  method  has  given  good  results  with  pure  sugar  solutions  and 
with  blood;  but  as  its  use  for  the  determination  of  sugar  in  the  urine  has  not 
been  sufficiently  tested,  we  have  only  given  the  chief  points  of  the  method. 

Bertrand's  2  Titration  Method  is  more  complicated  than  Bang's  method  and 
does  not  seem  to  have  any  special  advantages  over  this  latter,  at  least  in  regard 
to  the  determination  of  sugar  in  the  urine.  A  part  of  the  cuprous  oxide  here  also 
remains  in  solution  and  like  the  titration,  according  to  Fehling,  the  cuprous 
oxide  sometimes  settles  only  with  difficulty.  As  this  method  seems  to  be  used 
extensively  we  will  give  the  principles  of  the  method. 

The  method  consists  in  boiling  the  sugar  solution  (sugar  urine)  with  an  excess 
of  Fehling's  solution.  The  cuprous  oxide,  freed  from  copper  salt  by  decantation 
and  washing  (under  special  precautions),  is  dissolved  by  ferric  sulphate  in  sulphuric 
acid,  and  the  ferrous  sulphate  produced  is  determined  by  titration  with  potassium 
permanganate,  standardized  by  oxalic  acid.  The  equations  of  the  reactions  are 
as  follows: 

1.  Cu20+Fe2(S04)3+H2S04  =  H20-r-2CuS04+2FeS04 

2.  10FeSO4+2KMnO4+8H2SO4  =8H20+5Fe2(S04)3+2MnS04+K2S04. 

2  Cu  are  equivalent  to  2  Fe,  and  as  these  are  equivalent  to  1  mol.  oxalic  acid, 
then  from  the  amount  of  oxalic  acid  (ammonium  oxalate)  used  in  the  standardiza- 
tion of  the  potassium  permanganate  solution  the  quantity  of  copper  separated  as 
cuprous  oxide  can  be  readily  calculated.  The  corresponding  quantity  of  sugar 
may  be  found  in  a  special  table. 

For  exact  determinations  of  sugar  the  method  as  suggested  by  Allihn 
and  modified  by  Pfluger  3  is  the  best  suited. 


1  Bioch.  Zeitschr.,  49. 

1  Bulletin  de  la  Soc.  chim.,  (3),  35,  (1906). 

1  Pfluger's  Arch.,  66. 


812  URINE. 

The  titration  according  to  Knapp  depends  on  the  fact  that  mercuric 
cyanide  in  alkaline  solution  is  reduced  to  metallic  mercury  by  glucose.  The 
titration  liquid  should  contain  10  grams  of  chemically  pure  dry  mercuric  cyanide 
and  100  cc.  of  caustic-soda  solution  of  a  specific  gravity  of  1.145  per  liter.  When 
the  titration  is  performed  as  described  below  (according  to  Worm-Muller  and 
Otto),  20  cc.  of  this  solution  should  correspond  to  exactly  0.05  gram  of  glucose. 
If  the  process  is  carried  out  in  other  ways,  the  value  of  the  solution  is  different. 

In  this  titration  also,  the  quantity  of  sugar  in  the  urine  should  be  between 
\  and  1  per  cent,  and  the  extent  of  dilution  necessary  be  determined  by  a  pre- 
liminary test.  To  determine  the  end-reaction  as  described  below,  the  test  for  the 
excess  of  mercury  is  made  with  sulphuretted  hydrogen. 

In  performing  the  titration  allow  20  cc.  of  Knapp's  solution  to  flow  into  a 
flask  and  dilute  with  SO  cc.  of  water,  or  when  the  urine  contains  less  than  0.5 
per  cent  of  sugar  use  only  40-60  cc.  After  this  heat  to  boiling  and  allow  the  diluted 
urine  to  flow  gradually  into  the  hot  solution,  at  first  2  cc,  then  1  cc,  then  0.5  cc, 
then  0.2  cc,  and  lastly  0.1  cc.  After  each  addition  let  it  boil  \  minute.  When 
the  end-reaction  is  approaching,  the  liquid  begins  to  clarify  and  the  mercury 
separates  with  the  phosphates.  The  end-reaction  is  determined  by  taking  a 
drop  of  the  upper  layer  of  the  liquid  into  a  capillary  tube  and  then  blowing  it  out 
on  pure  white  filter-paper.  The  moist  spot  is  first  held  over  a  bottle  containing 
fuming  hydrochloric  acid  and  then  over  strong  sulphuretted  hydrogen.  The 
presence  of  a  minimum  quantity  of  mercury  salt  in  the  liquid  is  shown  by  the 
spot  becoming  yellowish,  which  is  best  seen  when  it  is  compared  with  a  second 
spot  that  has  not  been  exposed  to  the  gas.  The  end-reaction  is  still  clearer  when 
a  small  part  of  the  liquid  is  filtered,  acidified  with  acetic  acid,  and  tested  with 
sulphuretted  hydrogen  (Otto)1.  As  the  added  quantity  of  urine  contains  0.050 
gram  sugar  the  calculation  of  the  percentage  content  in  sugar,  bearing  in  mind 
the  extent  of  dilution,  is  very  simple. 

This  titration,  unlike  the  previous  one,  may  be  performed  equally  well  by 
daylight  and  by  artificial  light.  It  is  applicable  even  when  the  quantity  of  sugar 
in  the  urine  is  very  small  and  that  of  the  other  urinary  constituents  is  normal.  It 
is  more  easily  performed,  and  the  titration  liquids  may  be  kept  without  decom- 
posing for  a  long  time  (Worm-Muller  and  his  pupils  2) .  There  is  diversity  of 
opinion,  nevertheless,  among  investigators  on  the  value  of  this  titration  method. 

Estimation  of  the  Quantity  of  Sugar  by  Fermentation.  This 
may  be  done  in  various  ways:  the  simplest  method,  and  one  at  the  same 
time  sufficiently  exact  for  ordinary  cases,  is  that  of  Roberts.  This 
consists  in  determining  the  specific  gravity  of  the  urine  before  and  after 
fermentation.  In  the  fermentation  of  sugar,  carbon  dioxide  and  alcohol 
are  formed  as  chief  products,  and  the  specific  gravity  is  lowered,  partly 
on  account  of  the  disappearance  of  the  sugar  and  partly  on  account  of 
the  production  of  alcohol.  Roberts  found  that  a  decrease  of  0.001  in 
the  specific  gravity  corresponded  to  0.23  per  cent  sugar,  and  this  has  been 
substantiated  since  by  several  other  investigators  (Worm-Muller  and 
others).  If  the  urine,  for  example,  has  a  specific  gravity  of  1.030  before 
fermentation  and  1.008  after,  then  the  quantity  of  sugar  contained  therein 
was  22X0.23  =  5.06  per  cent. 

In  performing  this  test  the  specific  gravity  must  be  taken  at  the  same 
temperature  before  and  after  the  fermentation.  The  urine  must  be 
faintly  acid,  and  when  necessary  it  should  be  acidified  with  a  little  hydro- 


1  Journal  f.  prakt.  Chem.,  26. 

2  Pfluger'e  Arch.,  16  and  23. 


ESTIMATION  OF  SUGAR  IN  URINE.  813 

chloric  acid  or  sulphuric  acid.  The  activity  of  the  yeast  must,  when, 
necessary,  be  controlled  by  a  special  test.  Place  200  cc.  of  the  urine 
in  a  400  cc.  flask,  add  a  piece  of  compressed  yeast  the  size  of  a  pea,  and 
subdivide  the  yeast  through  the  liquid  by  Bhaking;  close  the  flask  with 
a  stopper  provided  with  a  finely- drawn-out  glass  tube,  and  allow  the  test 
to  stand  at  the  temperature  of  the  room  or,  still  better,  at  30-35°  C. 
After  twenty-four  hours  the  fermentation  is  ordinarily  ended,  but  this 
must  be  verified  by  the  bismuth  test.  After  complete  fermentation  filter 
through  a  dry  filter,  bring  the  filtrate  to  the  proper  temperature,  and 
determine  the  specific  gravity. 

If  the  specific  gravity  be  determined  with  a  good  pycnometer  sup- 
plied with  a  thermometer  and  an  expansion-tube,  this  method,  when  the 
quantity  of  sugar  is  not  less  than  0.4-0.5  per  cent,  gives,  according  to 
Worm-Muller,  very  exact  results,  but  this  has  been  disputed  by  Budde.1 
For  the  physician  the  method  in  this  form  is  not  serviceable.  Even  when 
the  specific  gravity  is  determined  by  a  delicate  urinometer  which  can 
give  the  density  to  the  fourth  decimal,  exact  results  are  not  obtained, 
because  of  the  ordinary  errors  of  the  method  (Budde);  but  the  errors 
are  usually  smaller  than  those  which  occur  in  titrations  made  by  unskilled 
hands. 

When  the  quantity  of  sugar  is  less  than  1.5  per  cent,  these  methods 
cannot  be  used.  Such  small  amounts  cannot,  as  already  mentioned, 
be  determined  by  titration  directly,  because  of  the  reducing  power  of  nor- 
mal urine.  In  such  cases,  it  is  better  to  first  determine  the  reducing  power 
of  the  urine  by  titration  according  to  Bang  or  Knapp,  then  ferment  the 
urine  with  the  addition  of  yeast  and  titrate  again.  The  difference 
found  between  the  two  titrations  calculated  as  sugar  gives  the  true 
quantity  of  the  latter. 

The  determination  of  the  sugar  by  fermentation  can  be  so  performed 
that  the  loss  in  weight  due  to  the  CO2  can  be  estimated,  or  the  volume 
of  the  gas  measured.  For  this  last  purpose  Lohnstein  2  has  constructed 
a  special  fermentation  saccharometer,  and  his  "  precision  saccharometer  " 
is  to  be  recommended.  Based  upon  Lohnstein 's  instrument,  Wag- 
ner3 has  constructed  a  "fermentation  saccharo-manometer,"  which 
has  certain  advantages  over  Lohnstein 's  apparatus. 

Estimations  of  Sugar  by  Polarization.  In  this  method  the 
urine  must  be  clear,  not  too  deeply  colored,  and,  above  all,  must  not 
contain  any  other  optically  active  substances  besides  glucose.  The 
urine  may  contain  several  levorotatory  substances  such  as  proteids, 
0-oxybutyric  acid,  conjugated  glucuronic  acids,  the  so-called  Leo's  sugar 
and  less  often  cystine,  all  of  which  are  unfermentable.  The  proteid  is 
removed  by  coagulation,  and  the  others  are  detected  by  the  polariscope 
after  complete  fermentation.  The  fermentable  fructose  is  detected  in  a 
special  manner  (see  below),  and  the  dextrorotatory  milk-sugar  differs 
from  glucose  in  its  not  fermenting  readily.     By  using  a  delicate  instru- 


1  Roberts,  The  Lancet,  1862;    Worm-Muller,  Pfluger's  Arch.,  33  and  37;    Budde, 
ibid.,  40,  and  Zeitschr.  f.  physiol.  Chem.,  13.     See  Lohnstein,  Pfluger's  Arch.,  62. 

2  Berlin,  klin.  Wochenschr.,  35,  and  Allg.  med.  Central-Ztg.,  1899;  Goldman,  Chem. 
Centralbl.,  1907,  1,  1149. 

'  Munch,  med.  Wochensch.,  1905. 


$14  URINE. 

ment  and  with  sufficient  practice  very  exact  results  can  be  obtained  by 
this  method.  The  value  of  this  procedure  consists  in  the  rapidity  with 
which  the  determination  can  be  made.  In  using  instruments  specially 
constructed  for  clinical  purposes  the  accuracy  is  less  than  with  the  less 
expensive  fermentation  test.  Under  such  circumstances,  and  as  the 
estimation  by  means  of  polarization  can  be  performed  with  exactitude 
only  by  specially  trained  chemists,  it  is  hardly  worth  while  to  give  this 
method  in  detail,  and  the  reader  is  referred  to  handbooks  for  hints  in  the 
use  of  the  apparatus. 

Hasselbach  and  Lindhard  1  have  recently  suggested  a  method  for  the 
quantitative  estimation  of  sugar  which  is  based  on  the  decolorization  of  an  alkaline 
safranin  solution  in  the  presence  of  sugar. 

Fructose  (levulose).  Levogyrate  urines  containing  sugar  have  been 
noted  by  several  investigators,  although  the  nature  of  the  sugar  was  not 
well  known  to  the  earlier  observers.  In  recent  years  several  positively 
authentic  cases  of  levulosuria  have  been  described,  and  also  cases  of 
diabetes  have  been  found  where  fructose  exists  in  the  urine  besides  glucose. 
Reports  on  this  subject  do  not  agree,  however.2 

Fructose  may  be  detected  as  follows:  The  urine  is  levorotatory,  and 
the  levorotatory  substance  ferments  with  yeast.  The  urine  gives  the 
ordinary  reduction  tests  and  the  ordinary  phenylglucosazone.  With 
methylphenylhydrazine  it  gives  the  characteristic  fructose  methyl- 
phenylosazone,  and  it  also  gives  Seliwanoff's  reaction  on  heating 
after  the  addition  of  an  equal  volume  of  hydrochloric  acid  and  a  little 
resorcin.  With  this  test  it  must  be  remarked  that  too  lengthy  or  too 
strong  heating  must  not  be  applied,  since  other  carbohydrates  may  also 
give  the  reaction  (see  page  218  and  the  works  of  Rosin  and  Umber3). 
In  the  presence  of  fructose  a  red  coloration  appears.  After  cooling  it 
can  be  neutralized  with  soda  and  shaken  out  with  amyl  alcohol,  (Rosin) 
or  with  acetic  ether  (Borchardt).  The  amyl  alcohol  removes  a  red 
pigment  which  gives  a  band  in  the  spectrum  between  E  and  b  and  on 
stronger  concentration  also  a  band  in  the  blue  at  F.  The  acetic  ether 
in  the  presence  of  fructose  becomes  yellow,  and  this  is  more  characteristic 
according  to  Borchardt  than  Rosin's  method,  which  has  certain 
fallacies.  The  simultaneous  presence  of  nitrites  and  indican  disturbs 
the  test,  and  in  this  case  first  remove  the  nitrous  acid  by  boiling  the 
urine,  acidified  with  acetic  acid  or  hydrochloric  acid  for  one  minute.  In 
order  to  remove  other  disturbing  pigments,  Malfatti  suggests  the 
oxidation  of  the  urine  with  a  little  hydrochloric  acid  and  potassium 
permanganate.  Jolles  4  has  suggested  a  method  for  detecting  fructose 
besides  glucose  by  means  of  a  diphenylamine  solution. 


1  Bioch.  Zeitschr.,  27. 

*See  Borchardt,  Zeitschr.  f.  physiol.  Chem.,  55  and  60;  W.  Voit,  ibid.,  58  and  61; 
Adler,  Pfluger's  Arch.,  139. 

a  Umber,  Salkowski's  Festschrift,  Berlin,  1904;  Rosin,  ibid.,  and  Zeitschr.  f. 
physiol.  Chem.,  38. 

4  Rosin,  1.  c;  Borchardt,  1.  c;  Malfatti,  Zeitschr.  f.  physiol.  Chem.,  58;  Jolles 
and  Mauthner,  Chem.  Centralbl.,  1910,  1,  483. 


LACTOSE  IN  URINE.  815- 

Maltose  sometimes  occurs  in  the  urine  according  to  Lepine  and  Boulud, 
Geelmuyden,1  who  also  held  this  view,  now  states  that  maltose  does  not  occur 
in  the  urine. 

Laiose  is  a  substance  named  by  Huppert  and  found  by  Leo  -  in  diabetic  urines 
in  certain  cases,  and  which  he  considers  as  a  sugar.  It  is  levogyrate,  amorphous, 
and  does  not  taste  sweet,  but  rather  sharp  and  salty.  Laiose  lias  a  reducing 
action  on  metallic  oxides,  does  not  ferment,  and  gives  a  non-crystalline,  yellowish- 
brown  oil  with  phenylhydrazine.  There  is  no  positive  proof  as  yet  that  this 
substance  is  a  sugar. 

Lactose.  The  appearance  of  lactose  in  the  urine  of  pregnant  women 
was  first  shown  by  the  observations  of  De  Sinety  and  F.  Hofmeister, 
and  this  has  been  substantiated  by  other  investigators.  After  the  inges- 
tion of  large  quantities  of  milk-sugar  some  lactose  may  be  found  in  the 
urine  (see  Chapter  VIII  on  absorption).  Langstein  and  Steinitz  have 
observed  the  passage  of  lactose  and  also  of  galactose  3  into  the  urine  of 
nurslings  with  disease  of  the  stomach.  The  passage  of  lactose  into  the 
urine  is  called  lactosuria. 

The  positive  detection  of  this  sugar  in  the  urine  is  difficult,  because 
it  is,  like  glucose,  dextrogyrate,  and  also  gives  the  usual  reduction  tests. 
If  urine  contains  a  dextrogyrate,  non-fermentable  sugar  which  reduces 
bismuth  solutions,  then  it  is  very  probable  that  it  contains  lactose.  It 
must  be  remarked  that  the  fermentation  test  for  lactose  is,  according  to 
the  experience  of  Lusk  and  Voit,4  best  performed  by  using  pure  cultivated 
yeast  (saccharomyces  apiculatus).  This  yeast  only  ferments  the  glucose 
while  it  does  not  decompose  the  milk-sugar.  Voit  claims  that  if  Rub- 
ner's  test  is  performed  without  heating  to  boiling,  but  only  to  80°  C, 
the  color  becomes  yellow  or  brown  in  the  presence  of  lactose,  instead 
of  red.  The  most  positive  means  for  the  detection  of  this  sugar  is  to 
isolate  the  sugar  from  the  urine.  This  may  be  done  by  the  method 
suggested  by  F.  Hofmeister.5 

R.  Bauer  6  detects  galactose  as  well  as  lactose  in  the  urine  by  oxidation  with 
concentrated  nitric  acid,  producing  mucic  acid. 

Cammidge's  reaction,  which  is  recommended  in  the  diagnosis  of  acute  diseases 
of  the  pancreas,  consists  in  that  certain  urines  do  not  give  the  phenylhydrazine 
reaction  directly,  but  only  after  boiling  with  an  acid.  The  reason  of  this  is  not 
known  and  the  reaction  is  partly  due  to  cane-sugar,  in  part  to  pentoses  or  gluco- 
ronic  acid  and  in  part  to  mixtures  of  bodies. 


1  Lepine  and  Boulud,  Compt.  Rend.,  132;   Geelmuyden,  Zeitschr.  f.  klin,  Med.,  70. 

2  Virchow's  Arch.,  107. 

3  Hofmeister,   Zeitschr.   f.   physiol.   Chem.,    1,   which  also   contains   the  pertinent 
literature.     See  also  Lemaire,  ibid.,  21;  Langstein  and  Steinitz,  Hofmeister's  Beitrage,  7. 

4  Carl  Voit,  Ueber  Die  Glycogenbiklung  nach  Aufnahme  verschiedener  Zuckeraten, 
Zeitschr.  f.  Biologic,  28. 

5  Hofmeister,   Zeitschr.   f.  physiol.  Chem.,   1,   which  also  contains  the  pertinent 
literature. 

6  Zeitschr.  f.  Physiol.  Chem.,  51. 


816  URINE. 

Pentoses.  Salkowski  and  Jastrowitz  first  found  in  the  urine  of 
persons  addicted  to  the  morphine  habit  a  variety  of  sugar  which  was  a 
pentose  and  yielded  an  osazone  which  melted  at  159°  C.  Since  this 
several  other  cases  of  pentosuria  have  been  observed,  and  according  to 
Kulz  and  Vogel  and  others  small  amounts  of  pentose  also  occur  in  the 
urine  of  diabetics,  as  also  in  the  urine  of  dogs  with  pancreatic  or  phlorhizin 
diabetes.1 

The  pentose  isolated  by  Neubeeg  from  the  urine  in  chronic  pentosuria 
was  cW-arabinose.  Luzzatto  and  Klercker  studied  cases  of  pento- 
suria and  found  Z-arabinose.  In  alimentary  pentosuria  the  £-arabinose 
of  the  plant  food  may  be  found  in  the  urine.  The  appearance  of  pentoses 
in  the  urine  after  eating  fruits  and  fruit-juices  has  been  repeatedly 
observed  by  Blumenthal  and  also  by  v.  Jaksch.  According  to  Comi- 
notti  2  pentoses  habitually  occur  in  human  urine  on  a  mixed  diet. 

A  urine  containing  pentose  reduces  bismuth  as  well  as  copper  solu- 
tions, although  the  reduction  is  not  so  rapid,  but  appears  gradually. 
If  only  pentose  is  present,  the  urine  does  not  ferment,  but  in  the  presence 
of  glucose  small  amounts  of  pentose  may  also  undergo  fermentation. 
The  preparation  of  the  osazone  serves  in  the  detection  of  pentoses  and 
when  obtained  from  the  urine  it  melts  at  156-160°  C.  The  phloroglucin 
or  orcin  tests  can  also  be  employed  (see  page  209).  Of  these  the  last  is 
most  preferable,  especially  as  it  excludes  a  confusion  with  the  conjugated 
glucuronic  acids. 

The  orcin  test  can  be  performed  as  follows:  5  cc.  of  the  urine  are  mixed 
with  an  equal  volume  of  HC1  sp.gr.  1.19,  a  small  amount  of  orcin  added  and 
the  whole  heated  to  boiling.  As  soon  as  a  greenish  cloudiness  appears 
cool  the  mixture  off  and  shake  carefully  with  amyl  alcohol.  The  amyl- 
alcohol  solution  is  used  in  the  spectroscopic  examination.  The  pre- 
cipitation of  a  bluish-green  pigment  is  in  itself  significant. 

Bial  a  uses  as  reagent  30  per  cent  hydrochloric  acid,  which  contains  1  gram 
of  orcin  and  25  drops  of  a  ferric-chloride  solution  (62.9  per  cent  of  the  crystalline 
salt)  in  500  cc.  of  the  acid.  4.5  cc.  of  the  reagent  are  heated  to  boiling  and  then  a 
few  drops  ("not  more  than  1  cc.)  of  the  urine  are  added  to  the  hot  but  not  boiling 
liquid.  In  the  presence  of  pentose  the  liquid  turns  a  beautiful  green.  The  use- 
fulness of  Bial's  reagent  is  questioned  by  several  experimenters.  The  delicacy 
is  too  great  and  the  possibility  of  confounding  with  other  carbohydrates  is  not 
excluded.  In  regard  to  the  numerous  modifications  of  this  test  and  to  Jolles' 
reaction  we  refer  to  page  209.     The  same  for  the  quantitative  estimation  of  pen- 


1  In  regard   to  the  literature,   see  footnote   1,   page  20S.     See  also   Blumenthal, 
"Die  Pentosurie,"  Deutsche  Klinik,  1902. 

2  Blumenthal,   Deutsche  Klinik,   1902;     v.   Jaksch,  Centralbl.   f.   innere  Medizin, 
1906;  Cominotti,  Bioch.  Zeitschr.,  22. 

'  Deutsch.  med.  Wochenschr.,  1903;  see  also  footnote  4,  page  209. 


CONJUGATED  GLUCURONIC  ACIDS.  817 

toses.  Jolles1  considers  the  preparation  of  the  osazonc  as  a  specially  conclusive 
test  and  the  distillation  of  the  osazone  with  hydrochloric  acid  and  testing  the 
distillate  with  Bial's  reagent. 

Rosenberger  2  believes  he  has  detected  a  heptose  in  the  urine  in  a  case  of 
diabetes.  According  to  him  and  to  Geelmuyden  3  probably  different  varieties 
of  sugar,  which  are  not  well  known,  can  possibly  occur  in  urine  of  diabetics. 

Conjugated  Glucuronic  Acids.  Certain  conjugated  glucuronic  acids 
such  as  menthol-  and  turpentine-glucuronic  acid  may  spontaneously 
decompose  in  the  urine,  and  in  this  case  they  may  readily  lead  to  a  con- 
fusion with  pentoses.  The  urine  should  always  be  fresh  as  possible 
for  these  examinations. 

A  confusion  of  the  glucuronic  acids,  which  have  a  reducing  power  on 
copper  or  bismuth  solutions,  with  glucose  and  fructose,  can  be  pre- 
vented by  the  fermentation  test.  They  may  also  be  distinguished  from 
glucose  by  their  optical  behavior,  as  the  conjugated  glucuronic  acids 
are  levogyrate.  On  boiling  with  an  acid,  dextrorotatory  glucuronic  acid 
is  produced  and  the  levorotation  is  changed  to  dextrorotation. 

The  conjugated  glucuronic  acids,  like  the  pentoses,  give  the  phloro- 
glucin-hydrochloric-acid  test.  On  the  contrary  they  do  not  give  the 
orcin  test  directly,  but  only  after  cleavage  with  the  setting  free  of  glucuronic 
acid.  On  using  Bial's  reagent  no  mistaking  for  pentoses  occurs,  although 
this  statement  requires  further  substantiation.  The  pentoses  may  also 
be  isolated  and  identified  by  their  osazones.  Certain  readily  decomposable 
glucuronic  acids  can  here  give  phenylhydrazine  compounds.  In  order  to 
detect  glucuronic  acid  in  the  osazone  precipitate,  we  can,  as  suggested 
by  Xeuberg  and  Saneyoshi4  take  a  knife  point  (about  8  milligrams) 
of  the  precipitate,  dissolve  in  4  cc.  strong  hydrochloric  acid,  dilute  with 
4  cc.  water,  heat  to  boiling,  add  at  least  0.1  gram  naphthoresorcin,  warm 
for  \  minute,  allow  to  slowly  cool  to  50°  and  shake  with  benzene.  In  the 
presence  of  glucuronic  acid  the  benzene  solution  is  violet  with  an  absorp- 
tion in  the  yellowish-green . 

The  occurrence  of  conjugated  glucuronic  acids  in  the  urine  is  shown 
when  the  urine  does  not  give  the  orcin-hydrochloric-acid  reaction  directly, 
but  only  after  boiling  with  the  acid.  The  naphthoresorcin  reaction, 
as  suggested  by  Tollens,  can  also  be  used.  To  5  cc.  urine  add  0.5  cc. 
of  a  1  per  cent  alcoholic  solution  of  naphthoresorcin  and  5  cc.  hydro- 
chloric acid  (sp.gr.  1.19),  boil  for  one  minute,  allow  to  stand  four  minutes, 


1  Jolles,  Bioch.  Zeitschr.,  2,  Centralbl.  f.  inn.  Med.,  1907  and  1912,  and  Zeitschr. 
f.  anal.  Chem.,  46. 

2  Zeitschr.  f.  physiol.  Chem.,  49. 

3  Rosenberger,  Centralbl.  f.  inn.  Med.,  28;    Geelmuyden,  Zeitschr.  f.  klin.  Med., 
58,  63,  and  70. 

4  Bioch.  Zeitschr.,  36. 


818  URINE. 

cool  and  shake  with  ether.  In  the  presence  of  glucuronic  acid  the  ether 
becomes  violet  or  blue,  and  shows  the  absorption  bands,  given  on  page 
223.  According  to  Neuberg  this  test,  which  is  not  specific  for  glucuronic 
acid,  is  best  performed  with  the  naphthoresorcin  in  substance.  This 
test  is  more  conclusive,  if,  as  suggested  by  Neuberg  and  Schewket,1 
the  residue  from  an  ethereal  extract  of  the  acidified  urine  is  used. 

The  surest  method  is  that  suggested  by  Mayer  and  Neuberg,  which 
consists  in  precipitating  the  urine  with  basic  lead  acetate,  decomposing 
the  precipitate  with  H2S,  boiling  with  dilute  sulphuric  acid  in  order  to 
split  the  conjugated  acid,  and  then  after  neutralizing  with  soda,  prepar- 
ing the  characteristic  bromphenylhydrazine  compound  of  glucuronic 
acid  (see  page  223)  with  ^-bromphenylhydrazine  hydrochloride  and 
sodium  acetate.  Hervieux  2  has  slightly  modified  this  method.  In 
regard  to  the  quantitative  estimation  of  glucuronic  acid  we  must  refer 
to  the  work  of  C.  Tollens.3 

Inosite  seems  to  be  a  normal  urinary  constituent,  although  it  occurs 
only  in  very  small  quantities  (Hoppe-Seyler,  Starkenstein  4) .  In 
diabetes  insipidus,  as  well  as  after  excessive  drinking  of  water,  it  occurs 
in  large  quantities  in  the  urine  because  of  a  more  abundant  washing- 
out  of  the  tissues. 

For  the  detection  of  inosite  we  make  use  of  the  method  given  on  page  581, 
with  the  modifications  suggested  by  Meillere  and  Starkenstein. 

Acetone  Bodies  (acetone,  acetoacetic  acid,  /3-oxybutyric  acid).  These 
bodies,  whose  occurrence  in  the  urine  and  formation  in  the  organism  have 
been  the  subject  of  numerous  investigations,  occur  in  the  urine  espe- 
cially in  diabetes  mellitus,  but  also  in  many  other  diseases.5  According 
to  v.  Jaksch  and  others,  acetone  is  a  normal  urinary  constituent,  though 
it  may  occur  only  in  very  small  amounts  (0.01  gram  in  twenty-four  hours). 

In  regard  to  the  origin  of  these  bodies  it  was  formerly  considered  that 
they  were  produced  by  an  increased  destruction  of  protein.  One  of  the 
various  reasons  for  this  was  the  increase  in  the  elimination  of  acetone 


1  B.  Tollens,  Ber.  d.  d.  chem.  Gesellsch.,  41,  1788,  and  C.  Tollens,  Zeitschr.  f.  physiol. 
Chem.,  56;  Neuberg,  Bioch.  Zeitschr.,  24;  Neuberg  and  Schewket,  ibid.,  44;  see  also 
Mandel  and  Neuberg,  ibid.,  13. 

2  Mayer  and  Neuberg,  Zeitschr.  f.  physiol.  Chem.,  29;  Hervieux,  Compt.  rend, 
eoc.  biol.,  63. 

3  Zeitschr.  f.  physiol.  Chem.,  61. 

4  Starkenstein,  Zeitschr.  f.  exp.  Path.  u.  Therap.,  5,  which  contains  the  literature. 

'■>  In  regard  to  the  extensive  literature  on  acetone  bodies  the  reader  is  referred  to- 
Huppcrt-N'eubauer,  Ham-Analyse,  10.  Aufl.,  and  v.  Noorden's  Lehrb.  d.  Pathol,  des 
Stoffwechsels.  Berlin,  1906,  and  for  recent  work,  Magnus-Levy,  Die  Azetonkorper,. 
Ergbn.  d.  inn.  Med.  u.  Kinderheilk.,  I. 


ACETONE  BODIES.  819 

and  acctoacetic  acid  during  inanition  (v.  Jaksch,  Fr.  Muller  :).  This 
also  stands  in  accord  with  the  observations  that  a  considerable  increase 
in  the  quantity  of  acetone  and  acctoacetic  acid  eliminated  is  observed 
in  such  diseases  as  fevers,  diabetes,  digestive  disturbances,  mental  dis- 
eases with  abstinence  and  cachexia,  where  the  body  protein  is  largely 
destroyed.  The  formation  of  acetone  bodies  from  protein  is  also  indi- 
cated by  the  fact  that  acetone  has  been  obtained  as  an  oxidation  prod- 
uct from  gelatin  and  protein  (Blumenthal  and  Neuberg,  Orgler2). 
The  investigations  of  Embden  and  collaborators  are  more  conclusive. 
After  Embden  and  Kalberlah  showed  that  the  liver  is  an  organ  where 
acetone  is  formed,  Embden,  Salomon  and  Schmidt3  showed  by  exper- 
iments on  extirpated  livers,  that  butyric  acid,  oxybutyric  acid,  leucine, 
tyrosine  and  in  fact  those  aromatic  bodies  which,  like  tyrosine,  phenyl- 
alanine, phenyl-a-lactic  acid  and  homogentisic  acid  contain  a  combustible 
benzene  nucleus,  are  transformed,  in  the  liver,  into  acetone.  Research, 
which  has  been  continued  further  by  Embden  and  his  collaborators 
and  substantiated  by  others,  such  as  Baer,  and  Blum,  Borchardt 
and  Lange,  Neubauer  and  Gross,  Schmitz  and  Fr.  Sachs  4  has  shown 
that  there  can  be  no  doubt  that  certain  amino-acids,  especially  leucine, 
are  strong  acetone  formers,  and  consequently  that  acetone  can  be  formed 
from  protein.  Protamines  and  histones  can  also  increase  the  acetone 
elimination  (Borchardt)  or,  as  we  say,  may  have  a  "  ketoplastic  " 
action,  and  it  is  therefore  possible  that  acetone  can  be  formed  from 
arginine  with  a-amino-valerianic  acid  as  intermediary  step  (Borchardt 
and  Lange). 

As  we  cannot  deny  the  possibility  of  a  formation  of  acetone  from  pro- 
teins, on  the  other  hand  we  have  observations  which  are  inconsistent  with 
the  origin  of  the  acetone  bodies  entirely  from  the  proteins.  Thus  no  par- 
allelism exists  between  the  acetone  bodies  and  the  nitrogen  excretion 
in  diabetics,  and  the  fact,  that  in  man  no  certain  relation  exists  between 
the  acetone  elimination  and  the  nitrogen  and  sulphur  excretion,  seems  to 
show  that  the  acetone  bodies  are  not  entirely  derived  from  the  proteins. 
In  man  the  excretion  of  acetone  does  not  increase  with  the  rise  in  the 


1  v.  Jaksch,  Ueber  Acetonurie  und  Diaceturie.  Berlin,  1885;  Fr.  Muller,  Bericht 
iiber  die  Ergebnisse  des  an  Cetti  ausgefiihrten  Hungerversuches.  Berlin,  klin.  Wochen- 
echr.,  1887. 

2  Blumenthal  and  Neuberg,  Deutsch.  med.  Wochenschr.,  1901;  Orgler,  Hofmeis- 
ter's  Beit  rage,  1. 

1  Hofmeister's  Beitriige,  8. 

4  Embden,  ibid.,  11,  with  Marx,  Engel,  Lattes  and  Michaud,  ibid.,  11;  Baer  and 
Blum,  Arch.  f.  exp.  Path.  u.  Pharm.,  55,  56,  and  62;  Borchardt,  ibid,  53,  with  Lange, 
Hofmeister"s  Beitriige,  8;  Neubauer  and  Gross,  Zeitschr.  f.  physiol.  Chem.,  67;  Schmitz, 
Bioch.  Zeitschr.,  28;  Fr.  Sachs,  ibid.,  27. 


820  URINE. 

quantity  of  protein,  and  an  increase  in  the  latter  above  the  average  causes 
a  diminution  in  the  elimination  of  acetone  (Rosenfeld,  Hirschfeld, 
Fr.  Voit1). 

The  carbohydrates  cannot  be  considered  as  material  for  the  forma- 
tion of  acetone  bodies.  It  is  generally  admitted  that  in  man  the 
exclusion  of  carbohydrates  from  the  food  or  the  diminution  in  their 
amount  or  their  assimilation  may  lead  to  more  or  less  increased  elimina- 
tion of  acetone  bodies.  This  behavior  may  occur  in  diabetes  as  well  as 
in  starvation  and  in  the  above-mentioned  diseased  conditions.  The 
increased  elimination  of  acetone  with  food  lacking  carbohydrates  also 
occurs  in  healthy  persons  with  a  fatty  diet  but  with  a  sufficient  supply  of 
calories  in  other  ways  (alimentary  acetonuria).  With  an  abundant 
supply  of  carbohydrates  the  elimination  of  acetone  bodies  may  be  greatly 
diminished  or  even  stopped  entirely.  The  carbohydrates  therefore 
act  "  antiketoplastic,"  and  a  similar  retarding  action  can  be  produced 
by  certain  other  substances,  such  as  glycerin  (Hirschfeld),  lactic  acid 
and  glutaric  acid  (Baer  and  Blum)  alanine  and  asparagin  (Forssner, 
Borchardt  and  Lange2).  Certain  bodies  like  glycerine,  lactic  acid, 
alanine,  asparagin,  which  cause  a  sugar  formation  or  increased  elimina- 
tion of  sugar,  act  in  the  same  way. 

It  must  not  be  overlooked  that  the  conditions  are  different  in  man 
and  in  other  carnivora  (Geelmuyden,  Fr.  Voit).  In  dogs  the  elimina- 
tion of  acetone  bodies  is  not  increased  in  starvation,  but  is  reduced;  it 
is  augmented  with  increased  quantities  of  meat,  runs  parallel  with  the 
nitrogen  excretion,  and  is  not  diminished  by  carbohydrates  (Fr.  Voit  3) . 
In  spite  of  this  divergent  behavior  an  unmistakable  relation  also  exists 
in  the  dog  between  the  elimination  of  acetone  bodies  and  the  carbo- 
hydrate metabolism,  because  in  phlorhizin  diabetes  the  acidosis  occurs 
only  after  the  glycogen  has  been  consumed  (Marum4). 

As  the  carbohydrates  cannot  be  acetone-formers,  then  a  second 
source  only  remains,  namely,  the  fats.  As  proof  of  this  there  are  certain 
cases  of  diabetes  with  strong  elimination  of  acetone  bodies  (/3-oxybutyric 
acid)  where  the  quantity  of  protein  transformed  was  too  small  to  account 
for  the  acetone  bodies  (Magnus-Levy).  The  free  elimination  of  acetone 
bodies  in  starvation  may  also  depend  upon  the  fact  that  a  great  part  of 


1  Hirschfeld,  Zeitschr.  f.  klin.  Med.,  28;  Geelmuyden,  see  Maly's  Jahresber.,  26, 
and  Zeitschr.  f.  physiol.  Chem.,  23  and  26;  Rosenfeld,  Centralbl.  f.  innere  Med.,  16; 
Voit,  Deutsch.  Arch.  f.  klin.  Med.,  66. 

2  Borchardt  and  Lange,  1.  c;  Hofmeister's  Beitrage,  9;  which  also  cites  other 
works;  Baer  and  Blum,  ibid.,  10;  Forssner,  Skand.  Arch.  f.  Physiol.,  25. 

1  See  footnote  1. 

4  Hofmeister's  Beitrage,  10. 


ACETONE  BODIES.  821 

the  body  fat  is  consumed,  and  in  several  cases  a  certain  relation  has 
been  found  between  the  fat  consumed  and  the  acetone  bodies  eliminated. 
Certain  investigators  (Geelmuyden,  Schwarz,  Waldvogel)  have 
also  observed  an  increase  in  the  acetonuria  on  partaking  of  fatty  food, 
and  Forssner  l  has  indeed  found  a  certain  parallelism  lei  ween  the 
acetone  elimination  and  the  fat  taken  up.  For  the  present  the  fats  are 
considered  as  the  most  important  source  of  the  acetone  bodies. 

The  three  acetone  bodies  occurring  in  the  urine,  as  above  stated,  are 
acetone,  acetoacetic  acid  and  /3-oxybutyric  acid,  and  this  last  is  considered 
as  the  mother-substance  of  the  other  two.  If  /3-oxybutyric  acid, 
CH3.CHOH.CH2.COOH,  is  introduced  into  the  animal  body,  it  is  burnt 
if  the  quantity  is  not  too  great,  while  if  in  excess  it  passes  into  the  urine 
as  acetoacetic  acid,  CH3.CO.CH2COOH.  This  acid  can  also  be  burnt, 
but  if  large  quantities  are  introduced  it  appears  in  part  in  the  urine  and 
readily  splits  into  acetone,  CH3.CO.CH3,  and  CO2.  Acetone  is  in  part 
burnt  in  the  animal  body,  but  a  part  is  eliminated  by  the  kidneys  and 
especially  by  the  lungs.  We  can  imagine  that  the  /3-oxybutyric  acid  is 
a  physiological  metabolic  product  which  normally  is  completely  changed 
into  acetoacetic  acid  and  acetone,  and  in  diabetes  and  especially  with 
lack  of  carbohydrates  is  formed  to  an  increased  extent,  or  its  combustion 
made  more  difficult,  so  that  in  the  first  place  acetone  and  acetoacetic 
acid  pass  into  the  urine  and  in  severe  cases  also  /3-oxybutyric  acid  (acidosis). 
In  this  connection  it  must  be  borne  in  mind  that,  because  of  the  previously- 
mentioned  (page  774)  reversibility  of  the  process,  the  direction  may  also 
be  reversed,  that  is  acetoacetic  acid  can  also  be  changed  into  /3-oxybutyric 
acid  in  the  animal  body  and  this  has  been  proven  by  perfusion  of  livers 
(Friedmann  and  Maase)  as  well  as  in  animals  (Dakin)  and  in  diabetics 
(O.  Neubauer)2. 

Since  leucine  in  perfusion  experiments  with  livers  yields  acetoacetic  acid 
(Embden  and  Engel)  and  also,  as  Baer  and  Blum3  found,  that  leucine  and  iso- 
valeric acid  increased  the  /3-oxybutyric  acid  elimination  in  diabetics,  it  has  been 
accepted  that  a  formation  of  /3-oxybutyric  acid  takes  place  from  the  leucine 
with  isovaleric  acid  as  an  intermediary  product : 

(leucine  CH3)2CH.CH2.CH(XH2).Co6h->(CH3)2CH.CH2.COOH,  isovaleric  acid). 
Valine  (a-amino-valeric  acid  (CH3)2CH.CH(NH2).COOH)  is,  on  the  contrary, 
not  an  acetone  former. 

1  Magnus-Levy,  Arch.  f.  exp.  Path.  u.  Pharm.,  42;  Geelmuyden,  1.  0.,  and  Norsk, 
Magasin  for  Laegevidenskaben,  1900;  see  also  Zeitschr.  f.  physiol.  Chem.,  41;  Schwarz, 
Deutsch.  Arch.  f.  klin.  Med.,  1903;  Waldvogel,  Centralbl.  f.  inn.  Med.,  20;  Forssner, 
jskand.  Arch.  f.  Physiol.,  22  and  23. 

2  Friedmann  and  Maase,  Bioch.  Zeitschr.,  27;  Dakin,  Journ.  of  biol.  Chem.,  8; 
Neubauer,  Maly's  Jahresb.,  40,  849. 

3  Arch.  f.  exp.  Path.  u.  Pharm.,  55  and  56;  Embden  and  Engel,  Hofme.ster's 
Beitrage,  11. 


822  URINE. 

In  regard  to  the  formation  of  acetone  bodies  from  fat  it  must  be 
remarked  that  glycerin  has  an  antiketoplastic  action,  and  that  the 
fatty  acids  can  only  be  considered.  As  to  the  behavior  of  these  in  the 
formation  of  acetone,  Embden  and  Marx1  have  shown  that  only  those 
normal  fatty  acids  which  contain  an  even  number  of  carbon  atoms  are 
acetone  formers,  while  those  with  an  uneven  number  of  carbon  atoms 
are  without  action  in  this  regard.  This  is  true  at  least  for  the  acids  from 
n-decanoic  acid  to  n-butyric  acid,  which  latter  is  a  strong  acetone  former. 
As  in  diabetics  a  greater  number  of  oxybutyric  acid  molecules  can  be 
eliminated  than  corresponds  to  the  number  of  fatty  acid  molecules  decom- 
posed, it  seems  as  if  more  than  one  molecule  of  /3-oxybutyric  acid  is  pro- 
duced from  one  molecule  of  fatty  acid.  We  cannot  therefore  admit 
of  a  simple  demolition  of  the  fatty  acids  to  butyric  acid  (by  consecutive 
oxidation  attacks  in  the  /3-position) ,  but  rather  a  destruction  of  the  fatty 
acid  molecules  into  several  parts,  and  these  take  part  in  the  formation  of 
/3-oxybutyric  acid. 

A  synthetical  formation  of  /3-oxybutyric  acid  has  been  accomplished  by  Geel- 
muyden  and  others,  but  especially  by  Magnus-Levy,  starting'  with  acetaldehyde, 
according  to  the  hypothesis  of  Spiro.  It  is  also  interesting  that  Friedmann  2 
has  shown  by  perfusion  experiments  with  livers  that  aldehyde  ammonia,  and  to 
a  greater  extent  aldol,  are  acetone  formers.  It  must  therefore  be  admitted  that 
first  a  condensation  of  the  aldehyde  to  aldol  takes  place,  CH3.COH-f  CH3.COH 
=  CH3.CH(OH).CH2.COH,  and  that  /3-oxybutryic  acid,  CH3.CH(OH).CH2.COOH, 
is  formed  from  this  by  oxidation. 

According  to  the  above-mentioned  perfusion  experiments  it  must 
be  admitted  that  the  liver  is  important  in  the  formation  of  acetone 
bodies,  and  Embden  and  Lattes  have  found  that  the  ability  of  the  liver 
of  the  dog  with  pancreas  diabetes  or  phloridzin  diabetes  to  produce 
acetone  is  much  greater  than  the  liver  of  the  normal  animal.  On  the 
other  hand,  as  shown  by  Embden  and  Michaud,3  in  dogs  and  oxen  the 
liver  also  has  a  strong  destructive  action  upon  acetoacetic  acid.  A 
similar  action  is  also  found  in  the  kidneys,  muscles  and  spleen  of  dogs 
and  pigs.  The  destructive  action  of  fresh  organs  is  much  stronger  upon 
acetoacetic  acid  than  upon  acetone.  They  could  not  find  any  special 
cleavage  products,  and  the  above-mentioned,  so-called  demolition  may 
therefore  perhaps  in  part  be  a  reformation  of  /3-oxybutyric  acid  from  the 
acetoacetic  acid. 

Acetone,  C.3HoO,  dimethylketone,  CH3.CO.CH3,  is  a  thin,  water- 
clear  liquid,  boiling  at  56.3°  and  possessing  a  pleasant  odor  of  fruit, 

1  Hofmeister's  Beitriige,  11. 

2  Geelmuyden,  Zeitschr.  f.  physiol.  Chem.,  23  and  26;  Magnus-Levy,  Arch.  f.  exp. 
Path.  u.  Pharm.,  42;  Friedmann,  Hofmeister's  Beitriige,  11. 

3  Embden  and  Lattes,  Hofmeister's  Beitriige,  11;  Embden  and  Michaud,  ibid.,  11. 


ACETONE.  823 

which  in  diabetes  gives  a  pomaceous  or  fruit  odor  to  the  urine  as  well  as 
the  expired  air.  It  is  Lighter  than  water,  with  which  it  mixes  in  all 
proportions,  also  with  alcohol  and  ether.  The  most  important  reactions 
for  acetone  are  the  following: 

Lieben's  Iodoform  Test.  When  a  watery  solution  of  acetone  is  treated 
with  alkali  and  then  with  some  iodo-potassium-iodide  solution  and  gently 
warmed,  a  yellow  precipitate  of  iodoform  is  produced,  which  is  known 
by  its  odor  and  by  the  appearance  of  the  crystals  (six-sided  plates  or  stars) 
under  the  microscope.  This  reaction  is  very  delicate,  but  it  is  not  char- 
acteristic of  acetone.  Gunning's  modification  of  the  iodoform  test  con- 
sists in  using  an  alcoholic  solution  of  iodine  and  ammonia  instead  of  the 
iodine  dissolved  in  potassium  iodide  and  alkali  hydroxide.  In  this  case, 
besides  iodoform,  a  black  precipitate  of  nitrogen  iodide  is  formed,  but 
this  gradually  disappears  on  standing,  leaving  the  iodoform  visible.  This 
modification  has  the  advantage  that  it  does  not  give  any  iodoform  with 
alcohol  or  aldehyde.  On  the  other  hand,  it  is  not  quite  so  delicate, 
but  still  it  detects  0.01  milligram  of  acetone  in  1  cc. 

Frommer's  l  Test.  This  reagent  is  a  10  per  cent  alcoholic  solution 
of  salicylaldehyde.  Add  1-2  cc.  of  this  solution  to  10  cc.  of  the  solution 
(urine)  and  add  to  this  mixture  1  gram  KOH  in  substance,  when  a 
carmine-red  color  will  be  observed.  If  necessary  warm  to  about  70°  C. 
This  reaction  is  just  as  delicate  as  the  above. 

Reynold's  Mercuric-oxide  Test  is  based  on  the  power  of  acetone  to 
dissolve  freshly  precipitated  HgO.  A  mercuric-chloride  solution  is  pre- 
cipitated by  alcoholic  caustic  potash.  To  this  add  the  liquid  to  be 
tested,  shake  well,  and  filter.  In  the  presence  of  acetone  the  filtrate 
contains  mercury,  which  may  be  detected  by  ammonium  sulphide. 
This  test  has  about  the  same  delicacy  as  Gunning's  test.  Aldehydes 
also  dissolve  appreciable  quantities  of  mercuric  oxide. 

Legal's  Sodium  Nitroprusside  Test.  If  an  acetone  solution  is  treated 
with  a  few  drops  of  a  freshly  prepared  sodium-nit roprusside  solution 
and  then  with  caustic-potash  or  soda  solution,  the  liquid  is  colored  ruby- 
red.  Creatinine  gives  the  same  color;  but  if  the  mixture  is  saturated 
with  acetic  acid,  the  color  becomes  carmine  or  purplish  red  in  the  presence 
of  acetone,  but  yellow  and  then  gradually  green  and  blue  in  the  presence 
of  creatinine.  With  this  test  paracresol  responds  with  a  reddish-yellow 
color,  which  becomes  light  pink  when  acidified  with  acetic  acid  and  can- 
not be  mistaken  for  acetone.  Rothera  2  has  suggested  a  modification 
which  is  more  delicate  by  using  ammonium  salts  and  ammonia. 

Penzoldt's  Indigo  Test  depends  on  the  fact  that  orthonitrobenzaldehyde 
in  alkaline  solution  with  acetone  yields  indigo.     A  warm  saturated  and 

1  Berl.  klin.  Wochenschr.,  1905.  2  Journ.  of  Physiol.,  37. 


824  URINE. 

then  cooled  solution  of  the  aldehyde  is  treated  with  the  liquid  to  be  tested 
for  acetone  and  next  with  caustic  soda.  In  the  presence  of  acetone  the 
liquid  first  becomes  yellow,  then  green,  and  lastly  indigo  separates;  and 
this  may  be  dissolved  with  a  blue  color  by  shaking  with  chloroform; 
1.6  milligrams  acetone  can  be  detected  by  this  test. 

Acetoacetic  Acid,  C4HGO3,  acetylacetic  acid,  diacetic  acid,  CH3.CO. 
CH2.COOH,  is  a  colorless,  strongly  acid  liquid  which  mixes  with  water, 
alcohol,  and  ether  in  all  proportions.  On  heating  to  boiling  with  water, 
and  especially  with  acids,  it  decomposes  into  carbon  dioxide  and  ace- 
tone, and  therefore  gives  the  above-mentioned  reactions  for  acetone. 
It  differs  from  acetone  in  that  it  gives  a  violet-red  or  brownish-red 
color  with  a  dilute  ferric-chloride  solution.  For  the  detection  of  this 
acid  we  make  use  of  the  following  reactions  which  may  be  applied  directly 
to  the  urine: 

Gerhardt's  Reaction.  Treat  10-15  cc.  of  the  urine  with  ferric- 
chloride  solution  until  it  fails  to  give  a  precipitate  filter,  and  add  some 
more  ferric  chloride.  In  the  presence  of  acetoacetic  acid  a  wine-red 
color  is  obtained.  The  color  becomes  paler  at  the  room  temperature 
within  twenty-four  hours,  but  more  quickly  on  boiling  (differing  from 
salicylic  acid,  phenol,  sulphocyanides) .  A  portion  of  the  urine  slightly 
acidified  and  boiled  does  not  give  this  reaction  on  cooling,  on  account 
of  the  decomposition  of  the  acetoacetic  acid. 

Arnold  and  Lipliawsky's  Reaction.  6  cc.  of  a  solution  contain- 
ing 1  gram  of  p-aminoacetophenone  and  2  cc.  of  concentrated  hydro- 
chloric acid  in  100  cc.  of  water  are  mixed  with  3  cc.  of  a  1  per  cent  potas- 
sium-nitrite solution  and  then  treated  with  an  equal  volume  of  urine. 
A  few  drops  of  concentrated  ammonia  are  now  added  and  violently 
shaken.  A  brick-red  coloration  is  obtained.  Then  take  10  drops  to 
2  cc.  of  this  mixture  (according  to  the  quantity  of  acetoacetic  acid  in 
the  urine),  add  15-20  cc.  HO  of  sp.gr.  1.19,  3  cc.  of  chloroform,  and 
2-4  drops  of  ferric-chloride  solution  and  mix  without  shaking.  In  the 
presence  of  acetoacetic  acid  the  chloroform  is  colored  violet  or  blue 
(otherwise  only  yellowish  or  faintly  red).  This  reaction  is  more  delicate 
than  the  preceding  test  and  reacts  with  0.04  p.m.  acetoacetic  acid.  Large 
amounts  of  acetone  (but  not  the  quantity  occurring  in  urines)  give  this 
reaction  according  to  Allard.1 

Bondi  and  Schwarz's  2  Reaction.  5  cc.  of  the  urine  is  treated  drop  by  drop 
with  iodine-potassium  iodide  solution  until  the  color  is  orange-red.  Then  warm 
gently  and  when  the  orange-red  color  has  disappeared  add  the  iodine  solution 
again  until  the  color  remains  permanent  on  warming.  Then  boil,  when  the  irritat- 
ing vapors  of  iodo-acetone  will  attack  the  eyes.     Acetone  does  not  give  this  reaction. 

1  Arnold,    Wicn.   klin.   Wochenschr.,    1899,   and  Centralbl.   f.   innere  Med.,    1900; 
Lipliawsky,  Deutsch.  med.  Wochenschr.,  1901;  Allard,  Berl.  klin.  Wochenschr.,  1901. 
"  Wxn.  klin.  Wochenschr.,  1906. 


ACETOACETIC  ACID.     (9-OXYBUTYRIC  ACID.  825 

Detection  of  Acetone  and  Acetoacetic  Acid  in  the  Urine.  Before  test- 
ing for  acetone  test  for  acetoacetic  acid;  as  this  acid  gradually  decom- 
poses on  allowing  the  urine  to  stand,  the  specimen  must  be  as  fresh  as 
possible.  In  the  presence  of  acetoacetic  acid  the  urine  gives  the  above- 
mentioned  tests.  In  testing  for  acetone  in  the  presence  of  acetoacetic 
acid  make  the  urine  slightly  alkaline  and  shake  in  a  separatory  funnel 
with  ether  free  from  alcohol  and  acetone.  Remove  the  ether  and  shake 
it  with  water,  which  takes  up  the  acetone,  and  test  for  acetone  in  the 
watery  solution. 

In  the  absence  of  acetoacetic  acid  the  acetone  may  be  tested  for 
directly  in  the  urine;  this  may  be  done  by  Frommer's  test  or  Legal's 
test.  These  tests,  which  are  only  approximate,  are  of  value  only  when 
the  urine  contains  a  considerable  amount  of  acetone. 

For  a  more  accurate  test  \vc  distill  at  least  250  cc.  of  the  urine  faintly  acidified 
with  sulphuric  acid,  care  being  taken  to  have  a  good  condensation.  Most  of  the 
acetone  is  contained  in  the  first  10-20  cc.  of  the  distillate.  A  better  result  may 
be  obtained  by  distilling  a  large  quantity  of  urine  until  about  tV  has  been  dis- 
tilled off,  acidify  the  distillate  with  hydrochloric  acid,  redistill  and  repeat  this 
several  times,  collecting  the  first  portion  of  each  distillation.  The  final  distillate 
is  used  for  the  above  reactions.1  Salkowski  and  Borchardt  have  called  attention 
to  the  fact  that  in  the  distillation  of  an  acidified  urine  containing  sugar  for  the 
detection  or  estimation  of  acetone,  a  substance  giving  iodoform  can  be  formed 
from  the  sugar  if  the  distillation  is  carried  too  far.  According  to  Borchardt  2 
the  urine  must  therefore  first  lie  diluted  with  water,  or  the  concentration  prevented 
by  the  addition  of  water  dropwise  during  distillation. 

The  quantitative  estimation  of  acetone  (also  that  formed  from  the 
acetoacetic  acid)  is  done  by  distilling  the  urine  after  the  addition  of  acetic 
acid  or  a  little  sulphuric  acid.  The  quantity  of  acetone  in  the  distillate 
can  be  determined,  according  to  the  Huppert-Messinger  method,  by 
converting  it  into  iodoform  by  means  of  potassium  iodide  and  then  titrat- 
ing the  quantity  of  iodine  used  in  the  formation  of  the  iodoform.  The 
precipitation  of  the  acetone  as  p-nitrophenylhydrazone-acetone  by  means 
of  p-nitrophenylhydrazine  in  acetic  acid  solution  can  also  be  used  for 
determining  the  acetone  in  the  distillate  (v.  Ekenstein  and  Blanksma 
and  Moller).  In  regard  to  these  methods  we  refer  to.3  Embden  and 
Schliep  and  Folin  4  have  suggested  methods  for  determining  the  quan- 
tity of  acetone  and  acetoacetic  acid  separately.  In  regard  to  these 
estimations  we  must  refer  to  the  work  of  Embden  and  Schmitz.5 

0-Oxybutyric  Acid,  C,4H8U3,  =  CH?.CH(OH).CH2.COOH,  ordinarily 
forms  an  odorless  syrup,  but  may  also  be  obtained  as  crystals.  It  is  readily 
soluble  in  water,  alcohol,  and  ether.     It  is  levorotatory ;    (a)D=—  24.12° 


1  See  also  Salkowski,  Pfluger's  Arch.,  56. 
*  Hofmeister's  Beitnige,  8. 

3  Hoppe-Seyler,  Thierfelder,  8.  Aufl.,  617  and  618. 

4  Embden  and  Schliep,  Centralbl.  f.  d.  ges.  Phys.  u.  Path.  d.  Stoffwechsel,  1907; 
Folin,  Journ.  of  biol.  Chem.,  3.  In  regard  to  the  quantitative  estimation  of  acetone 
see  also  0.  Sammet,  Zeitschr.  f.  physiol.  Chem.,  83. 

8  Abderhalden's  Handbuch  der  biochemischen  Arbeitsmethoden,  Bd.  3. 


826  URINE. 

for  solutions  of  1-11  per  cent  and  has  a  disturbing  action  upon  the  deter- 
mination of  sugar  by  means  of  the  polariscope.  It  is  not  precipitated 
by  basic  lead  acetate  or  by  ammoniacal  lead  acetate,  neither  does  it 
ferment.  On  boiling  with  water,  especially  in  the  presence  of  a  mineral 
acid,  this  acid  decomposes  into  a-crotonic  acid,  which  melts  at  71-72° 
C,  and  water,  CH:,.CH(OH).CH2.COOH  =  H20-r-CH3.CH:CH.COOH. 
It  yields  acetone  on  oxidation  with  a  chromic-acid  mixture. 

Detection  of  0-Oxybutyric  Acid  in  the  Urine.  If  a  urine  is  still  levo- 
gyrate  after  fermentation  with  yeast,  the  presence  of  oxybutyric  acid 
is  probable.  A  further  test  may  be  made,  according  to  Rulz,  by  evaporat- 
ing the  fermented  urine  to  a  syrup  and,  after  the  addition  of  an  equal 
volume  of  concentrated  sulphuric  acid,  distilling  directly  without  cool- 
ing, a-crotonic  acid  is  produced,  which  distills  over,  and,  after  collect- 
ing in  a  test-tube,  crystals  which  melt  at  +72°  C.  separate  on  cooling. 
If  no  crystals  are  obtained,  shake  the  distillate  repeatedly  with  ether  and 
let  this  spontaneously  evaporate.  The  crystals  which  separate  out 
can  be  purified  according  to  Embden  and  Schmitz  by  redissolving  in  ether, 
evaporating  the  chief  part  of  the  ether  and  precipitating  with  petroleum- 
ether,  which  removes  the  volatile  fatty  acids  and  benzoic  acid. 

The  quantitative  estimation  is  done  by  complete  extraction  of  the 
P-oxy butyric  acid  by  ether  and  determining  the  specific  rotation.  The 
extraction  can  be  done  according  to  Magnus-Levy  l  or  according  to 
Bergell.2  Other  methods  of  estimating  /3-oxybutyric  acid  have  been 
suggested  by  Darmstadter,  Boekelman  and  Bouma.3  In  regard  to 
the  quantitative  estimation  we  refer  to  Embden  and  Schmitz.4 

Ehrlich's  5  Urine  Test.  Mix  250  cc.  of  a  solution  which  contains  50  cc.  of 
HC1  and  1  gram  of  sulphanilic  acid  in  one  liter,  with  5  cc.  of  a  \  per  cent  solution 
of  sodium  nitrite  (which  produces  very  little  of  the  active  body,  sulphodiazo- 
benzene).  In  performing  this  test  treat  the  urine  with  an  equal  volume  of  this 
mixture  and  then  supersaturate  with  ammonia.  Normal  urine  will  become 
yellow  or  orange  after  the  addition  of  ammonia  (aromatic  oxyacids  may  after  a  cer- 
tain time  give  red  azo  bodies  which  color  the  upper  layer  of  the  phosphate  sediment). 
In  pathological  urines  there  sometimes  occurs  (and  this  is  the  characteristic 
diazo  reaction)  a  primary  yellow  coloration,  with  very  marked  secondary  red 
coloration  on  the  addition  of  ammonia,  and  the  froth  is  also  tinged  with  red. 
The  upper  layer  of  the  sediment  becomes  greenish.  The  body  which  gives  this 
reaction  is  unknown,  but  it  especially  occurs  in  the  urine  of  typhoid  patients 
(Ehrlich).  Opinions  differ  in  regard  to  the  significance  of  this  reaction.  If 
the  urine  is  made  alkaline  with  sodium  carbonate  instead  of  ammonia  and  treated 

1  See  Hoppe-Seyler,  Thierfelder's  Handbuch,  8.  Aufl.,  G19,  and  Geelmuyden,  Ham- 
marsten's  Festschr.,  1906. 

5  Zeitschr.  f.  physiol.  Chem.,  33. 

a  Darmstadter,  ibid.,  37;  Boekelman  and  Bouma,  see  Maly's  Jahresber,  31. 

1  Bee  footnote  •",,  page  825. 

'Ehrlich,  Zeitschr.  f.  klin.  Med.,  5.  See  also  Clemens,  Deutsch.  Arch.  f.  klin. 
Med.,  (53  (literature).     Kutscher  and  Engeland,  footnote  1,  page  758. 


CYSTINE.  827 

with  a  freshly  prepared  solution  of  diazobenzene  sulphonic  acid  made  alkaline 
with  sodium  carbonate,  normal  urine  also  rives  an  orange  or  Bordeaux-red 
coloration.  The  known  normal  urinary  constituents  which  give  the  diazo  reac- 
tion are  the  aromatic  oxyacidfl,  antoxyproteic  acid  and  the  imidazole  derivative 
found  by  Engeland  (see  page  758). 

Ehrlich's  reaction  with  p-dime1 hylaminobenzaldehyde  has  already  been 
discussed  in  connection  with  urobilinogen. 

Hosenbach's  urine  test,  which  consists  in  adding  nitric  acid  drop  by  drop 
to  the  boiling-hot  urine  and  obtaining  a  claret-red  coloration  and  a  bluish-red 
foam  on  shaking,  depends  upon  the  formation  of  indigo  substances,  especially 
indigo-red.1 

Fat  in  the  Urine.  The  elimination  of  a  urine  which  in  appearance  and  rich- 
ness in  fat  resembles  chyle  is  called  chyluria.  It  habitually  contains  a  proteid 
and  often  fibrin.  Chyluria  occurs  mostly  in  the  inhabitants  of  the  tropics. 
Lipuria,  or  the  elimination  of  fat  with  the  urine,  may  appear  in  apparently  healthy 
persons,  sometimes  with  and  sometimes  without  albuminuria,  in  pregnancy,  and 
also  in  certain  diseases,  as  in  diabetes,  poisoning  with  phosphorus,  and  fatty 
degeneration  of  the  kidneys. 

Fat  is  usually  detected  by  the  microscope.  It  may  also  be  dissolved  with 
ether,  and  may  invariably  be  detected  by  evaporating  the  urine  to  dryness  and 
extracting  the  residue  with  ether. 

Cholesterin  is  also  sometimes  found  in  the  urine  in  chyluria  and  in  a  few  other 
cases. 

Amino-acids.  Leucine  and  tyrosine  have  been  repeatedly  found  in  urine  by 
the  older  methods,  especially  in  acute  yellow  atrophy  of  the  liver,  in  acute  phos- 
phorus poisoning,  and  in  severe  cases  of  typhoid  and  smallpox.  Since  (3-naphtha- 
lene  sulphochloride  has  been  used  in  the  detection  of  amino-acids  these  bodies  have 
not  only  been  repeatedly  found  in  normal  urine  (glycocoll,  see  page  756),  but  also 
in  pathological  urines.2 

Cystine.  Baumann  and  Goldmann  claim  that  a  substance  similar 
to  cystine  occurs  in  very  small  amounts  in  normal  urine.  This  substance 
occurs  in  large  quantities  in  the  urine  of  dogs  after  poisoning  with  phos- 
phorus. Cystine  itself  is  only  found  with  positiveness,  and  even  then 
very  rarely,  in  urinary  calculi  and  in  pathological  urines,  from  which 
it  may  separate  as  a  sediment.  Cystinuria  occurs  oftener  in  men  than 
in  women.  Baumann  and  v.  Udranszky  found  in  urine  in  cystinuria 
the  two  diamines,  cadaverine  (pentamethylendiamine)  and  putrescine 
(tetramethylendiamine),  which  are  produced  in  the  putrefaction  of 
proteins.  Cases  of  cystinuria  may  occur  with  or  without  the  occurrence 
of  diamines  in  the  urine,  and  only  rarely  are  the  diamines  found  in  the 
urine  as  well  as  in  the  feces,  which  perhaps  depends  upon  the  fact,  as 
found  by  Cammidge  and  Garrod  3  in  one  case,  that  the  diamines  occur 

1  See  Rosin,  Virchow's  Arch.,  123. 

2  Ignatowski,  Zeitschr.  f.  physiol.  Chem.,  42;  Abderhalden  and  Schittenhelm, 
ibid.,  45;  Abderhalden  and  Barker,  ibid.,  42.     See  also  footnote  5,  page  756,  and  2,  757. 

3  Baumann,  Zeitschr.  f.  physiol.  Chem.,  8.  In  regard  to  the  literature  on  cystinuria 
6ee  Brenzinger,  ibid.,  16;  Baumann  and  Goldmann,  ibid.,  12;  Baumann  and  v. 
Udranszky,  ibid.,  13;  Stadthagen  and  Brieger,  Berlin,  klin.  Wochenschr.,  1889; 
Cammidge  and  Garrod,  Journ.  of  Path,  and  Bacteriol.,  1900  (literature  on  diamines 
in  the  urine  and  feces);  Loewy  and  Neuberg,  Bioeh.  Zeitschr.,  2;  Wolf  and  Schaffer, 
Journ.  of  biol.  Chem.,  4;  Williams  and  Wolf,  ibid.,  6. 


828  UKINE. 

only  from  time  to  time  in  the  feces.  Cystinuria  is  generally  admitted  as 
rather  an  anomaly  in  the  protein  metabolism  where  the  cystine  for 
unknown  reasons  is  not  destroyed  as  ordinarily.  It  is  remarkable 
that  the  cystine  of  the  food-proteins  is  eliminated  by  the  urine,  while  in 
cystinurics,  at  least  sometimes,  such  cystine  introduced  is  quantitatively 
transformed.1  Certain  observations,  such  as  the  appearance  of  lysine 
in  the  urine  of  cystinurics  (Ackermann  and  Kutscher  2),  make  it  probable 
that  the  demolition  of  other  amino-acids  is  diminished  in  cystinuria. 
The  properties  and  reactions  of  cystine  have  been  given  on  pages  148 
and  149. 

Cystine  is  easily  prepared  from  cystine  calculi  by  dissolving  them 
in  alkali  carbonate,  precipitating  the  solution  with  acetic  acid,  and  redis- 
solving  the  precipitate  in  ammonia.  The  cystine  crystallizes  on  the 
spontaneous  evaporation  of  the  ammonia.  The  cystine  dissolved  in 
the  urine  is  detected,  in  the  absence  of  proteid  and  sulphuretted  hydrogen, 
by  boiling  with  alkali  and  testing  with  a  lead  salt  or  sodium  nitroprusside. 
To  isolate  cystine  from  the  urine,  acidify  the  urine  strongly  with  acetic 
acid.  The  precipitate  containing  cystine  is  collected  after  twenty-four 
hours  and  digested  with  hydrochloric  acid,  which  dissolves  the  cystine 
and  calcium  oxalate,  leaving  the  uric  acid  undissolved.  Filter,  supersat- 
urate the  filtrate  with  ammonium  carbonate,  and  treat  the  precipitate 
with  ammonia,  which  dissolves  the  cystine  and  leaves  the  calcium  oxalate. 
Filter  again  and  precipitate  with  acetic  acid.  The  precipitated  cystine 
is  identified  by  the  microscope  and  the  above-mentioned  reactions. 
Cystine  as  a  sediment  is  identified  by  the  microscope.  It  must  be  purified 
by  dissolving  in  ammonia  and  precipitating  with  acetic  acid;  it  is  then 
further  tested.  Traces  of  dissolved  cystine  may  be  detected  by  the 
production  of  benzoyl-cystine,  according  to  Baumann  and  Goldmann. 
For  the  detection  and  estimation  of  cystine  we  can  proceed  to  advantage 
in  the  following  manner,  suggested  by  Gaskell.3  The  urine  freed  from 
oxalates  and  phosphates  by  means  of  ammonia  and  calcium  chloride  is 
treated  with  an  equal  volume  of  acetone  and  with  acetic  acid.  The 
crystals  which  precipitate  are  dissolved  in  ammonia  and  then  purified 
by  reprecipitation  with  acetone. 

VH.     URINARY   SEDIMENTS  AND   CALCULI. 

Urinary  sediment  is  the  more  or  less  abundant  deposit  which  is  found 
in  the  urine  after  standing.  This  deposit  may  consist  partly  of  organized 
and  partly- of  non-organized  constituents.  The  first,  consisting  of  cells 
of  various  kinds,  yeast-fungi,  bacteria,  spermatozoa,  casts,  etc.,  must 
be  investigated  by  means  of  the  microscope,  and  the  following  only 
applies  to  the  non-organized  deposits. 

1  See  Wolf  and  Schaffer,  Journ.  of  biol.  Chem.,  4;  and  Hele,  Journ.  of  Physiol..  39. 

2  Zeitschr.  f.  Biol.,  57. 

8  Journ.  of  Physiol.,  36. 


URINARY  SEDIMENTS.  829 

As  previously  mentioned  (page  674),  the  urine  of  healthy  individuals 
may  sometimes,  even  on  voiding,  be  cloudy  on  account  of  the  phosphates 
present,  or  become  so  after  a  little  while  because  of  the  separation  of 
urates.  As  a  rule,  urine  just  voided  is  clear,  and  after  cooling  shows 
only  a  faint  cloud  (nubecula)  which  consists  of  urine  mucoid,  a  few  epithe- 
lium-cells, mucous  corpuscles,  and  urate  particles.  If  an  acid  urine  is 
allowed  to  stand,  it  will  gradually  change;  it  becomes  darker  and  deposits 
a  sediment  consisting  of  uric  acid  or  urates,  and  sometimes  also  calcium- 
oxalate  crystals,  in  which  yeast-fungi  and  bacteria  are  often  to  be  seen. 
This  change,  which  the  earlier  investigators  called  "  acid  fermenta- 
tion of  the  urine,"  is  generally  considered  as  an  exchange  of  the  dihy- 
drogen  alkali  phosphates  with  the  urates  of  the  urine.  Monohydrogen 
phosphates  besides  acid  urates,  quadriurates  (page  708)  or  free  uric  acid 
or  a  mixture  of  both,  according  to  conditions,1  are  thus  formed. 

Sooner  or  later,  sometimes  only  after  several  weeks,  the  reaction 
of  the  original  acid  urine  changes  and  becomes  neutral  or  alkaline.  The 
urine  has  now  passed  into  the  "  alkaline  fermentation,"  which  con- 
sists in  the  decomposition  of  the  urea  into  carbon  dioxide  and  ammonia 
by  means  of  lower  organisms,  micrococcus  ureae,  bacterium  ureae,  and 
other  bacteria.  Museums 2  has  isolated  an  enzyme  from  the  micro- 
coccus ureae  which  decomposes  urea,  which  is  soluble  in  water  and  is  called 
urease.  During  the  alkaline  fermentation  volatile  fatty  acids,  especially 
acetic  acid,  may  be  produced,  chiefly  by  the  fermentation  of  the  car- 
bohydrates of  the  urine  (Salkowski  3).  A  fermentation  by  which 
nitric  acid  is  reduced  to  nitrous  acid,  and  another  where  sulphuretted 
hydrogen  is  produced,  may  sometimes  occur. 

When  the  alkaline  fermentation  has  advanced  only  so  far  as  to  render 
the  reaction  neutral,  there  often  occur  in  the  sediment  fragments  of  uric- 
acid  crystals,  sometimes  covered  with  prismatic  crystals  of  alkali  urate; 
dark-colored  spheres  of  ammonium  urate,  crystals  of  calcium  oxalate, 
and  sometimes  crystallized  calcium  phosphate  are  also  found.  Crystals 
of  ammonium-magnesium  phosphate  (triple  phosphate)  and  spherical 
ammonium  urate  are  specially  characteristic  of  alkaline  fermentation. 
The  urine  in  alkaline  fermentation  becomes  paler  and  is  often  covered 
with  a  fine  membrane  which  contains  amorphous  calcium  phosphate 
and  glistening  crystals  of  triple  phosphate  and  numerous  micro-organisms. 


1  See  Huppert-Xeubauer,  10.  Aufl.,  and  A.  Ritter,  Zeitschr.  f.  Biologie,  35. 

5  Museulus,  Pfliiger's  Arch.,  12. 

1  Salkowski,  Zeitschr.  f.  Physiol.  Chem.,  13. 


830  URINE. 

Non-Organized  Sediments. 

Uric  Acid.  This  acid  occurs  in  acid  urines  as  colored  crystals  which 
are  identified  partly  by  their  form  and  partly  by  their  property  of  giving 
the  murexid  test.  On  warming  the  urine  they  are  not  dissolved.  On 
the  addition  of  caustic  alkali  to  the  sediment  the  crystals  dissolve,  and 
when  a  drop  of  this  solution  is  placed  on  a  microscope-slide  and  treated 
with  a  drop  of  hydrochloric  acid,  small  crystals  of  uric  acid  are  obtained 
which  can  be  easily  seen  under  the  microscope. 

Acid  Urates.  These  occur  only  in  the  sediment  of  acid  or  neutral 
urines.  They  are  amorphous,  clay-yellow,  brick-red,  rose-colored,  or 
brownish-red.  They  differ  from  other  sediments  in  that  they  dissolve 
on  warming  the  urine.  They  give  the  murexid  test,  and  small  micro- 
scopic crystals  of  uric  acid  separate  on  the  addition  of  hydrochloric 
acid.  Crystalline  alkali  urates  occur  very  rarely  in  the  urine,  and  as  a 
rule  only  in  such  as  have  become  neutral  but  not  alkaline,  by  alkaline 
fermentation.  The  crystals  are  somewhat  similar  to  those  of  neutral 
calcium  phosphate;  they  are  not  dissolved  by  acetic  acid,  however, 
but  give  a  cloudiness  therewith  due  to  small  crystals  of  uric  acid. 

Ammonium  urate  may  indeed  occur  as  a  sediment  in  a  neutral  urine 
which  at  first  was  strongly  acid  and  has  become  neutralized  by  the  alkaline 
fermentation,  but  it  is  only  characteristic  of  ammoniacal  urines.  This 
sediment  consists  of  yellow  or  brownish  rounded  spheres  which  are  often 
covered  with  thorny-shaped  prisms  and,  because  of  this,  are  rather 
large  and  resemble  the  thorn-apple.  It  reacts  to  the  murexid  test.  It 
is  dissolved  by  alkalies  with  the  development  of  ammonia,  and  crystals 
of  uric  acid  separate  on  the  addition  of  hydrochloric  acid  to  this  solution. 

Calcium  oxalate  occurs  in  the  sediment  generally  as  small,  shining, 
strongly  refractive  quadratic  octahedra,  which  on  microscopical  examina- 
tion remind  one  of  a  letter-envelope.  The  crystals  can  only  be  mistaken 
for  small,  not  fully  developed  crystals  of  ammonium-magnesium  phos- 
phate. They  differ  from  these  by  their  insolubility  in  acetic  acid.  The 
oxalate  may  also  occur  as  flat,  oval,  or  nearly  circular  disks  with  central 
cavities  which  from  the  side  appear  like  an  hour-glass.  Calcium  oxalate 
may  occur  as  a  sediment  in  an  acid  as  well  as  in  a  neutral  or  alkaline 
urine.  The  quantity  of  calcium  oxalate  separated  from  the  urine  as 
sediment  depends  not  only  upon  the  amount  of  this  salt  present,  but 
also  upon  the  acidity  of  the  urine.  The  solvent  for  the  oxalate  in  the 
urine  seems  to  be  the  diacid  alkali  phosphate,  and  the  greater  the  quan- 
tity of  this  salt  in  the  urine  the  greater  the  quantity  of  oxalate  in  solu- 
tion. When,  as  previously  mentioned  (page  829),  the  simple-acid  phos- 
phate is  formed  from  the  diacid  phosphate,  on  allowing  the  urine  to 
stand,  a  corresponded  part  of  the  oxalate  may  be  separated  as  sediment. 


URINARY   SEDIMENTS.  831 

Calcium  carbonate  occurs  in  considerable  quantities  as  sediment  in 
the  urine  of  herbivora.  It  occurs  in  but  small  quantities  as  a  sediment 
in  human  urine,  and  in  fact  only  in  alkaline  urines.  It  either  has  the 
same  appearance  as  amorphous  calcium  oxalate  or  it  occurs  as  some- 
what larger  spheres  with  concentric  bands.  It  dissolves  in  acetic  acid 
with  the  generation  of  gas,  which  differentiates  it  from  calcium  oxalate. 
It  is  not  yellow  or  brown  like  ammonium  urate,  and  does  not  give  the 
murexid  test. 

Calcium  sulphate  occurs  very  rarely  as  a  sediment  in  strongly  acid  urine.  It 
appears  as  long,  thin,  colorless  needles,  or  generally  as  plates  grouped  together. 

Calcium  Phosphate.  The  calcium  triphosphate,  Ca3(P04)2,  which 
occurs  only  in  alkaline  urines,  is  always  amorphous  and  occurs  partly 
as  a  colorless,  very  fine  powder,  and  partly  as  a  membrane  consisting  of 
very  fine  granules.  It  differs  from  the  amorphous  urates  in  that  it  is 
colorless,  dissolves  in  acetic  acid,  but  remains  undissolved  on  warming 
the  urine.  Calcium  Diphosphate,  CaHP04+2H20,  occurs  in  neutral 
or  only  in  very  faintly  acid  urine.1  It  is  found  sometimes  as  a  thin 
film  covering  the  urine  and  sometimes  as  a  sediment.  In  crystallizing, 
the  crystals  may  be  single,  or  they  may  cross  one  another,  or  they  may 
be  arranged  in  groups  of  colorless,  wedge-shaped  crystals  whose  wide 
end  is  sharply  defined.  These  crystals  differ  from  crystalline  alkali 
urates  in  that  they  dissolve  without  a  residue  in  dilute  acids  and  do  not 
give  the  murexid  test. 

Ammonium-magnesium  phosphate,  triple  phosphate,  may  separate 
from  an  amphoteric  urine  in  the  presence  of  a  sufficient  quantity  of  ammo- 
nium salts,  but  it  is  generally  characteristic  of  a  urine  which  is  ammo- 
niacal  through  alkaline  fermentation.  The  crystals  are  so  large  that  they 
may  be  seen  with  the  unaided  eye  as  colorless  glistening  particles  in  the 
sediment,  on  the  walls  of  the  vessel,  and  in  the  film  on  the  surface  of  the 
urine.  This  salt  forms  large  prismatic  crystals  of  the  rhombic  system 
(coffin-shaped)  which  are  easily  soluble  in  acetic  acid.  Amorphous 
magnesium  triphosphate,  Mg3(P04)2,  occurs  with  calcium  triphosphate 
in  urines  rendered  alkaline  by  a  fixed  alkali.  Crystalline  magnesium 
phosphate,  Mg3(P04)2+22H20,  has  been  observed  in  a  few  cases  in 
human  urine  (also  in  horse's  urine)  as  strongly  refractive,  long  rhombic 
plates. 

As  more  rare  sediments  we  fine*  cystine,  tyrosine,  hippuric  acid,  xanthine,  hama- 
toidine.  In  alkaline  urine  blue  crystals  of  indigo  may  also  occur,  due  to  a  decom- 
position of  indoxyl-glucuronic  acid. 

1  In  regard  to  the  conditions  for  the  appearance  of  these  sediments  in  urines  see 
C.  Th.  Morner,  Zeitschr.  f.  physiol.  Chem.,  58. 


832  UEINE. 


Urinary  Calculi. 

Besides  certain  pathological  constituents  of  the  urine,  all  those  urinary- 
constituents  which  occur  as  sediments  take  part  in  the  formation  of 
urinary  calculi.  Ebstein1  considers  the  essential  difference  between  an 
amorphous  and  crystalline  sediment  in  the  urine  on  the  one  side  and  urinary 
sand  or  large  calculi  on  the  other  to  be  the  occurrence  of  an  organic 
frame  in  the  latter.  As  the  sediments  which  appear  in  normal  acid  urine 
and  in  a  urine  alkaline  through  fermentation  are  diverse,  so  also  are  the 
urinary  calculi  which  appear  under  corresponding  conditions. 

.If  the  formation  of  the  calculus  and  its  further  development  take  place 
in  an  un  decomposed  urine,  it  is  called  a  primary  formation.  If,  on  the 
contrary,  the  urine  has  undergone  alkaline  fermentation  and  the  ammonia 
formed  thereby  has  given  rise  to  a  calculus  formation  by  precipitating 
ammonium  urate,  triple  phosphate,  and  earthy  phosphates,  then  it  is 
called  a  secondary  formation.  Such  a  formation  takes  place,  for 
instance,  when  a  foreign  body  in  the  bladder  produces  catarrh  accom- 
panied by  alkaline  fermentation. 

We  discriminate  between  the  nucleus  or  nuclei — if  such  can  be  seen — 
and  the  different  layers  of  the  calculus.  The  nucleus  may  be  essentially 
different  in 'different  cases,  for  quite  frequently  it  consists  of  a  foreign 
body  introduced  in  the  bladder.  The  calculus  may  have  more  than 
one  nucleus.  In  a  tabulation  made  by  Ultzmann  of  545  cases  of  vesic- 
ular calculi,  the  nucleus  in  80.9  per  cent  of  the  cases  consisted  of  uric 
acid  (and  urates);  in  5.6  per  cent,  of  calcium  oxalate;  in  8.6  per  cent, 
of  earthy  phosphates;  in  1.4  per  cent,  of  cystein;  and  in  3.5  per  cent, 
of  some  foreign  body. 

During  the  growth  of  a  calculus  it  often  happens  that,  for  some  reason 
or  other,  the  original  calculus-forming  substance  is  covered  with  another 
layer  of  a  different  substance.  A  new  layer  of  the  original  substance  may 
deposit  on  the  outside  of  this,  and  this  process  may  be  repeated.  In 
this  way  a  calculus  consisting  originally  of  a  simple  stone  may  be  con- 
verted into  a  so-called  compound  stcne  with  several  layers  of  different 
substances.  Such  calculi  are  always  formed  when  a  primary  is  changed 
into  a  secondary  formation.  By  the  continued  action  of  an  alkaline 
urine  containing  pus,  the  primary  constituents  of  a  primary  calculus 
may  be  partly  dissolved  and  be  replaced  by  phosphates.  Metamor- 
phosed urinary  calculi  are  formed  in  this  way. 


Die  Natur  unci  Behandlung  der  Harnsteine.     Wiesbaden,  1884. 


URINARY  CALCULI.  833 

Uric-acid  calculi  are  very  abundant.  They  are  variable  in  size  and 
form.  The  size  of  the  bladder-stone  varies  from  that  of  a  pea  or  bean  to 
that  of  a  goose-egg.  Uric  acid  stones  are  always  colored;  generally 
they  are  grayish-yellow,  yellowish-brown,  or  pale  red-brown.  The  upper 
surface  is  sometimes  entirely  even  or  smooth,  sometimes  rough  or  uneven. 
Next  to  the  oxalate  calculus  the  uric-acid  calculus  is  the  hardest.  The 
fractured  surface  shows  regular  concentric,  unequally  colored  layers 
which  may  often  be  removed  as  shells.  These  calculi  are  formed  pri- 
marily. Layers  of  uric  acid  sometimes  alternate  with  other  layers  of 
primary  formation,  most  frequently  with  layers  of  calcium  oxalate. 
The  simple  uric-acid  calculus  leaves  very  little  residue  when  burnt  on 
a  platinum  foil.  It  gives  the  murexid  test,  but  there  is  no  material 
development  of  ammonia  when  acted  on  by  caustic  soda. 

Ammonium  urate  calculi  occur  as  primary  calculi  in  new-born  or  nurs- 
ing infants,  rarely  in  grown  persons.  They  often  occur  as  a  secondary 
formation.  The  primary  stones  are  small,  with  a  pale  yellow  or  dark- 
yellowish  surface.  When  moist  they  are  almost  like  dough;  in  the 
dry  state  they  are  earthy,  easily  crumbling  into  pale  powder.  They 
give  the  murexid  test  and  develop  much  ammonia  with  caustic  soda. 

Calcium-oxalate  calculi  are,  next  to  uric-acid  calculi,  the  most  abundant. 
They  are  either  smooth  and  small  (hemp-seed  calculi)  or  larger,  of  the 
size  of  a  hen's  egg,  with  rough,  uneven  surface,  or  their  surface  is  cov- 
ered with  prongs  (mulberry  calculi).  These  calculi  produce  bleeding 
easily,  and  therefore  they  often  have  a  dark-brown  surface  due  to  decom- 
posed blood-coloring  matters.  Among  the  calculi  occurring  in  man 
these  are  the  hardest.  They  dissolve  in  hydrochloric  acid  without 
developing  gas,  but  are  not  soluble  in  acetic  acid.  After  gently  heating 
the  powder,  it  dissolves  in  acetic  acid  with  frothing.  With  more  intense 
heat  it  becomes  alkaline,  due  to  the  production  of  quicklime. 

Phosphate  Calculi.  These,  which  consist  mainly  of  a  mixture  of  the 
normal  phosphate  of  the  alkaline  earths  with  triple  phosphate,  may  be 
very  large.  They  are  as  a  rule  of  secondary  formation  and  contain 
besides  these  phosphates  also  some  ammonium  urate  and  calcium  oxalate. 
These  calculi  ordinarily  consist  of  a  mixture  of  three  constituents — 
earthy  phosphate,  triple  phosphate,  and  ammonium  urate — surrounding 
a  foreign  body  as  a  nucleus.  Their  color  is  variable — white,  dingy  white, 
pale  yellow,  sometimes  violet  or  lilac-colored  (from  indigo  red).  The 
surface  is  always  rough.  Calculi  consisting  of  triple  phosphate  alone 
are  seldom  found.  They  are  ordinarily  small,  with  granular  or  radiated 
crystalline  fracture.  Stones  of  mono-acid  calcium  phosphate  are  also 
seldom  obtained.  They  are  white  and  have  beautiful  crystalline  texture. 
The  phosphatic  calculi  do  not  burn  up,  the  powder  dissolves  in  acid 
without   effervescence,   and   the   solution   gives  the   reactions  for  phos- 


834  URINE. 

phoric  acid  and  the  alkaline  earths.     The  triple-phosphate  calculi  generate 
ammonia  on  the  addition  of  an  alkali. 

Calcium-carbonate  ^calculi  occur  chiefly  in  herbivora.  They  are  seldom  found 
in  man.  They  have  mostly  chalky  properties,  and  are  ordinarily  white.  They 
are  completely  or  in  great  part  dissolved  by  acids  with  effervescence. 

Cystine  calculi  occur  but  seldom.  They  are  of  primary  formation,  of  various 
sizes,  sometimes  as  large  as  a  hen's  egg.  They  have  a  smooth  or  rough  surface, 
are  white  or  pale  yellow,  and  have  a  crystalline  fracture.  They  are  not  very 
hard  and  are  consumed  almost  entirely  on  the  platinum  foil,  burning  with  a  bluish 
flame.     They  give  the  above-mentioned  reactions  for  cystine. 

Xanthine  calculi  are  very  rarely  found.  They  are  also  of  primary  formation. 
They  vary  from  the  size  of  a  pea  to  that  of  a  hen's  egg.  They  are  whitish,  yel- 
lowish-brown or  cinnamon-brown  in  color,  of  medium  hardness,  with  amorphous 
fracture,  and  on  rubbing  appear  like  wax.  They  burn  up  completely  when  heated 
on  a  platinum  foil.  They  give  the  xanthine  reaction  with  nitric  acid  and  alkali, 
but  this  must  not  be  mistaken  for  the  murexid  test. 

Urostealith  calculi  have  been  observed  only  a  few  times.  In  the  moist  state 
they  are  soft  and  elastic  at  the  temperature  of  the  body,  but  in  the  dry  state 
they  are  brittle,  with  an  amorphous  fracture  and  waxy  appearance.  They  burn 
with  a  luminous  flame  when  heated  on  platinum  foil  and  generate  an  odor  similar  to 
resin  or  shellac.  Such  a  calculus,  investigated  by  Krukenberg,1  consisted  of 
paraffin  derived  from  a  paraffin  bougie  used  as  a  sound  on  the  patient.  Perhaps 
the  urostealith  calculi  observed  in  other  cases  had  a  similar  origin,  although  the 
substances  of  which  they  consisted  have  not  been  closely  studied.  Horbaczew- 
ski  has  recently  analyzed  a  case  of  urostealith  which,  to  all  appearances,  was 
formed  in  the  bladder.  This  calculus  contained  25  p.  m.  water,  8  p.  m.  inorganic 
bodies,  117  p.  m.  bodies  insoluble  in  ether,  and  850  p.  m.  organic  bodies  soluble 
in  ether,  among  which  were  515  p.  m.  free  fatty  acids,  335  p.  m.  fat,  and  traces  of 
cholesterin.  The  fatty  acids  consisted  of  a  mixture  of  stearic,  palmitic,  and 
probably  myristic  acids.     - 

Horbaczewski  2  has  also  analyzed  a  bladder  stone  which  contained  958.7 
p.  m.  cholesterin. 

Fibrin  calculi  sometimes  occur.  They  consist  of  more  or  less  changed  fibrin- 
coagulum.     On  burning  they  develop  an  odor  of  burnt  horn. 

The  chemical  investigation  of  urinary  calculi  is  of  great  practical  impor- 
tance. To  make  such  an  examination  actually  instructive  it  is  necessary 
to  investigate,  separately,  the  different  layers  which  constitute  the  cal- 
culus. For  this  purpose  saw  the  calculus,  previously  wrapped  in  paper, 
with  a  fine  saw  so  that  the  nucleus  becomes  accessible.  Then  peel  off  the 
different  layers,  or,  if  the  stone  is  to  be  kept,  scrape  off  enough  of  the 
powder  from  each  layer  for  examination.  This  powder  is  then  tested  by 
heating  on  the  platinum  foil.  It  must  not  be  forgotten  that  a  calculus 
is  never  entirely  burnt  up,  and  also  that  it  is  never  so  free  from  organic 
matter  that  on  heating  it  does  not  carbonize.  Do  not,  therefore,  lay  too 
great  stress  on  a  very  insignificant  unburnt  residue  or  on  a  very  small 


lChem.  Untersuch.  z.  wissensch.  Med.,  2.     Cited  from  Maly's  Jahresber.,  19,  422. 
2  Zeitschr.  f.  physiol.  Chem.,  18. 


URINARY  CALCULI.  835 

amount  of  organic  matter,  but  consider  the  calculus  in  the  former  case 
as  completely  burnt  and  in  the  latter  as  unaffected. 

When  tlif  powder  is  in  great  part  burnt  up,  but  a  significant  quantity 
of  unburnt  residue  remains,  then  the  powder  in  question  contains  as  a 
rule  urat<-  mixed  with  inorganic  bodies.  In  such  cases  remove  the  urate 
with  boiling  water  and  then  test  the  filtrate  for  uric  acid  and  the  sus- 
pected  bases.  The  residue  is  then  tested  according  to  the  following 
scfu  me  of  Heller,  which  is  well  adapted  to  the  investigation  of  urinary 
calculi.  In  regard  to  the  more  detailed  examination  the  reader  is 
referred  to  special  works  on  the  subject. 


836 


URINE. 

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CHAPTER  XV 
THE  SKIN  AND  ITS  SECRETIONS 

In  the  structure  of  the  skin  of  man  and  vertebrates  many  different 
kinds  of  substances  occur  which  have  already  been  considered,  such  as 
the  constituents  of  the  epidermal  formation,  the  connective  and  fatty 
tissues,  the  nerves,  muscles,  etc.  Among  these  the  different  horn  struc- 
tures, the  hair,  nails,  etc.,  whose  chief  constituent,  keratin,  has  been 
spoken  of  in  another  chapter  (Chapter  II),  are  of  special  interest. 

The  cells  of  the  horny  structure  show,  in  proportion  to  their  age,  a 
different  resistance  to  chemical  reagents,  especially  fixed  alkalies.  The 
younger  the  horn-cell  the  less  resistance  it  has  to  the  action  of  alkalies; 
with  advancing  age  the  resistance  becomes  greater,  and  the  cell-mem- 
branes of  many  horn-formations  are  nearly  insoluble  in  caustic  alkalies. 
Keratin  (or  the  keratins)  occurs  in  the  horn  structure  mixed  with  other 
bodies,  from  which  it  is  isolated  with  difficulty.  These  are  detected  by 
microchemical  investigations,  and  according  to  Unna  l  three  different 
substances  can  be  detected  in  the  horn  substance,  designated  by  him  A-, 
B-  and  C-keratin. 

The  il-keratin,  which  forms  the  envelope  of  the  horn  and  hair  cells  and  the 
outer  layer  of  the  hair,  is  the  purest  keratin.  It  is  not  dissolved  by  fuming  nitric 
acid  at  the  ordinary  temperature  and  does  not  give  the  xanthoproteic  reaction, 
and  its  keratin  nature  is  doubtful.  The  5-keratin,  which  occurs  as  the  contents 
of  the  nail  cells,  gives  the  xanthoproteic  reaction  like  the  C-keratin  occurring  in 
hair,  but  differs  from  the  C-keratin  by  being  soluble  in  fuming  nitric  acid. 

Besides  these  substances,  which  have  been  called  keratins,  the  horn 
structure  also  contains  other  proteins  which  are  soluble  in  pepsin- 
hydrochloric  acid.  Among  these  we  find  residue  of  nuclei  and  the 
so-called  trichohyalin  in  the  hair,  which  is  a  substance  of  unknown 
constitution  and  characterized  by  great  insolubility.  From  these 
statements  it  is  evident  that  we  are  here  dealing  with  a  mixture  of  dif- 
ferent substances  and  for  this  reason  it  is  unnecessary  to  give  the  older 
elementary  analyses  of  the  various  epidermoidal  structures. 

1  Monatsch.  f.  prakt.  Dermat.,  44. 

837 


838  THE  SKIN  AND  ITS  SECRETIONS. 

The  quantity  of  sulphur  and  of  mineral  bodies  is  of  certain  interest. 
The  sulphur  and  cystine  content  of  these  structures  can  be  found  on 
pages  113,  114  and  in  this  connection  it  must  be  mentioned  that,  accord- 
ing to  the  investigations  of  Rutherford  and  Hawk,1  the  sulphur  content 
of  human  hair  is  higher  in  men  than  in  women,  at  least  for  the  Caucasian 
race,  and  also  that  red  hair  has  the  highest  sulphur  content  irrespective 
of  race  or  gender.  Hair  on  incineration  leaves  considerable  ash,  which 
in  human  hair  varies  between  2.6  and  16  p.  m.,  and  in  animal  hair 
is  still  greater,  even  up  to  71  p.  m.  in  the  hair  of  the  deer.  The  ash 
consists  of  large  amounts  of  alkali  and  calcium  sulphate,  and  its  sulphur 
probably  originates  from  the  organic  substance,  which  make  the  state- 
ments as  to  the  composition  of  the  ash  of  hair  of  little  value.  Calcium 
occurs  in  larger  amounts,  especially  phosphate  as  well  as  carbonate,  and  is 
most  abundant  in  white  hair.  The  amount  of  iron  oxide  in  1000  grams 
of  the  ash  of  human  hair  varies  between  42.2  grams  in  blond  and  108.7 
grams  in  brown  hair,  and  silicic  acid  between  66.1  grams  in  black  and 
424.6  grams  in  red  hair  (Baudrimont).  The  nails  are  rich  in  calcium 
phosphate,  and  the  feathers  rich  in  silicic  acid,  especially  the  feathers 
of  grain-eating  birds.  According  to  v.  Gorup-Besanez  2  the  quantity 
of  silicic  acid  in  grain-eating  birds  was  400  p.  m.,  and  in  meat,  berries 
and  insect-eating  birds  the  amount  was  only  270  p.  m.  of  the  total  ash. 
Drechsel  claims  that  at  least  a  part  of  the  silicic  acid  exists  in  the 
feathers  in  organic  combination  as  an  ester  while  according  to  Cerny  3  it 
exists  only  as  an  accidental  contamination. 

According  to  Gautier  and  Bertrand4  arsenic  also  occurs  in  the 
epidermal  formations.  Gautier  says  that  arsenic  is  of  importance  in 
the  formation  and  growth  of  the  formations,  and  on  the  other  hand  the 
hair,  nails,  and  epidermis-cells  are  of  great  importance  in  the  excretion 
of  arsenic. 

The  ability  of  the  skin  to  take  up  chlorides  as  observed  by  Wahlgren 
aDd  by  Padtberg,5  is  remarkable.  According  to  them  the  skin  is  an 
important  chloride  depot,  which  stores  up  chlorides  when  supplied  in 
excess  and  gives  them  up  when  necessary. 

The  skin  of  invertebrates  has  been  the  subject,  in  a  few  cases,  of 
chemical  investigation,  and  in  these  animals  various  substances  have 
been  found,  of  which  a  few,  though  little  studied,  are  worth  discussing. 
Among  them  tunicin,  which  is  found  especially  in  the  mantle  of  the 


1  Journ.  of  Biol.  Chem.,  3. 

1  Lehr.  d.  physiol.  Chem.,  4.  Aufl.,  660,  661;  Baudrimont,  ibid. 

*  Drechsel,  Centralbl.  f.  Physiol.,  11,  361;  Cerny,  Zeitschr.  (.  physiol.  Chem.,  62. 

♦Gautier,  Compt.  Rend.,  129,  130,  131;  Bertrand,  ibid.,  134. 

'  Wahlgren,  Arch.  f.  exp.  Path.  u.  Pharm.,  61;  Padtberg,  ibid.,  63. 


TUNICIN.     CHITIN.  839 

tunicata,  and  the  widely  diffused  chitin,  found  in  the  cuticle-formation  of 
invertebrates,  arc  of  interest. 

Tunicin.  Cellulose  seems,  from  the  investigations  of  Amukonn,  to  occur 
rather  extensively  in  the  animal  kingdom  in  the  aithropoda  and  the  mollusks. 
It  has  been  known  for  a  long  time  as  the  mantle  of  the  tunicata,  and  this  animal 
cellulose  was  called  tunicin  by  Berthelot.  According  to  the  investigations 
of  Winterstein  there  does  not  seem  to  exist  any  marked  difference  between 
tunicin  and  ordinary  vegetable  cellulose.  On  boiling  with  dilute  acid,  tunicin 
yields  glucose,  as  shown  first  by  Franchimont  and  later  confirmed  by  Win- 
terstein. By  the  action  of  acetic  acid  anhydride  and  sulphuric  acid,  upon 
tunicate-cellulose,  Abderhalden  and  Zemplen  1  obtained  octoacetyl-cellobiose, 
which  also  indicates  the  relationship  with  the  plant  cellulose. 

Chitin  is  not  found  in  vertebrates.  In  invertebrates  chitin  is  alleged 
to  occur  in  several  classes  of  animals;  it  occurs  chiefly  in  cephalopods 
(sepia  scales)  and  especially  in  the  arthropods,  in  which  it  forms  the 
chief  organic  constituent  of  the  shells,  etc.  It  has  been  found  in  the 
plant  kingdom  as  in  fungi  (Gilson,  Winterstein2).  The  question 
whether  there  are  two  or  more  chitins  or  whether  there  is  only  one 
is  still  disputed  (Krawkow,  Zander,  Wester3).  No  formula  can  be 
given  for  the  same  reasons  (Sundwik,  Araki,  Brach  4). 

Chitin  is  decomposed  on  boiling  with  mineral  acids  and  yields,  as 
shown  by  Ledderhose,  glucosamine  and  acetic  acid.  Hoppe-Seyler 
and  Araki  found,  on  heating  with  alkali  and  a  little  water  to  180°,  that 
chitin  was  split  into  a  new  substance,  chitosan,  and  acetic  acid,  and  that 
this  chitosan  contained  acetyl  groups  as  well  as  glucosamine.  Frankel 
and  Kelly  as  well  as  Offer  5  have  obtained  acetylglucosamine, 
(C6Hi2N05)COCH3  and  acetyldiglucosamine  (Ci2H23N2Cq)COCH3  as 
cleavage  products  of  chitin,  and  they  consider  chitin  as  a  polymeric 
monacetyldiglucosamine. 

The  chitosan  which  v.  Furth  and  Russo  6  have  obtained  as  a  crys- 
talline hydrochloric  acid  combination  and  which  E.  Loewy  has  obtained 
as  a  crystalline  sulphate  is,  according  to  the  latter,  a  polymeric  monacetyl- 
diglucosamine with  at  least  two  monacetyldiglucosamine  groups.     Accord- 

1  Ambronn,  Maly's  Jahresber.,  20;  Berthelot,  Annal.  de  Chim.  et  Phys.,  56,  Compt. 
Rend.,  47;  Winterstein,  Zeitschr.  f.  physiol.  Chem.,  18;  Franchimont,  Ber.  d.  deutsch. 
chem.  Gesellsch.,  12;   Abderhalden  and  Zemplen,  Zeitschr.  f.  physiol.  Chem.,  72. 

2  Gilson,  Compt.  Rend.,  120;   Winterstein,  Ber.  d.  d.  chem.  Gesellsch.,  27  and  28. 
'Krawkow,   Zeitschr.   f.   Biol.,   29;    Zander,  Pfliiger's  Arch.,   66;  Wester,   Chem. 

Centralbl.,  1909,  II. 

4  Sundwik,  Zeitschr.  f.  physiol.  Chem.,  5;  Araki,  ibid.,  20;  Brach,  Bioch.  Zeitschr., 
38. 

5  Ledderhose,  Zeitschr.  f.  physiol.  Chem.,  2  and  4;  Araki,  1.  c,  Frankel  and  Kelly, 
Monatshefte  f.  Chem.,  23;  Offer,  Bioch.  Zeitschr.,  7. 

frv.  Furth  and  Russo,  Hofmeister's  Beitrage,  8;  Loewy,  Bioch.  Zeitschr.,  23;  Brach, 
1.  c. 


840  THE   SKIN  AND  ITS  SECRETIONS. 

ing  to  v.  Furth  and  Russo  on  cleavage  it  yields  25  per  cent  acetic  acid 
and  60  per  cent  glucosamine.  The  formula  is  (C28H5oN40i9)z  according 
to  v.  FtiRTH  and  collaborators  and  splits  according  to  the  equation: 
(C28H5oN40i9)4+5:cH20  =  43(C6Hi3N05)+2x(CH3COOH).  According 
to  Brach,  who  admits  of  at  least  four  glucosamine  groups  in  chitosan, 
the  formula  for  chitin  is  (C32H54N402i)x  and  contains  4  acetyl  for  every 
4  nitrogen  atoms.  The  transformation  into  chitosan  consists  in  a 
rupture  of  one-half  of  the  acetic  acid  groups  in  the  chitin. 

In  a  dry  state  chitin  forms  a  white,  brittle  mass  retaining  the  form 
of  the  original  tissue.  It  is  insoluble  in  boiling  water,  alcohol,  ether, 
acetic  acid,  dilute  mineral  acids,  and  dilute  alkalies.  It  is  soluble  in 
concentrated  acids.  It  is  dissolved  without  decomposing  in  cold  con- 
centrated hydrochloric  acid,  but  is  decomposed  by  boiling  hydrochloric 
acid.  According  to  Krawkow  the  various  chitins  behave  differently 
with  iodine  or  with  sulphuric  acid  and  iodine,  in  that  some  are  colored 
reddish  brown,  blue,  or  violet,  while  others  are  not  colored  at  all.  Accord- 
ing to  Wester  chitin  free  from  chitosan  is  not  colored  by  iodine. 

Chitin  may  be  easily  prepared  from  the  wings  of  insects  or  from  the  shells 
of  the  lobster  or  the  crab,  the  last-mentioned  having  first  been  extracted  by  an 
acid  so  as  to  remove  the  lime  salts.  The  wings  or  shells  are  boiled  with  caustic 
alkali  until  they  are  white,  afterward  washed  with  water,  then  with  dilute  acid 
and  water.  The  pigments  remaining  can  be  destroyed  by  permanganate.  The 
excess  of  this  last  can  be  removed  by  a  dilute  solution  of  bisulphite,  washed  with 
water  and  then  extracted  with  alcohol  and  ether. 

Hyalin  is  the  chief  organic  constituent  of  the  walls  of  hydatid  cysts.  From  a 
chemical  point  of  view  it  stands  close  to  chitin,  or  between  it  and  protein.  In 
old  and  more  transparent  sacs  it  is  tolerably  free  from  mineral  bodies,  but  in 
younger  sacs  it  contains  a  great  quantity  (16  per  cent)  of  lime  salts  (carbonate, 
phosphate,  and  sulphate). 

According  to  Lucke  l  its  composition  is'. 

C 

From  old  cysts 45  3 

From  young  cysts. 44 . 1 

It  differs  from  keratin  on  the  one  hand  and  from  proteins  on  the  other  by  the 
absence  of  sulphur,  also  by  its  yielding,  when  boiled  with  dilute  sulphuric  acid, 
a  variety  of  sugar  in  large  quantities  (50  per  cent),  which  is  reducing,  fermentable, 
and  dextrogyrate.  It  differs  from  chitin  by  the  property  of  being  gradually 
dissolved  by  caustic  potash  or  soda,  or  by  dilute  acids;  also  by  its  solubility  on 
heating  with  water  to  150°  C. 

The  coloring  matters  of  the  skin  and  horn-formations  are  of  different 
kinds,  but  have  not  been  extensively  studied.  Those  occurring  in  the 
stratum  Malpighii  of  the  skin,  especially  of  the  negro,  and  the  black 
or  brown  pigment  occurring  in  the  hair,  belong  to  the  group  of  those 
substances  which  have  received  the  name  melanins. 

1  Virchow'e  Arch.,  19. 


H 

N 

O 

6.5 

5.2 

43.0 

6.7 

4.5 

44.7 

MELANINS.  841 

Melanins.  This  group  includes  several  different  varieties  of  amorphous 
black  or  brown  pigments  which  are  insoluble  in  water,  alcohol,  ether. 
chloroform,  and  dilute  acids,  and  which  occur  in  the  skin,  hair,  chorioidia, 
in  sepia,  in  certain  pathological  formations,  and  in  the  blood  and  urine 
in  disease.  From  the  true  native  melanins  we  must  differentiate  the 
humus-like  products  obtained  on  boiling  proteins  with  mineral  acids 
and  which  have  been  called  melanoidins  or  melanoidic  acid  (Schmiedeberg) 
and  whose  relation  to  the  true  melanins  is  still  unknown. 

The  melanoidins  are  readily  soluble  in  dilute  alkali  while  the  melanins 
show  a  different  behavior  in  this  regard.  Of  the  melanins  a  few  such  as 
Schmiedeberg's  sarcomelanin,  and  that  from  the  melanotic  sarcomata 
of  horses,  the  hippomelanin  (Nencki,  Sieber,  and  Berdez),  which  are 
soluble  with  difficulty  in  alkalis,  while  others,  such  as  the  coloring  matter 
of  certain  pathological  swellings  in  man,  the  phymatorhusin  (Nencki  and 
Berdez)  are  readily  soluble  in  alkalies.  The  melanins,  as  above  stated, 
are  in  general  insoluble  in  dilute  mineral  acids;  from  black  sheep- 
wool  Gortner  l  has  isolated  a  melanin  which  was  soluble  in  acetic  acid 
and  in  dilute  mineral  acids  (see  below). 

Among  the  melanins  there  are  a  few,  for  example  the  choroid  pig- 
ment, which  are  free  from  sulphur  (Landolt  and  others);  others,  on  the 
contrary,  as  sarcomelanin  and  the  pigment  of  the  hair  (Sieber)  are  rather 
rich  in  sulphur  (2-4  per  cent),  while  the  phymatorhusin  found  in  cer- 
tain swellings  and  in  the  urine  (Nencki  and  Berdez,  K.  Morner)  is 
very  rich  in  sulphur  (8-10  per  cent).  Whether  any  of  these  pigments, 
especially  the  phymatorhusin,  contains  any  iron  or  not  is  an  important 
though  disputed  point,  for  it  leads  to  the  question  whether  these  pigments 
are  formed  from  the  blood-coloring  matters. 

According  to  Nencki  and  Berdez  the  pigment,  phymatorhusin,  isolated  by 
them  from  a  melanotic  sarcoma  did  not  contain  any  iron,  and  according  to  them 
is  not  a  derivative  of  haemoglobin.  K.  Morner  and  later  also  Brandl  and  L. 
Pfeiffer  found,  on  the  contrary,  that  this  pigment  did  contain  iron,  and  they 
consider  it  as  a  derivative  of  the  blood-pigments.  The  sarcomelanin  (from  a  sar- 
comatous liver)  analyzed  by  Schmiedeberg  contained  2.7  per  cent  iron  which 
was  partly  in  organic  combination  and  could  not  be  completely  removed  by 
dilute  hydrochloric  acid.  The  sarcomelanic  acid  prepared  by  Schmiedeberg 
by  the  action  of  alkali  on  this  melanin  contained  1.07  per  cent  iron.  The  sar- 
comelanin investigated  by  Zdarek  and  v.  Zeynek  also  contained  0.4  per  cent  iron. 
Recently  Wolff  2  prepared  two  pigments  from  a  melanotic  liver,  of  which  one 
was  no  doubt  modified.     The  other,  which  was  soluble  in  a  soda  solution,  con- 

1  Gortner,  Journ.  of  biol.  Chem.,  8,  and  Bioch.  Bulletin,  1,  1911. 

i  Zdarek  and  v.  Zeynek,  Zeitschr.  f.  physiol.  Chem.,  36;  Wolff,  Hofmeister's  Beitrage, 
5.  The  literature  on  the  melanins  may  be  found  in  Schmiedeberg,  "  Elementarformeln 
einiger  Eiweisskorper,  etc."  Arch.  f.  exp.  Path.  u.  Pharm.,  39;  also  in  Robert,  Wiener 
Klinik,  27  (1901),  and  Spiegler,  Hofmeister's  Betrage,  4,  and  especially  v.  Furth, 
Centralbl.  f.  allg.  Path.  u.  Path.  Anat.,  ,15,  1907,  617. 


842  THE  SKIN  AND  ITS  SECRETIONS. 

tained  2.51  per  cent  sulphur  and  2.63  per  cent  iron,  which  was  in  great  part 
split  off  by  20  per  cent  hydrochloric  acid.  From  another  liver  he,  on  the  con- 
trary, obtained  melanin  free  from  iron  but  with  1.67  per  cent  sulphur.  From 
this  melanin  he  obtained,  by  treatment  with  bromine,  a  hydro-aromatic  body 
which  was  related  to  xyliton  (a  condensation  product  of  acetone).  A  similar 
product  could  not  be  obtained  from  the  pigment  of  the  hair  (Spiegler)  nor  from 
hippomelanin  (v.  Furth  and  Jerusalem  1). 

The  difficulties  which  attend  the  isolation  and  purification  of  the 
melanins  have  not  been  overcome  in  certain  cases,  while  in  others  it  is 
questionable  whether  the  final  product  obtained  has  not  another  com- 
position from  the  original  coloring  matter,  owing  to  the  energetic  chemical 
processes  resorted  to  in  its  purification.  The  elementary  composition 
shows  widely  varying  results  in  the  different  melanins,  namely,  48-60 
per  cent  carbon,  and  8-14  per  cent  nitrogen.  Under  these  circumstances, 
and  as  no  doubt  we  have  a  large  number  of  melanins  having  different 
composition,  it  seems  that  a  tabulation  of  the  analyses  of  the  different 
preparations  can  only  be  of  secondary  importance. 

Gortner  differentiates  between  two  different  groups  of  melanins. 
The  one,  to  which  the  melanin  isolated  by  him  from  sheeps-wool  belongs, 
is  soluble  in  very  dilute  acid,  has  a  protein  nature  and  is  called  melano- 
protein.  By  the  action  of  strong  alkali  the  nitrogen  and  hydrogen  con- 
tent is  much  reduced  and  the  quantity  of  carbon  increased.  The  melanin 
is  now  insoluble  in  dilute  acids,  like  the  second  group  of  melanins.  The 
melanoprotein  on  hydrolysis  with  hydrochloric  acid  yields  besides  amino- 
acids,  a  black  pigment,  rich  in  carbon  and  insoluble  in  acids.  The 
melanin  isolated  by  Piettre2  form  sarcomatous  horse  tumors,  on  alkali 
hydrolysis,  yielded  amino-acids  and  a  melanin  much  richer  in  carbon 
and  poorer  in  nitrogen,  a  melainin.  The  sepia  melanin  and  also  the 
artificially  prepared  melanin  by  means  of  tyrosinase,  had  a  similar  behavior. 
The  melanin  is,  therefore,  according  to  Piettre,  composed  of  a  protein 
group  and  a  pigment  residue,  which  is  insoluble  in  acids. 

So  little  is  known  about  the  structural  products  of  the  melanins  or 
melanoids  that  it  is  impossible  to  give  the  origin  of  these  bodies.  As 
undoubtedly  there  are  several  distinct  melanins,  their  origin  must  also  be 
distinct.  The  ferruginous  melanins  should  be  considered  as  originating 
from  the  blood-pigments  until  further  research  proves  otherwise.  Others, 
on  the  contrary,  cannot  have  this  origin;  for  example,  the  pigments 
of  the  hair  and  choroid,  which  are  free  from  iron  and  which  do  not  yield 
hamopyrrol  according  to  Spiegler.     Several  melanins — and  this  is  also 


1  Wolff,    Hofmeister's   Beitrage,    5;  Speigler,   ibid.,    10;  v.    Furth   and   Jerusalem, 
ibid.,  10. 

2  Gortner,  1.  c.  and  Bull.  Soc.  Chim.  de  France  (4)  11;    Piettre,  Compt.  Rend.,  153. 
and  Congres,  internat.,  de  Path.  Comparee,  Paris,  1912. 


MELANINS.  843 

true  of  the  melanoids  produced  from  proteins  on  cleavage  with  acids 
(Samuely  !) — yield  indol  or  skatol  and  a  pyrrol  substance  on  fusion 
with  alkali,  while  hippomelanin,  according  to  v.  Furth  and  Jebubalem, 
gives  a  fecal  odor  on  this  treatment,  but  does  not  yield  any  indol  or  skatol. 
More  characteristic  than  the  two  last  mentioned  bodies  is  a  phenol-like 
substance,  which  occurs  to  a  slight  extent,  and  gives  a  bluish-black 
color  with  ferric  chloride  (v.  Furth). 

The  cyclic  complexes  of  the  proteins  are  rightly  considered  as  the 
mother-substance  of  the  melanins  (Samuely  and  v.  Furth  and  others), 
and  this  view  has  received  support  by  the  behavior  of  tyrosine  with 
oxidases.  It  has  been  found  that  by  the  action  of.  a  plant  oxidase, 
Bertrand's  tyrosinase,2  upon  tyrosine,  colored  products  and  then 
melanin-like  substances  are  formed,  v.  Furth  with  Schneider  and 
Pribram,  Gessard,  Neuberg,  Dewitz  and  others3  have  shown  that 
similar-acting  tyrosinases  also  occur  in  the  animal  kingdom,  in  insects 
and  sepia,  in  melanotic  tumors  and  in  pigmented  skin,  and  v.  Furth 
and  Jerusalem  have  prepared  an  artificial  melanin  from  tyrosine  which 
shows  great  similarity  to  hippomelanin.  Finally  Neuberg  and  Jager  4 
have  also  prepared  extracts  from  melanotic  growths  which  formed  a  dark- 
brown  pigment  from  adrenalin.  As  indicated  above,  we  tend  more  and 
more  to  accept  the  view  that  the  melanins  are  derived  from  the  cyclic 
components  of  the  proteins. 

In  addition  to  the  coloring  matters  of  the  human  skin  it  is  in  place  here  to 
treat  of  the  pigments  found  in  the  skin  or  epidermal  formation  of  animals. 

The  beautiful  color  of  the  feathers  of  many  birds  depends  in  certain  cases  on 
purely  physical  causes  (interference-phenomena),  but  in  other  cases  on  coloring 
matters  of  various  kinds.  Such  a  coloring  matter  is  the  amorphous  reddish- 
violet  turacin,  which  contains  7  per  cent  copper  and  whose  spectrum  is  very  similar 
to  that  of  oxyhemoglobin.  It  must  be  remarked  that  according  to  Laidlaw  5 
turacin  or  at  least  a  pigment  with  the  same  properties  can  be  obtained  on  boiling 
lurmatoporphyrin  in  dilute  ammonia  with  ammoniacal  copper  solution.  Kruken- 
berg  6  found  a  large  number  of  coloring  matters  in  bird's  feathers,  namely  zooery- 
thnn,  zoofulrin  turacoverdin,  zoorubin  psittacofulvin,  and  others  which  cannot  be 
enumerated  here. 

Tetronerythrin,  so  named  by  Wurm,  is  a  red  amorphous  pigment  which  is 
soluble  in  alcohol  and  ether,  and  which  occurs  in  the  red  warty  spots  over  the  eyes 
of  the  heathcock  and  the  grouse,  and  which  is  very  widely  spread  among  the 
invertebrates  (Halliburton,  De  Merejkowski  MacMunn).     Besides  tetronery- 


1  Hofmeister's  Beitrage,  2. 

2  Compt.  Rend.,  122. 

3  The  literature  can  be  found  in  v.  Furth  and  Jerusalem,  Hofmeister's   Beitrage, 
10. 

4  Neuberg,  Virchow's  Arch.,  192;  Jager,  ibid.,  198. 
6  Journ.  of  Physiol.,  31. 

s  Vergleichende  physiol.  Studien,  Abth.  5,  and  (2.  Reihe)   Abth.  1,  151,  Abth.  2,  1, 
and  Abth.  3,  128. 


844  THE  SKIN  AND  ITS  SECRETIONS. 

thrin  MacMunn  found  in  the  shells  of  crabs  and  lobsters  a  blue  coloring  matter, 
cyanocrystallin,  which  turns  red  with  acids  and  by  boiling  water.  Hcematoporphyrin, 
according  to  MacMunn,  also  occurs  in  the  integuments  of  certain  of  the  lower 
animals.  The  blue  pigment  occurring  in  the  fins  of  the  fish,  crenilabrus  pavo, 
is  according  to  v.  Zeynek  *  a  chromoprotein. 

In  certain  butterflies  (the  pieridinse)  the  white  pigment  of  the  wings  consists, 
as  shown  by  Hopkins,2  of  uric  acid,  and  the  yellow  pigment  of  a  uric-acid  deriva- 
tive, lepidotic  acid,  which  yields  a  purple  substance,  lepidoporphyrin,  on  warming 
with  dilute  sulphuric  acid.  The  yellow  and  red  pigment  of  the  Vanessa  are, 
according  to  Linden,3  of  an  entirely  different  kind.  In  this  case  we  are  dealing 
with  a  compound  between  protein  and  a  pigment  which  is  allied  to  bilirubin  or 
urobilin,  i.e.,  a  compound  similar  to  haemoglobin. 

In  addition  to  the  coloring  matters  thus  far  mentioned  a  few  others  found  in 
certain  animals  (though  not  in  the  skin)  will  be  spoken  of. 

Carminic  Acid,  or  the  red  pigment  of  the  cochineal,  gives  on  oxidation,  accord- 
ing to  Liebermann  and  Voswinckel,4  cochenillic  acid,  CioH807,  and  coccinic  acid. 
C6H80t,  the  first  being  the  tri-carboxylic  acid,  and  the  other  the  di-carboxylic 
acid,  of  m-cresol.  The  beautiful  purple  solution  of  ammonium  carminate  has  two 
absorption-bands  between  D  and  E  which  are  similar  to  those  of  oxyhemoglobin. 
These  bands  lie  nearer  to  E  and  closer  together  and  are  less  sharply  defined.  Pur- 
ple is  the  evaporated  residue  from  the  purple-violet  secretion,  caused  by  the  action 
of  the  sunlight,  upon  the  so-called  "  purple  gland  "  of  the  mantle  of  certain  species 
of  murex  and  purpura.  According  to  Friedlander  5  the  pigment  is  a  bromine 
derivative  of  indigo  and  indeed  di-bromindigo. 

Among  the  remaining  coloring  matters  found  in  invertebrates  may  be  men- 
tioned blue  stentorin,  actiniochrom,  bonellin,  poly pery thrin,  pentacrinin,  antedonin, 
crustaceorubin,  janthinin,  and  chlorophyll. 

Sebum  when  freshly  secreted  is  an  oily  semi-fluid  mass  which  solidifies 
on  the  upper  surface  of  the  skin,  forming  a  greasy  coating.  Rohmann 
and  Linser  hold  that  sebum  is  a  mixture  of  the  secretion  of  the  sebaceous 
glands  and  of  the  constituents  of  the  epidermis.  Hoppe-Seyler  found, 
in  the  sebum,  a  body  similar  to  casein  besides  albumin  and  fat,  while 
Rohmann  and  Linser  claim  that  true  fat  occurs  only  to  a  very  slight 
extent.  On  saponification  the  sebum  gives  an  oil,  dermohin,  which 
combines  readily  with  iodine,  and  another  body,  dermocerin,  which 
melts  at  64-65°  and  which  occurs  to  a  considerable  extent  in  dermoid 
cysts,  and  which  is  perhaps  identical  with  the  constituent  of  cysts, 
called  cetyl  alcohol  by  v.  Zeynek.  According  to  Ameseder  this  der- 
mocerin is  not  a  pure  substance,  and  the  cetyl  alcohol,  obtained  from 
the  fat  of  dermoid  cysts  is  an  eicosyl  alcohol,  C20H42O,  corresponding  to 
arachinic  acid.     Cholesterin    is    found    in  especially   large  quantities  in 


1  Wurm,  cited  from  Maly's  Jahresber.,  1;  Halliburton,  Journ.  of  Physiol.,  6;  Merej- 
kowski,  Compt.  Rend.,  93;  MacMunn,  Proc.  Roy.  Soc,  1883,  and  Journ.  of  Physiol.,  7; 
v.  Zeynek,  Zeitschr.  f.  physiol.  Chem.,  34  and  36,  and  Wien.  Sitz.-Ber.  121,  1912. 

'  Phil.  Trans.,  186. 

'  Pfluger's  Arch.,  98. 

4  Ber.  d.  deutsch.  chem.  Gesellsch.,  30. 

*  Ibid.,  42. 


SEBUM.     CERUMEN.  845 

the  verniz  caseosa.  Ruppel  l  found  on  an  average  in  the  vernix  caseosa 
348.52  p.  m.  water  and  138.72  p.  m.  ether  extractives,  and  also  mentions 
the  presence  of  isocholesterin.  These  claims  are  disputed  by  Unna.2 
In  his  experience  isocholesterin  does  not  occur  in  the  vernix  fat  nor  in 
the  sebum  of  man,  although  all  kinds  of  sebum  contain  cholesterin. 

According  to  Unna  and  Golodetz3  the  fat  secretion  (of  the  skin), 
as  the  fat  of  the  ball  of  the  foot,  and  sebum  are  rich  in  oxycholesterin, 
while  the  cell  fats  of  the  outer  skin  does  not  contain  any  oxycholesterin. 
The  nails,  which  are  rather  rich  in  oxycholesterin,  are  an  exception. 

On  account  of  the  opinion  generally  held  that  the  wax  of  the  plant 
epidermis  serves  as  protection  for  the  inner  parts  of  the  fruit  and  plant, 
Liebreich  4  has  suggested  that  these  combinations  of  fatty  acids  with 
monatomic  alcohols  are  the  cause  of  the  waxes  having  a  greater  resistance 
as  compared  with  the  glycerin  fats.  He  also  considers  that  the  choles- 
terin fats  play  the  role  of  a  protective  fat  in  the  animal  kingdom,  and  he 
has  been  able  to  detect  cholesterin  fat  in  human  skin  and  hair,  in  vernix 
caseosa,  whalebone,  tortoise-shell,  cow's  horn,  the  feathers  and  beaks 
of  several  birds,  the  spines  of  the  hedgehog  and  porcupine,  the  hoofs  of 
horses,  etc.  He  draws  the  following  conclusion  from  this,  namely, 
that  the  cholesterin  fats  always  appear  in  combination  with  the  keratinous 
substance,  and  that  the  cholesterin  fat,  like  the  wax  of  plants,  serves 
as  protection  for  the  skin-surface  of  animals.  Of  the  sebum  fats  inves- 
tigated by  Unna  all  contained,  with  the  exception  of  the  epidermis  fat, 
besides  cholesterin,  greater  or  smaller  amounts  of  cholesterin  ester.  The 
epidermis  fat,  on  the  contrary,  was  almost  free  from  esters  and  consisted 
chiefly  of  free  cholesterin. 

In  the  fatty  protective  substance  secreted  by  the  Psylla  altii,  Sundvik  5 
found  psylla-alcohol,  CsnHesO,  which  exists  there  as  an  ester  in  combination  with 
peyllic  acid,  C32H65COOH.  This  alcohol  has  also  been  found  in  the  wax  of  the 
humble-bee. 

Cerumen  is  a  mixture  of  the  secretion  of  the  sebaceous  and  sweat 
glands  of  the  cartilaginous  part  of  the  outer  passages  of  the  ear.  It 
chiefly  contains  soaps  and  fat,  fatty  acids,  cholesterin  and  protein,  and. 
besides  these  a  red  substance  easily  soluble  in  alcohol  and  with  a  bitter- 
sweet taste.6 

1  Hoppe-Seyler,  Physiol.  Chem.,  760;  Linser  with  Rohmann,  Centralbl.  f.  Physiol., 
19,  317;  see  also  reference  in  ibid.,  18,  from  Deutsch.  Arch.  f.  klin.  Med.,  1904;  Riippel,. 
Zeitschr.  f.  physiol.  Chem.,  21;  Ameseder,  ibid.,  52;  Zumbusch,  ibid.,  59. 

2  Monatsch.  f.  prakt.  Dermat.,  45. 

3  Bioch.  Zeitschr.,  20. 

4  Virchow's  Arch.,  121. 

5  Zeitschr.  f.  physiol.  Chem.,  17,  25,  32,  53,  54  and  72. 
•  See  Lamois  and  Martz,  Maly's  Jahresber.,  27,  40. 


S46  THE  SKIN  AND  ITS  SECRETIONS. 

The  preputial  secretion,  smegma  prceputii,  contains  chiefly  fat,  also 
cholesterin  and  ammonium  soaps,  which  probably  are  produced  from 
decomposed  urine.  The  hippuric  acid,  benzoic  acid,  and  calcium  oxalate 
found  in  the  smegma  of  the  horse  probably  have  the  same  origin. 

We  may  also  consider  as  a  preputial  secretion  the  castoreum,  which  is  secreted 
by  two  peculiar  glandular  sacs,  in  the  prepuce  of  the  beaver.  The  castoreum  is  a 
mixture  of  proteins,  fats,  resins,  traces  of  phenol  (volatile  oil),  and  a  non-nitrog- 
enous body,  castorin,  crystallizing  from  alcohol  in  four-sided  needles,  insoluble 
in  cold  water,  but  somewhat  soluble  in  boiling  water,  and  whose  composition  is 
little  known. 

In  the  secretion  from  the  anal  glands  of  the  skunk,  butyl  mercaptan  and  alkyl 
sulphides  have  been  found  (Aldrich,  E.  Beckmann  l). 

Wool-fat,  or  the  so-called  fat-sweat  of  sheep,  is  a  mixture  of  the  secretion  of 
the  sudoriparous  and  sebaceous  glands.  There  is  found  in  the  watery  extract  a 
large  quantity  of  potassium  which  is  combined  with  organic  acid,  volatile  and  non- 
volatile fatty  acids,  benzoic  acid,  phenol-sulphuric  acid,  lactic  acid,  malic  acid, 
succinic  acid,  and  others.  The  fat  contains,  among  other  bodies,  abundant  quan- 
tities of  ethers  of  fatty  acids  with  cholesterin  and  isocholesterin.  Darmstadter 
and  Lifschutz  have  found  other  alcohols  in  wool-fat  besides  myristic  acid,  also 
two  oxyfatty  acids,  lanoceric  acid,  CjoHeoCj,  and  lanopalmitic  acid,  C16H32O3. 
Isocholesterin,  oxycholesterin  and  carnaubyl  alcohol,  C24H49OH,  are  besides  the 
two  last-mentioned  acids,  substances  that  are  characteristic  of  wool-fat.  Accord- 
ing to  Rohmann  2  wool-fat  contains  a  body  lanocerin,  which  is  the  internal  anhy- 
dride of  the  above-mentioned  lanoceric  acid. 

The  secretion  of  the  coccygeal  glands  of  ducks  and  geese  contains  a  body  similar 
to  casein,  besides  albumin,  nuclein,  lecithin,  and  fat,  but  no  sugar  (De  Jonge). 
The  chief  constituent  is  octadecyl  alcohol,  Ci8Hs80,  which  represents  40-45  per 
cent  of  the  ethereal  extract  (Rohmann).  The  fatty  acids  are  oleic  acid,  small 
amounts  of  caprylic  acid,  palmitic  acid,  and  stearic  acid,  and  optical  isomers  of 
lauric  and  myristic  acid.  The  fatty  acids  are  in  great  part  combined  with  the 
octadecylic  alcohol,  and  this  is  probably  formed  by  the  reduction  of  stearic  acid  or 
oelic  acid.  The  secretion  also  contains  a  substance  related  to  lanocerin  which 
Rohmann  calls  pennacerin.  Poisonous  bodies  have  been  found  in  the  secretion 
of  the  skin  of  the  salamander  and  the  toad,  namely,  samandarin  (Zaleski,  Faust) 
and  bufidin  (Jornara  and  Casali),  bufotalin  and  the  disputed  bodies  bufonin 
and  bvjotenin  (Faust,  Bertrand  and  Phisalix  3).  The  active  constituents  in 
the  poison  of  the  rattle-snake  and  cobra,  the  crotalotoxin  and  the  ophiotoxin  have 
been  isolated  and  studied  by  Faust.4  They  are  free  from  nitrogen  and  have  a 
similar  composition,  namely,  C34H54O21  and  C34H62O20  and  are  classified  in  the 
pharmacological  group  of  sapotoxins  by  Faust.  Thalassin  is  the  crystalline  body 
discovered  by  Richet  5  which  is  the  poisonous  constituent  of  the  feelers  of  the 
sea  nettle. 


1  Aldrich,  Journ.  of  Exp.  Med.,  1;  Beckmann,  Maly's  Jahresber.,  26,  566. 

2  Darmstader  and  Lifschutz,  Ber.  d.  d.  Chem.,  Gesellsch.,  29  and  31;  Rohmann, 
Hofmeister's  Beitrage,  5,  and  Centralbl.  f.  Physiol.,  19,  317.  See  also  Unna,  1.  c,  45; 
and  Lifschutz  and  Unna,  ibid.,  p.  234. 

a  De  Jonge,  Zeitschr.  f.  physiol.  Chem.,  3;  Rohmann,  1.  c;  Zaleski,  Hoppe-Seyler's 
Med.-chem.  (Jntersuch.,  p.  85;  Faust,  Arch.  f.  exp.  Path.  u.  Pharm.,  41;  Jornara  and 
Casali,  Maly's  Jahresbr.,  3;  Faust,  Arch.  f.  exp.  Path.  u.  Pharm.,  47  and  49;  Bertrand, 
Compt.  Rend.,  135;  Bertrand  and  Phisalix,  ibid. 

4  Arch.  f.  exp.  Path.  u.  Pharm.,  56  and  64. 

6  Pfluger's  Arch.,  108. 


PERSPIRATION.  847 

The  Perspiration.  Of  the  bodies  secreted  by  the  skin,  whose  quantity 
amounts  to  about  ^j  of  the  weight  of  the  body,  a  disproportionately 
large  part  consists  of  water.  Next  to  the  kidneys,  the  skin,  in  man,  is 
the  most  important  means  for  the  elimination  of  water.  As  the  glands 
of  the  skin  and  the  kidneys  stand  near  to  each  other  in  regard  to  their 
functions,  they  may  to  a  certain  extent  act  vicariously. 

The  circumstances  which  influence  the  secretion  of  perspiration  are  numerous, 
and  the  quantity  of  sweat  secreted  must  consequently  vary  considerably.  The 
secretion  differs  in  different  parts  of  the  skin,  and  it  has  been  stated  that  the  per- 
spiration of  the  cheek,  that  of  the  palm  of  the  hand,  and  that  under  the  arm  stand 
to  each  other  as  100:90:45.  From  the  unecjual  secretion  on  different  parts  of  the 
body  it  follows  that  no  results  as  to  the  quantity  of  secretion  for  the  entire  surface 
of  the  body  can  be  calculated  from  the  quantity  secreted  by  a  small  part  of  the 
skin  in  a  given  time.  In  determining  the  total  quantity  a  stronger  secretion  is  as 
a  rule  produced,  and  as  the  glands  can  with  difficulty  work  for  a  long  time  with 
the  same  energy,  it  is  hardly  correct  to  estimate  the  quantity  of  secretion  per  day 
from  a  strong  secretion  during  only  a  short  time. 

The  perspiration  obtained  for 'investigation  is  never  quite  pure,  but 
contains  cast-off  epidermis-cells,  also  cells  and  fat-globules  from  the 
sebaceous  glands.  Filtered  perspiration  is  a  clear,  colorless  fluid  with 
a  salty  taste  and  of  different  odors  from  different  parts  of  the  body.  The 
physiological  reaction  is  acid,  according  to  most  reports.  Under  certain 
conditions  an  alkaline  sweat  may  be  secreted  (Trumpy  and  Luchsinger, 
Heuss).  An  alkaline  reaction  may  also  depend  on  a  decomposition 
with  the  formation  of  ammonia.  According  to  a  few  investigators  the 
physiological  reaction  is  alkaline,  and  an  acid  reaction  depends  upon 
an  admixture  of  fatty  acids  from  the  sebum.  Camerer  found  that 
the  reaction  of  human  perspiration  in  certain  cases  was  acid  and  in 
others  alkaline.  Moriggia  found  that  the  sweat  from  herbivora  was 
ordinarily  alkaline,  while  that  from  carnivora  was  generally  acid. 
Smith  j  showed  that  horse's  sweat  is  strongly  alkaline. 

Kittsteiner,2  who  has  found  that  human  perspiration  is  nearly  always 
acid,  has  also  found  that  the  perspiration  from  the  vola  manus,  when 
not  contaminated  with  sebum,  is  acid  in  reaction  and  that  an  acid  reac- 
tion is  not  necessarily  dependent  upon  an  admixture  with  sebum. 

The  specific  gravity  of  human  perspiration  varies  between  1.001 
and  1.010.  It  contains  977.4-995.6  p.  m.,  average  about  982  p.  m. 
water.  The  solids  are  4.4-22.6  p.  m.  The  molecular  concentration 
also  varies  widely  and  the  freezing-point  depression  depends  essentially 

1  Trumpy  and  Luchsinger,  Pfli'iger's  Arch.,  18;  Heuss,  Maly's  Jahresbcr.,  22; 
Camerer,  Zeitschr.  f.  Biologie,  41;  Moriggia,  Moleschott's  Untersuch.  zur  Xaturlehre, 
11;  Smith,  Journ.  of  Physiol.,  11.  In  regard  to  the  older  literature  on  perspiration, 
Bee  Hermann's  Handbuch,  5,  Thl.  1,  421  and  543. 

2  Arch.  f.  Hyg.,  73  and  78. 


848  THE  SKIN  AND  ITS  SECRETIONS. 

upon  the  content  of  NaCl.  Ardin-Delteil  found  A  =  —0.08-0.46°, 
average— 0.327°.  Brieger  and  Disselhorst  found  with  perspiration 
containing  2.9,  7.07  and  13.5  p.  m.  NaCl  that  the  A  was  equal  to-0.322°, 
—  0.608°  and  —1.002°,  respectively.  Tarugi  and  Tomasinelli  *  found  A 
to  be  0.52°  as  an  average.  Kittsteiner2  found  that  perspiration  had  an 
average  specific  gravity  of  1.0046  and  the  average  quantities  of  nitrogen 
and  sulphur  were  0.5  and  0.08  p.  m.  respectively.  The  NaCl  content 
increased  with  the  rapidity  of  secretion  while  the  nitrogen  content 
.  diminished.  The  organic  bodies  are  neutral  fats,  cholesterin,  volatile  fatty 
acids,  traces  of  protein  (according  to  Leclerc  and  Smith  always  in 
horses,  and  according  to  Gaube  regularly  in  man,  while  Leube  3  claims 
only  occasionally  after  hot  baths,  in  Bright's  disease,  and  after  the  use 
of  pilocarpin),  creatinine  (Capranica),  aromatic  oxyacids,  ethereal-sulphuric 
acids  of  phenol  and  skatoxijl  (Kast4),  sometimes  also  of  indoxyl,  serine 
(page  145)  and  lastly  urea.  The  quantity  of  urea  has  been  determined  by 
Argutinsky.  In  two  steam-bath  experiments,  in  which  in  the  course  of 
£  and  |  hour  respectively  he  obtained  225  and  330  cc.  of  perspiration,  he 
found  1.61  and  1.24  p.  m.  urea.  Of  the  total  nitrogen  of  the  perspiration 
in  these  two  experiments  68.5  per  cent  and  74.9  per  cent  respectively 
belong  to  the  urea.  From  Argutinsky's  experiments,  and  also  from 
those  of  Cramer,5  it  follows  that  of  the  total  nitrogen  a  portion,  not  to 
be  disregarded,  is  eliminated  by  the  perspiration.  This  portion  was 
indeed  12  per  cent,  in  an  experiment  of  Cramer,  at  high  temperature 
and  powerful  muscular  activity,  and  Zuntz  and  his  collaborators  find 
indeed  more  than  13  per  cent  in  high  altitudes.  Cramer  also  found 
ammonia  in  the  perspiration.  In  uraemia  and  in  anuria  in  cholera, 
urea  may  be  secreted  in  such  quantities,  by  the  sweat-glands,  that  crystals 
deposit  upon  the  skin.  The  mineral  bodies  consist  chiefly  of  sodium 
chloride  with  some  potassium  chloride,  alkali  sulphate  and  phosphate. 
The  relative  quantities  of  these  in  perspiration  differ  materially  from 
the  amount  in  the  urine  (Favre,  Kast6).  The  relation,  according  to 
Kast,  is  as  follows: 


Chlorine 

In  perspiration 

In  urine 


Phosphate 
0.001.5 
0.1320 


Sulphate: 
0.009 
0.397 


1  Ardin-Delteil,    Maly's  Jahresber.,   30;  Brieger   and    Disselhorst,   Deutsch.   med. 
Wochenschr.,  29;  Tarugi  and  Tomasinelli,  cited  in  Physiol.  Centralbl.,  22,  748. 

2  I.e. 

3  Leclerc,   Compt.   Rend.,    107;  Gaube,    Maly's  Jahresber.,   22;  Leube,  Virchow's 
Arch.,  48  and  50,  and  Arch.  f.  klin.  Med.,  7. 

*  Capranica,  Maly's  Jahresber.,  12;  Kast,  Zeitschr.  f.  physiol.  Chem.,  11. 
5  Argutinsky,  Pfluger's  Arch.,  46;     Cramer,  Arch.  f.  Hygiene,  10. 
•Compt.  Rend.,  35,  and  Arch,  gener.  de  Med.  (5),  2;  Kast,  1.  c. 


EXCHANGE  OF  GAS  THROUGH  THE  SKIN.  849 

Kast  found  that  the  proportion  of  ethereal-sulphuric  acid  to  the 
sulphate-sulphuric  acid  in  perspiration  was  1:12.  After  the  administra- 
tion of  aromatic  substances  the  ethereal-sulphuric  acid  does  not  increase 
to  the  same  extent  in  the  perspiration  as  in  the  urine  (see  Chapter  XIV). 
The  quantity  of  mineral  substances  was  on  an  average  7  p.  m. 

Sugar  may  pass  into  the  perspiration  in  diabetes,  but  the  passage  of  the  bile- 
coloring  matters  has  not  been  positively  shown  in  this  secretion.  Benzoic  acid, 
succinic  acid,  tartaric  acid,  iodine,  arsenic,  mercuric  chloride  and  quinine  pass 
into  the  perspiration.  Uric  acid  has  also  been  found  in  the  perspiration  in  gout 
and  cystine  in  cystinuria. 

Chromnidrosia  is  the  name  given  to  the  secretion  of  colored  perspiration. 
Sometimes  perspiration  has  been  observed  to  be  colored  blue  by  indigo  (Bizio), 
by  pyocyanin,  or  by  ferro-phosphate  (Collmann  l).  True  blood-sweat,  in  which 
blood-corpuscles  exude  from  the  opening  of  the  glands,  has  also  been  observed. 

The  exchange  of  gas  through  the  skin  is  of  great  importance  for  non- 
scaly  amphibians;  in  mammalia,  birds  and  human  beings  it  is  of  little 
importance  compared  with  the  exchange  of  gas  by  the  lungs.  The 
absorption  of  oxygen  by  the  skin,  which  was  first  shown  by  Regnault 
and  Reiset,  is  small,  and  according  to  Zuelzer  amounts  under  the 
most  favorable  circumstances  to  Tfo-  of  the  oxygen  absorbed  by  the 
lungs.  The  quantity  of  carbon  dioxide  eliminated  by  the  skin  increases 
with  the  rise  of  temperature  (Aubert,  Rohrig,  Fubini  and  Ronchi, 
Barratt  and  according  to  Willebrand  beginning  at  33°)2.  It  especially 
increases  with  hyperemia  of  the  skin  and  in  particular  after  muscular 
activity.  It  is  also  greater  in  light  than  in  darkness.  It  is  greater  dur- 
ing digestion  than  when  fasting,  and  greater  after  a  vegetable  than  after 
an  animal  diet  (Fubini  and  Ronchi).  The  quantity  calculated  by  differ- 
ent investigators  for  the  entire  skin  surface  in  twenty-four  hours  varies 
between  2.23  and  32.8  grams.  According  to  Schierbeck  and  Wille- 
brand 3  the  average  quantity  is  7.5-9  grams,  and  it  is  ordinarily  given  as 
about  1.5  per  cent  of  the  quantity  eliminated  by  the  lungs.  In  a  horse, 
Zuntz,  with  Lehmann  and  Hagemann,4,  found  for  twenty-four  hours 
an  elimination  of  carbon  dioxide  by  the  skin  and  intestine  which  amounted 
to  nearly  3  per  cent  of  the  total  respiration.  Less  than  four-fifths  of 
this  carbon  dioxide  came  from  the  skin  respiration.  The  same  investi- 
gators found  that  the  skin  respiration  equals  2\  per  cent  of  the  simulta- 
neous lung  respiration. 

1  Bizio,  Wien.  Sitzungsber.,  39;  Collmann,  cited  from  v.  Gorup-Besanez's  Lehrbuch, 
4.  Aufl.,  555. 

2  Zuelzer,  Zeitschr.  f.  klin.  Med.,  53;  Aubert,  Pfluger's  Arch.,  6;  Rohrig,  Deutsch. 
Klin.,  1872,  209;  Fubini  and  Ronchi,  Moleschott's  Untersuch.  z.  Naturlehre,  12; 
Barratt,  Journ.  of  Physiol.,  21;   Willebrand,  Skand.  Arch.  f.  Physiol.,  13. 

1  See  Hoppe-Seyler,  Physiol.  Chem.,  580;  Schierbeck,  Arch.  f.  (Anat.  u.)  Physiol., 
1892;  Willebrand,  1.  c. 

4  Arch.  f.  (Anat.  u.)  Physiol.,  1894,  and  Maly's  Jahresber.,  24. 


CHAPTER  XVI. 
RESPIRATION  AND  OXIDATION. 

During  life  a  constant  exchange  of  gases  takes  place  between  the 
animal  body  and  the  surrounding  medium.  Oxygen  is  inspired  and 
carbon  dioxide  expired.  This  exchange  of  gases,  which  is  called  respira- 
tion, is  brought  about  in  man  and  vertebrates  by  the  nutritive  fluids, 
blood  and  lymph,  which  circulate  in  the  body  and  which  are  in  constant 
communication  with  the  outer  medium  on  one  side  and  the  tissue-elements 
on  the  other.  Such  an  exchange  of  gaseous  constituents  may  take  place 
wherever  the  anatomical  conditions  offer  no  obstacle,  and  in  man  it  may 
go  on  in  the  intestinal  tract,  through  the  skin,  and  in  the  lungs.  As 
compared  with  the  exchange  of  gas  in  the  lungs,  the  exchange  already 
mentioned,  which  occurs  in  the  intestine  and  through  the  skin,  is  very 
insignificant.  For  this  reason  we  will  discuss  in  this  chapter  only  the 
exchange  of  gas  between  the  blood  and  the  air  of  the  lungs  on  one  side 
and  the  blood  and  lymph  and  the  tissues  on  the  other.  The  first  is  often 
designated  as  external  respiration,  and  the  other,  internal  respiration. 
Besides  this  we  will  discuss  the  oxidation  processes  caused  by  the  internal 
respiration. 

I.    THE   GASES   OF  THE  BLOOD. 

Since  the  pioneer  investigations  of  Magnus  and  Lothar  Meyer,  the 
gases  of  the  blood  have  formed  the  subject  of  repeated  careful  investiga- 
tions by  prominent  experimenters,  among  whom  must  be  mentioned  first 
C.LuDwiGand  his  pupils,  and  E.  Pfluger  and  his  school;  and  C.  Bohr. 
By  these  investigations  not  only  has  science  been  enriched  by  a  mass  of 
facts,  but  also  the  methods  themselves  have  been  made  more  perfect 
and  accurate.  In  regard  to  these  methods,  as  also  in  regard  to  the  laws 
of  the  absorption  of  gases  by  liquids,  dissociation,  and  related  questions, 
the  reader  is  referred  to  text-books  on  physiology,  on  physics,  and  on 
gasometric  analysis. 

The  gases  occurring  in  blood  under  physiological  conditions  are 
oxygen,  carbon  dioxide  and  nitrogen,  and  traces  of  argon,  and  perhaps 
also  carbon  monoxide.  Traces  of  hydrogen  and  marsh-gas  also  some- 
times occur.  The  nitrogen  is  found  only  in  very  small  quantities,  on  an 
average  1.2  vols,  per  cent.     The  quantity  is  here,  as  in  all  following 

850 


GASES  OF  THE  BLOOD.  851 

experiments,  calculated  for  0°  C.  and  760  mm.  mercury  pressure.  The 
nitrogen  seems  to  be  simply  absorbed  by  the  blood,  at  least  in  great 
part.  It  appears,  like  argon,  to  play  no  direct  part  in  the  processes  of 
life,  and  its  quantity  varies  but  slightly  in  the  blood  of  different  blood- 
vessels. 

The  oxygen  and  carbon  dioxide  behave  otherwise,  as  their  quantities 
have  significant  variations,  not  only  in  the  blood  from  different  blood- 
vessels, but  also  because  many  factors,  such  as  a  difference  in  the  rapidity 
of  circulation  and  the  ventilation  of  the  lungs,  a  different  temperature, 
alkalinity  of  the  blood,  rest  and  activity  cause  a  change.  In  regard  to  the 
gases  they  contain,  the  greatest  difference  is  observable  between  the  blood 
of  the  arteries  and  that  cf  the  veins. 

The  quantity  of  oxygen  in  the  arterial  blood  (of  dogs)  is  on  an  average 
22  vols,  per  cent  (Pfluger,  Bohr  and  Henriques).  In  human  blood 
Setschenow  found  about  the  same  quantity,  namely,  21. G  vols,  per 
cent.  Leowy  in  another  manner  has  determined  the  quantity  of  oxygen 
which  the  blood  can  take  up  by  first  shaking  human  venous  blood  with 
air  and  then  calculating  from  this  the  quantity  of  oxygen  in  human 
arterial  blood.  He  calculates  the  average  amount  as  18  vols,  per  cent. 
Lower  figures  have  been  found  for  the  blood  of  herbivora  (such  as  horse, 
sheep,  rabbits)  and  birds  (hen  and  ducks)  namely,  14-10.7  per  cent  (Zuntz 
and  Hagemann,  Sczelkow,  Walter,  Jolyet).  Venous  blood  in  dif- 
ferent vascular  regions  has  variable  quantities  of  oxygen.  By  sum- 
marizing a  great  number  of  analyses  by  different  experimenters,  Zuntz 
has  calculated  that  the  venous  blood  of  the  right  side  of  the  heart  con- 
tains on  an  average  7.15  per  cent  less  oxygen  than  the  arterial  blood. 

The  quantity  of  carbon  dioxide  in  the  arterial  blood  (of  dogs)  is  about 
40  vols,  per  cent  (Ludwig,  Setschenow,  Pfluger,  P.  Bert,  Bohr 
and  Henriques  and  others),  or  a  little  above.  In  herbivora  and  the 
above-mentioned  birds  the  quantity  of  carbon  dioxide  in  the  arterial 
blood  is  higher  than  in  the  carnivorous  dog.  Setschenow  found  40.3 
vols,  per  cent  in  human  arterial  blood.  The  quantity  of  carbon  dioxide 
in  venous  blood  varies  still  more  (Ludwig,  Pfluger,  and  their  pupils, 
P.  Bert,  Mathieu  and  Urbain,  and  others).  According  to  the  calcula- 
tions of  Zuntz,  the  venous  blood  of  the  right  side  of  the  heart  contains 
about  8.2  per  cent  more  carbon  dioxide  than  the  arterial.  The  average 
amount  may  be  put  down  as  50  vols,  per  cent.  Holmgren  found  in 
blood  after  asphyxiation  even  69.21  vols,  per  cent  carbon  dioxide.1 

1  All  the  figures  given  above  may  be  found  in  Zuntz 's  "  Die  Gase  des  Blutes  "  in 
Hermann's  Handbuch  d.  Physiol.,  4,  Thl.  2,  33-43,  which  also  contains  detailed  state- 
ments and  the  pertinent  literature,  and  Bohr  in  Xagel's  Handbuch  der  Physiologie  dcs 
Menschen,  Bd.  1,  Hefte  1,  1905,  and  in  Loewy,  Handb.  d.  Bioch.  of  C.  Oppenheimer, 
Bd.  4. 


852  RESPIRATION  AND  OXIDATION. 

Oxygen  is  dissolved  only  in  a  small  extent  by  the  plasma,  whose 
absorbability  for  oxygen  is  97.5  per  cent  of  that  of  water,  according  to 
Bohr.  The  greater  part  or  nearly  all  of  the  oxygen  is  loosely  combined 
with  the  haemoglobin.  The  quantity  of  the  oxygen  which  is  contained  in 
the  blood  of  the  dog  corresponds  closely  to  the  quantity  which,  from  the 
activity  of  the  haemoglobin,  we  should  expect  to  combine  with  oxygen 
and  from  the  quantity  of  haemoglobin  contained  therein.  It  is  difficult 
to  ascertain  how  far  the  circulating  arterial  blood  is  saturated  with  oxygen, 
as  immediately  after  bleeding  a  loss  of  oxygen  always  takes  place.  Still 
it  seems  to  be  unquestionable  that  it  is  not  quite  completely  saturated 
with  oxygen,  in  life.  The  laws  which  regulate  the  binding  of  the  oxygen 
in  the  blood  will  be  found  in  the  discussion  of  the  gas  exchanged  between 
the  blood  and  the  air  of  the  lungs. 

The  carbon  dioxide  of  the  blood  occurs  in  part,  and  indeed,  accord- 
ing to  the  investigations  of  Alex.  Schmidt,1  Zuntz,2  and  L.  Fredericq,3 
to  the  extent  of  at  least  one-third  in  the  blood-corpuscles,  also  in  part, 
and  in  fact  the  greatest  part,  in  the  plasma  or  serum.  Bohr4  claims 
that  about  30  mm.  may  be  considered  as  the  average  pressure  of  the 
carbon  dioxide  in  the  organism,  and  with  such  a  pressure  the  quantity  of 
physically  dissolved  CO2  in  100  cc.  of  the  blood  amounts  to  2.01  cc.  As 
the  blood  with  this  tension  takes  up  about  40  vols,  per  cent  CO2,  there- 
fore about  5  per  cent  of  the  total  carbon  dioxide  is  simply  dissolved. 
Under  the  assumption  that  the  blood  corpuscles  make  up  about  one- 
third  of  the  volume  of  the  blood,  of  the  physically  dissolved  CO2,  0.59 
cc.  exists  with  the  corpuscles  and  1.42  cc.  with  the  plasma. 

As  the  blood  corpuscles  in  100  cc.  blood  as  above  stated  take  up  at 
the  above  pressure  about  14  cc.  CO2,  only  a  small  part  of  its  CO2  is  physi- 
cally dissolved.  The  chief  mass  of  the  CO2  is  loosely  combined  and  the 
constituent  of  these  cells  which  unites  with  the  CO2  seems  to  be  the 
alkali  combined  with  phosphoric  acid,  oxyhemoglobin  or  haemoglobin, 
and  globulin  on  one  side  and  the  haemoglobin  itself  on  the  other.  That 
in  the  red  blood-corpuscles  alkali  phosphate  occurs  in  such  quantities 
that  it  may  be  of  importance  in  the  combination  with  carbon  dioxide 
is  not  to  be  doubted ;  and  it  must  be  allowed  that  from  the  diphosphate, 
by  a  greater  partial  pressure  of  the  carbon  dioxide,  monophosphate  and 
alkali  carbonate  are  formed,  while  by  a  lower  partial  pressure  of  the 
carbon  dioxide,  the  mass  action  of  the  phosphoric  acid  again  comes  into 
play,  so  that,  with  the  carbon  dioxide  becoming  free,  a  reformation  of 

1  Ber.  d.  k.  sachs.  Gesellsch.  d.  Wissensoh.  math.-phys.  Klasse,  1867. 
•Centralbl.  f.  d.  med.  Wissensch.,  1867,  529. 

3  Recherches  sur  la  constitution  du  Plasma  sanguin,  1878,  50,  51. 

4  In  regard  to  the  work  of  Bohr  we  will  refer  here  and  in  future  to  Nagel's  Handbuch 
der  Physiologje  des  Menschen,  Bd.  1. 


CARBON  DIOXIDE  IN  THE  BLOOD.  853 

alkali  diphosphate  takes  place.  It  is  generally  admitted  that  the  blood- 
coloring  matters,  especially  the  oxyhemoglobin,  which  can  expel  carbon 
dioxide  from  sodium  carbonate  in  vacuo,  acts  like  an  acid,  and  as  the 
globulins  also  act  similarly  (see  below),  these  bodies  may  also  occur  in 
the  blood-corpuscles  as  an  alkali  combination.  The  alkali  of  the  blood- 
corpuscles  must,  therefore,  according  to  the  law  of  mass  action,  be  divided 
between  the  carbon  dioxide,  phosphoric  acid,  and  the  other  constituents 
of  the  blood-corpuscles  which  possess  acidic  properties,  and  among  these 
especially  the  blood  pigments,  because  the  globulin  can  hardly  be  of 
importance  on  account  of  its  small  quantity.  By  greater  mass  action 
or  greater  partial  pressure  of  the  carbon  dioxide,  bicarbonate  must  be 
formed  at  the  expense  of  the  diphosphates  and  the  other  alkali  combina- 
tions, while  at  a  diminished  partial  pressure  of  the  same  gas,  with  the 
escape  of  carbon  dioxide,  the  alkali  diphosphate  and  the  other  alkali 
combinations  must  be  reformed  at  the  cost  of  the  bicarbonate. 

Haemoglobin  must  nevertheless,  as  the  investigations  of  Setschenow  l 
and  Zuntz,  and  especially  those  of  Bohr  and  Torup,2  have  shown,  be 
able  to  hold  the  carbon  dioxide  loosely  combined  even  in  the  absence 
of  alkali.  Bohr  has  also  found  that  the  dissociation  curve  of  the  car- 
bon dioxide  haemoglobin  corresponds  essentially  to  the  curve  of  the 
absorption  of  carbon  dioxide,  on  which  ground  he  and  Torup  consider 
the  haemoglobin  itself  as  of  importance  in  the  binding  of  the  carbon 
dioxide  of  the  blood,  and  not  its  alkali  combinations.  According  to 
Bohr  the  haemoglobin  takes  up  the  two  gases,  oxygen  and  carbon  dioxide, 
simultaneously  by  the  oxygen  uniting  with  the  pigment  nucleus  and  the 
carbon  dioxide  with  the  protein  component.  But  as  according  to  the 
researches  of  Zuntz  3  the  combination  of  haemoglobin  with  the  alkali  is 
first  split  to  any  great  extent  with  a  carbon  dioxide  tension  of  more  than 
70  mm.,  it  must  be  admitted  that  with  the  ordinary  CO2  pressure  in 
the  organism,  the  combination  of  the  carbon  dioxide  in  the  blood  cor- 
puscles does  not  essentially  take  place  through  the  agency  of  the  alkali 
but  chieriy  by  means  of  the  haemoglobin. 

The  chief  part  of  the  carbon  dioxide  of  the  blood  is  found  in  the 
blood-plasma  or  the  blood-serum,  which  follows  from  the  fact  that  the 
serum  is  richer  in  carbon  dioxide  than  the  corresponding  blood  itself. 
By  experiments  with  the  air-pump  on  blood-serum  it  has  been  found 
that  the  chief  part  of  the  carbon  dioxide  contained  in  the  serum  is  given 
off  in  a  vacuum,  while  a  smaller  part  can  be  removed  only  after  the 


1  Centralbl.  f.  d.  med.  Wissench.,  1877.     See  also  Zuntz  in  Hermann's  Handbuch, 
76. 

1  Zuntz,  1.  c,  76;  Bohr,  Maly's  Jahresber.,  17;  Torup,  ibid. 
1  Centralbl.  f.  d.  med.  Wissensch.,  1867. 


854  RESPIRATION  AND  OXIDATION. 

addition  of  an  acid.  The  red  blood-corpuscles  also  act  as  an  acid,  and 
therefore  in  blood  all  the  carbon  dioxide  is  expelled  in  vacuo.  Hence 
a  part  of  the  carbon  dioxide  is  in  firm  chemical  combination  in  the  serum. 

Absorption  experiments  with  blood-serum  have  shown  us  further  that 
the  carbon  dioxide  which  can  be  pumped  out  is  in  greater  part  loosely 
chemically  combined,  and  from  this  loose  combination  of  the  carbon 
dioxide  it  necessarily  follows  that  the  serum  must  also  contain  simply 
absorbed  carbon  dioxide.  For  the  form  of  binding  of  the  carbon  dioxide 
contained  in  the  serum  or  the  plasma,  there  are  the  three  following  pos- 
sibilities: 1.  A  part  of  the  carbon  dioxide  is  simply  absorbed;  2.  Another 
part  is  in  loose  chemical  combination;  3.  A  third  part  is  in  firm  chemical 
combination. 

The  quantity  of  physically  dissolved  carbon  dioxide  in  the  serum 
cannot  be  higher  than  about  2  vols,  per  cent,  as  the  quantity  of  carbon 
dioxide  in  the  plasma  corresponding  to  100  cc.  of  blood  is  given  above 
as  1.42  cc. 

The  quantity  of  carbon  dioxide  in  the  blood-serum  which  is  combined 
as  a  firm  chemical  union  depends  upon  the  quantity  of  simple  alkali 
carbonate  in  the  serum.  This  amount  is  not  known,  and  it  cannot  be 
determined  either  by  the  alkalinity  found  by  titration,  nor  can  it  be  cal- 
culated from  the  excess  of  alkali  found  in  the  ash,  because  the  alkali  is  not 
only  combined  with  carbon  dioxide,  but  also  with  other  bodies,  especially 
with  protein.  The  quantity  of  carbon  dioxide  in  firm  chemical  combi- 
nation cannot  be  ascertained  after  pumping  out  in  vacuo  without  the 
addition  of  acid,  because  to  all  appearances  certain  active  constituents 
of  the  serum,  acting  like  acids,  expel  carbon  dioxide  from  the  simple 
carbonate.  The  quantity  of  carbon  dioxide  not  expelled  from  dog- 
serum  by  vacuum  alone  without  the  addition  of  acid  amounts  to  4.9 
to  9.3  vols,  per  cent,  according  to  the  determinations  of  Pfluger.1 

From  the  occurrence  of  simple  alkali  carbonates  in  the  blood-serum 
it  naturally  follows  that  a  part  of  the  loosely  combined  carbon  dioxide 
of  the  serum  which  can  be  pumped  out  must  exist  as  bicarbonate.  The 
occurrence  of  this  combination  in  the  blood-serum  has  also  been  directly 
shown.  In  experiments  with  the  pump,  as  well  as  in  absorption  experi- 
ments, the  serum  behaves  in  other  ways  differently  from  a  solution  of  bicar- 
bonate, or  carbonate  of  a  corresponding  concentration;  and  the  action 
of  the  loosely  combined  carbon  dioxide  in  the  serum  can  be  explained 
only  by  the  occurrence  of  bicarbonate  in  the  serum.  By  means  of  a 
vacuum,  the  serum  always  allows  much  more  than  one-half  of  the  carbon 
dioxide  to  be  expelled  and  it  follows  from  this  that  in  the  pumping  out 

1  E.  Pflvip;er's  Ueber  die  Kohlensiiure  des  Blutes,  Bonn,  1864,  11.  Cited  from 
Zuntz  in  Hermann's  Handbuch,  65. 


CARBON  DIOXIDE  IN  THE  BLOOD.  855 

not  only  may  a  dissociation  of  the  bicarbonate  take  place,  but  also  a 
conversion  of  the  double  sodium  carbonate  into  a  simple  salt.  As  we 
know  of  no  other  carbon-dioxide  combination,  besides  the  bicarbonate, 
in  the  serum,  from  which  the  carbon  dioxide  can  be  set  free  by  simple 
dissociation  in  vacuo,  it  must  be  assumed  that  the  serum  contains  other 
weak  acids,  in  addition  to  the  carbon  dioxide,  which  contend  with  it  for 
the  alkalies,  and  which  expel  the  carbon  dioxide  from  simple  carbonates 
in  vacuo.  The  carbon  dioxide  which  is  expelled  by  means  of  the  pump, 
and  which,  without  regard  to  the  quantity  merely  absorbed,  is  generally 
designated  as  "  carbon  dioxide  in  loose  chemical  combination,"  is  thus 
only  obtained  in  part  in  dissociable  loose  combinations;  in  part  it  origi- 
nates from  the  simple  carbonates,  from  which  it  is  expelled,  in  vacuo,  by 
other  weak  acids. 

These  weak  acids  are  thought  to  be  in  part  phosphoric  acid  and  in 
part  globulins.  The  importance  of  the  alkali  phosphates  in  the  car- 
bon dioxide  combination  has  been  shown  by  the  investigations  of  Fernet; 
but  the  quantity  of  these  salts  in  the  serum  is,  at  least  in  certain  kinds 
of  blood,  for  example,  in  ox-serum,  so  small  that  it  can  hardly  be  of 
importance.  In  regard  to  the  globulins,  Setschenow  is  of  the  opinion 
that  they  do  not  act  as  acids  themselves,  but  form  a  combination  with 
carbon  dioxide,  producing  carboglobulinic  acid,  which  unites  with  the 
alkali.  According  to  Sertoli,1  whose  views  have  found  a  supporter 
in  Torup,  the  globulins  themselves  are  the  acids  which  are  combined 
with  the  alkali  of  the  blood-serum.  In  both  cases  the  globulins  would 
form,  directly  or  indirectly,  that  chief  constituent  of  the  plasma  or  of 
the  blood-serum  which,  according  to  the  law  of  mass  action,  contends 
with  the  carbon  dioxide  for  the  alkalies.  By  a  greater  partial  pressure 
of  the  carbon  dioxide  the  latter  deprives  the  globulin  alkali  of  a  part  of 
its  alkali,  and  bicarbonate  is  formed;  by  low  partial  pressure  carbon 
dioxide  is  set  free  and  it  is  abstracted  from  the  bicarbonate  by  the 
globulin  alkali.  It  must  also  be  added  that  the  above-mentioned  car- 
boglobulinic acid  can  perhaps  be  considered  as  a  dissociable  combination 
of  carbonic  acid  and  protein. 

The  assumption  that  the  proteins  of  the  blood  are  bodies  active  in 
combining  with  the  carbon  dioxide  has  received  some  support  from  the 
investigations  of  Siegfried2  on  the  combination  of  carbon  dioxide  with 
amphoteric  amino  bodies.  Siegfried  has  found  that  amino-acids  com- 
bine   with    carbon    dioxide,    thereby   being   converted   into   carbamino- 


1  Hoppe-Seyler,  Med.  chem.  Untersuch.,  350. 

2  Zeitschr.  f.  physiol.  Chem.,  44  and  46. 


856  RESPIRATION  AND  OXIDATION. 

H 

I 
acids— glycocoll  for  example,  into  carbamino  acetic  acid,  CH2 — N — COOH, 

COOH 

and  that  the  carbon  dioxide  can  be  readily  split  off  from  these  compounds. 
The  peptones  and  serum  proteins  in  the  presence  of  calcium  hydroxide 
may  also  act  in  the  same  manner  as  amino-acids.  Protein  carbamino- 
acids  are  formed,  and  the  possibility  of  such  a  binding  of  carbon  dioxide 
must  also  be  considered. 

In  the  foregoing  it  has  been  assumed  that  the  alkali  is  the  most  essen- 
tial and  important  constituent  of  the  blood-serum,  as  well  as  of  the  blood 
in  general,  in  uniting  with  the  carbon  dioxide.  The  fact  that  the  quan- 
tity of  carbon  dioxide  in  the  blood  greatly  diminishes  with  a  decrease 
in  the  quantity  of  alkali  strengthens  this  assumption.  Such  a  condi- 
tion is  found,  for  example,  after  poisoning  with  mineral  acids.  Thus 
Walter  found  only  2-3  vols,  per  cent  carbon  dioxide  in  the  blood  of 
rabbits  into  whose  stomachs  hydrochloric  acid  had  been  introduced.  In 
the  comatose  state  of  diabetes  mellitus  the  alkali  of  the  blood  seems  to 
be  in  great  part  saturated  with  acid  combinations,  /3-oxybutyric  acid 
(Stadelmann,  Minkowski),  and  Minkowski  x  found  only  3.3  vols, 
per  cent  carbon  dioxide  in  the  blood  in  diabetic  coma. 

Gases  of  the  Lymph  and  Secretions. 

The  gases  of  the  lymph  are  the  same  as  in  the  blood-serum,  and  the 
lymph  stands  close  to  the  blood-serum  in  regard  to  the  quantity  of  the 
various  gases,  as  well  as  to  the  kind  of  carbon-dioxide  combination.  The 
investigations  of  Daenhardt  and  Hensen  2  on  the  gases  of  human 
lymph  are  at  hand,  but  it  still  remains  a  question  whether  the  lymph 
investigated  was  quite  normal.  The  gases  of  normal  dog-lymph  were 
first  investigated  by  Hammarsten.3  This  gas  contained  traces  of 
oxygen  and  consisted  of  37.4-53.1  per  cent  CO2  and  1.6  per  cent  N  at  0° 
C.  and  760  mm.  Hg  pressure.  About  cne-half  of  the  carbon  dioxide  was 
in  firm  chemical  combination.  The  quantity  was  greater  than  in  the 
serum  from  arterial  blood,  but  smaller  than  from  venous  blood. 

The  remarkable  observation  of  Buchner,  that  the  lymph  collected 
after  asphyxiation  is  poorer  in  carbon  dioxide  than  that  of  the  breathing 


1  Walter,  Arch.  f.  exp.  Path.  u.   Pharm.,    7;  Stadelmann,  ibid.,   17;  Minkowski, 
Mittheil  a.  d.  med.  Klinik  in  Konigsberg,  1888. 

2  Virchow's  Arch.,  37. 

1  Ber.  d.  k.  sachs.  Gesellsch.  d.  Wissensch.,  math.-phys.  Klasse,  23. 


GASES  OF  THE  LYMPH  AND  .SECRETIONS.  857 

animal,  is  explained  by  Zuntz  '  by  the  formation  of  acid  in  the  tissues, 
ami  especially  in  the  lymphatic  glands,  immediately  after  death,  and 
this  acid  in  pari  decomposes  the  alkali  carbonates  <  f  the  Lymph. 

The  secretions,  with  the  exception  of  the  saliva,  in  which  Pfluger 
and  Kulz  found  respectively  0.6  per  cent  and  1  per  cent  oxygen,  are 
almost  free  from  oxygen.  The  quantity  of  nitrogen  is  the  same  as  in  blood, 
and  the  chief  mass  of  the  gases  consists  of  carbon  dioxide.  The  quantity 
of  this  gas  is  chiefly  dependent  upon  the  reaction,  i.e.,  upon  the  quan- 
tity of  alkali.  This  follows  from  the  analyses  of  Pfluger.  He  found 
19  per  cent  carbon  dioxide  removable  by  the  air-pump  and  54  per  cent 
firmly  combined  carbon  dioxide  in  a  strongly  alkaline  bile,  but,  on  the 
contrary,  G.6  per  cent  carbon  dioxide  removable  by  the  air-pump  and 
0.8  per  cent  firmly  combined  carbon  dioxide  in  a  neutral  bile.  Alkaline 
saliva  is  also  very  rich  in  carbon  dioxide.  As  average  for  two  analyses 
made  by  Pfluger  of  the  submaxillary  saliva  of  a  clog  we  have  27.5  per 
cent  carbon  dioxide  removable  by  the  air-pump  and  47.4  per  cent  chem- 
ically combined  carbon  dioxide,  making  a  total  of  74.9  per  cent.  Kulz  2 
found  a  maximum  of  65.78  per  cent  carbon  dioxide  for  the  parotid  saliva, 
of  which  3.31  per  cent  was  removable  by  the  air-pump  and  62.7  per 
cent  was  firmly  combined.  From  these  and  other  reports  as  to  the 
quantity  of  carbon  dioxide  removable  by  the  air-pump  and  chemically 
combined  in  the  alkaline  secretions  it  follows  that  bodies  occur  in  them, 
although  not  in  appreciable  quantities,  which  are  analogous  to  the  pro- 
tein bodies  of  the  blood-serum  and  which  act  like  weak  acids. 

The  acid  or  at  any  rate  non-alkaline  secretions,  urine  and  milk,  con- 
tain, on  the  contrary,  considerably  less  carbon  dioxide,  which  is  almost 
all  removable  by  the  air-pump,  and  a  part  seems  to  be  loosely  combined 
with  the  sodium  phosphate.  The  figures  found  by  Pfluger  for  the 
total  quantity  of  carbon  dioxide  in  milk  and  urine  are  10  and  18.1-19.7 
per  cent  respectively. 

Ewald  3  made  investigations  on  the  quantity  of  gas  in  pathological 
transudates.  He  found  only  traces,  or  at  least  only  very  insignificant 
quantities  of  oxygen  in  these  fluids.  The  quantity  of  nitrogen  was  about 
the  same  as  in  blood;  that  of  carbon  dioxide  was  greater  than  in  the 
lymph  (of  dogs),  and  in  certain  cases  even  greater  than  in  the  blood  after 
asphyxiation  (dog's  blood).  The  tension  of  the  carbon  dioxide  was 
greater  than  in  venous  blood.  In  exudates  the  quantity  of  carbon 
dioxide,  especially  that  firmly  combined,  increases  with  the  age  of  the 


1  Buchner,  Arbeiten  aus  der  physiol.  Anstalt  zu  Leipzig,  1876;    Zuntz,  1.  c,  85. 

2  Pfluger,  Pfltiger'a  Arch.,  1  and  2;  Kitlz,  Zcitschr.  f.  Biologie,  23.     It  seems  as  if 
Kiilz's  results  were  not  calculated  at  760  millimeters  Hg.  but  rather  at  1  meter. 

3C.  A.  Ewald,  Arch.  f.  (Anat.  u.)  Physiol.,  1873  and  1876. 


858  KESPIEATION  AND  OXIDATION. 

fluid,  while,  on  the  contrary,  the  total  quantity  of  carbon  dioxide,  and 
especially  the  quantity  firmly  combined,  decreases  with  the  quantity 
of  pus-corpuscles. 

H.  THE  EXCHANGE  OF  GAS  BETWEEN  THE  BLOOD,  ON  THE  ONE  HAND, 
AND  PULMONARY  AH*  AND  THE  TISSUES,  ON  THE  OTHER. 

In  Chapter  I  (page  42)  it  was  stated  that  we  are  to-day  of  the 
opinion,  derived  especially  from  the  researches  of  Pfluger  and  his 
pupils,  that  the  oxidations  of  the  animal  body  do  not  take  place  in  the 
fluids  and  juices,  but  are  connected  with  the  form-elements  and  tissues. 
It  is  nevertheless  true  that  oxidations  take  place  in  the  blood  itself,  al- 
though, only  to  a  slight  extent;  but  these  oxidations  depend,  it  seems, 
upon  the  form-elements  of  the  blood,  hence  it  does  not  contradict  the 
above  statement  that  the  oxidations  exclusively  occur  in  the  cells  and 
chiefly  in  the  tissues. 

The  gaseous  exchange  in  the  tissues,  which  has  been  designated 
internal  respiration,  consists  chiefly  in  that  the  oxygen  passes  from  the 
blood  in  the  capillaries  to  the  tissues,  while  the  great  bulk  of  the  carbon 
dioxide  of  the  tissues  originates  therein  and  passes  into  the  blood  of  the 
capillaries.  The  exchange  of  gas  in  the  lungs,  which  is  called  external 
respiration,  consists,  as  is  seen  by  a  comparison  of  the  inspired  and 
expired  air,  in  the  blood  taking  oxygen  from  the  air  in  the  lungs  and  giving 
off  carbon  dioxide.  This  does  not  exclude  the  fact  that  in  the  lungs,  as  in 
every  other  tissue,  an  internal  respiration  takes  place,  namely,  a  com- 
bustion with  a  consumption  of  oxygen  and  formation  of  carbon  dioxide. 
According  to  Bohr  and  Henriques  x  the  lungs  take  a  variable  but 
sometimes  a  very  important  part  in  the  total  metabolism.  This  part, 
which  on  an  average  is  33  per  cent,  but  may  even  rise  above  60  per 
cent  of  the  total  metabolism,  depends,  these  experimenters  say,  upon 
the  fact  that  the  intermediary  metabolic  products  formed  in  the  tissues 
are  burnt  in  the  lungs.  It  is  also  in  part  represented  by  the  specific 
work  of  the  lungs. 

What  kind  of  processes  take  part  in  this  double  exchange  of  gas? 
Is  the  gaseous  exchange  simply  the  result  of  an  unequal  tension  of  the 
blood  on  one  side  and  the  air  in  the  lungs  or  tissues  on  the  other?  Do 
the  gases  pass  from  a  place  of  higher  pressure  to  one  of  a  lower,  according 
to  the  laws  of  diffusion,  or  are  other  forces  and  processes  active? 

These  questions  are  closely  related  to  that  of  the  tension  of  the 
oxygen  and  carbon  dioxide  in  the  blood  and  in  the  air  of  the  lungs  and 
tissues. 

1  Centralbl.  f.  Physiol.,  6,  and  Maly's  Jahresber.,  27. 


GAS  EXCHANGE.     OXYGEN  TENSION.  859 

Oxygen  occurs  in  the  blood  in  a  disproportionately  large  part  as 
oxyhemoglobin,  and  the  law  of  the  dissociation  of  oxyhemoglobin  is 
of  fundamental  importance  in  the  study  of  the  tension  of  the  oxygen 
in  the  blood. 

Attempts  have  been  made  to  prove  this  law  by  investigations  on 
pure  solutions  of  haemoglobin,  and  Hufner  *  has  made  very  careful  and 
important  determinations  on  such  solutions.  Recent  investigations 
of  Bohr  2  and  his  pupils,  as  well  as  of  Loewy  and  Zuntz,3  have  shown 
that  the  conditions  in  the  blood  are  different  from  a  pure  hemoglobin 
solution,  which,  in  part,  may  be  due  to  a  change  in  the  hemoglobin 
brought  about  in  its  preparation.  A  hemoglobin  solution  in  which 
alcohol  is  used  in  preparing  it,  combines  more  firmly  with  oxygen  than 
the  blood,  and  the  dissociation  tension  of  the  oxygen  is  greater  in  blood 
than  in  such  a  hemoglobin  solution. 

The  oxygen  tension  may  be  variable,  as  Loewy  4  has  shown,  with 
different  individuals,  and  it  is  not  the  same  in  the  blood  of  different 
animals  with  the  same  oxygen  pressure;  for  example,  it  is  less  in  cat's 
blood  than  in  the  dog,  horse  and  human  blood.  The  temperature  is  also 
of  great  importance,  as  the  dissociation  tension  increases  with  a  rise  in 
temperature,  and  with  the  same  pressure  the  blood  combines  with  less 
oxygen  at  a  high  temperature  than  at  a  low  temperature.  The  influence 
of  the  concentration  of  the  hemoglobin  manifests  itself  in  that  in  dilute 
solutions  the  oxygen  is  more  firmly  combined  (Hufner,  Loewy  and  Zuntz, 
Bohr)  and  that  consequently  blood  made  laky  with  water  has  a  lower 
dissociation  tension  and  a  firmer  binding  of  the  oxygen  than  undiluted 
blood. 

Of  especial  interest  is  the  finding  of  Bohr,  Hasselbalch  and  Krogh  5 
that  the  CO2  present  also  influences  the  oxygen  taken  up,  in  that  as  the 
carbon  dioxide  tension  (also  within  physiological  limits)  increases  the 
oxygen  taken  up  diminishes.  The  laws  of  oxygen  absorption  must 
be  determined  by  determinations  upon  blood  itself,  at  the  same  time 
observing  the  temperature  and  the  carbon  dioxide  tension.  A  series  of 
determinations  made  by  Krogh  6  upon  horse's  blood  at  38°,  and  a  con- 
stant carbon  dioxide  tension,  is  given  below.  In  calculating  the  results 
in  column  4  the  quantity  of  oxygen  chemically  combined  at  150  mm. 
oxygen  pressure  is  equal  to  100. 


1  Arch.  f.  (Anat,  u.)  Physiol.,  1890  and  1894. 

2  See  Nagel's  Handbuch,  and  Krogh,  Skand.  Arch.  f.  Physiol.,  16. 
»Arch.  f.  (Anat.  u.)  Physiol.,  1904. 

«  Ibid. 

6Centralbl.  f.  Physiol.,  17,  and  Skand.  Arch.  f.  Physiol.,  16. 

6  Skand.  Arch.  f.  Physiol.,  16. 


860  RESPIRATION  AND  OXIDATION. 

In  100  cc.  blood  Oxygen  taken  up 


Oxygen  Chemically  ,9xypn,  ?er  cen,^        Dissolved  in 

tension  in  mm.         combined  dissolved  chemically    1QQ  laama 

v^uatuu  iii  ii.»u.  oxygen  in  plasma  combined  i^ac^xa 

10                 6.0  0.020  30.0  0.030 

20  12.9  0.041  64.7  0.061 

30  16.3  0.061  81.6  0.091 

40  18.1  0.081  90.4  0.121 

50  19.1  0.101  95.4  0.152 

60  19.5  0.121  97.6  0.182 

70  19.8  0.141  98.8  0.212 

80  19.9  0.162  99.5  0.243 

90  19.95  0.182  99.8  0.273 

150  20.00  0.303  100.0  0.455 

From  the  above  table  we  see  that  even,  with  an  oxygen  tension  which 
amounts  to  only  one-half  of  the  oxygen  pressure  in  the  air,  the  haemoglobin 
is  saturated  in  greatest  part  with  oxygen.  The  dissociation  is  hence  at 
70-80  mm.  pressure  only  slightly  more  than  with  a  pressure  of  150  mm. 
and  indeed  even  with  as  low  a  pressure  as  40-30  mm.,  still  90-80  per  cent 
of  the  entire  quantity  of  oxygen  taken  up  chemically  at  150  mm.  is  com- 
bined with  the  haemoglobin. 

Frcm  these  and  other  observations  it  follows  that  the  oxygen  partial 
pressure  may  sink  to  one-half  of  that  existing  in  the  atmospheric  air 
without  markedly  influencing  the  oxygen  content  of  the  blood.  This 
also  coincides  with  the  experience  of  Frankel  and  Geppert  x  on  the 
action  of  low  air  pressures  upon  the  oxygen  content  of  the  blood  of  dogs. 
With  an  air  pressure  of  410  mm.  Hg,  they  found  that  the  oxygen  content 
of  arterial  blood  was  normal.  With  an  air  pressure  of  378-3G5  mm. 
it  was  slightly  diminished,  and  only  on  reducing  the  pressure  to  300 
mm.  was  a  noticeable  decrease  observed.  A.  Loewy  2  found  that 
the  lowest  oxygen  pressure  of  the  alveolar  air  wherein  the  exchange 
of  material  can  go  on  normally  both  qualitatively  and  quantitatively, 
is  equal  to  30  mm.  Hg. 

In  regard  to  the  above-mentioned  action  of  low  air  pressure  it  must 
be  remarked  that  the  alveolar  oxygen  tension  is  regulated  by  changes 
in  the  respiration,  so  that  with  great  diminution  in  the  quantity  of  oxygen 
of  the  inspired  air,  the  alveolar  air  contains  the  same  quantity  of  oxygen 
as  with  a  higher  oxygen  partial  pressure  of  the  inspired  air  (Loewy). 
For  example,  Loewy  found  the  same  quantity  of  oxygen,  namely,  6.1 
per  cent,  in  the  alveolar  air  with  16  and  with  10.5  per  cent  oxygen  in 
the  inspired  air,  because  the  respiration  in  the  latter  case  was  8.5  liters 
per  minute  against  only  4.9  liters  in  the  first  case. 

It  may  be  concluded  from  the  large  quantity  of  oxygen  or  oxyhaemo- 

1  Ueber  die  Wirkungen  der  verdiinnten    Luft  auf  den  Organismus,  Berlin,   1883. 
1  A.  Loewy,  Untersuch.  iiber  die  Respiration  und    Zirculation,  etc.,  Berlin,  1895; 
also  Centralbl.  f.  Physiol.,  13,  449,  and  Arch.  f.  (Anat.  u.)  Physiol.,  1900. 


OXYGEN  TENSION  IN  BLOOD.  861 

globin  in  the  arterial  blood  that  the  tension  of  the  oxygen  in  the  arterial 
blood  must  be  relatively  higher.  This  is  substantiated  by  the  earlier 
observations  of  Bert  and  Hufner,  as  well  as  by  the  determination.-, 
of  Herter,  Fredericq  and  others,1  using  aerotonometric  methods, 
which  will  be  mentioned  below  in  connection  with  the  carbon  dioxide 
tension.  Herter  found  the  oxygen  tension  in  the  arterial  blood  of 
dogs  to  be  equal,  on  an  average,  to  a  pressure  of  78.7  mm.  Hg  and  Fre- 
dericq, by  a  better  method,  found  that  it  was  equal  to  91-99  mm.  Hg. 

The  oxygen  tension  of  the  venous  blood  of  dogs  has  been  found  by 
aerotonometric  means  to  be  equal  to  20.6-27.7  mm.  (Strassburg, 
Falloise),  and  by  means  of  the  lung-catheter  (see  below)  equal  to  25.5-27 
mm.  (Wolfberg,  Nussbaum).  For  human  venous  blood  Loewy  and  v. 
Schrotter2  found  an  average  of  37.68  mm.  Concerning  the  question 
as  to  the  mechanism  of  taking  up  oxygen  in  the  lungs  these  figures  are  of 
less  interest  than  the  oxygen  tension  in  the  arterial  blood,  that  is,  that 
which  has  left  the  lungs,  whose  tension  is  estimated  as  90  to  about  100 
mm.  Hg  as  given  above. 

These  results  do  not  coincide  with  the  investigations  of  Bohr,3  who 
in  many  cases  obtained  essentially  higher  figures  for  the  oxygen  tension 
in  arterial  blood. 

He  experimented  on  dogs,  allowing  the  blood,  whose  coagulation  had  been 
prevented  by  the  injection  of  peptone  solution  or  infusion  of  the  leech,  to  flow 
from  one  bisected  carotid  to  the  other,  or  from  the  femoral  artery  to  the  femoral 
vein,  through  an  apparatus  called  by  him  an  haemat aerometer.  The  apparatus, 
which  is  a  modification  of  Ludwig's  rheometer  (stromuhr),  allowed,  according 
to  Bohr,  of  a  complete  interchange  between  the  gases  of  the  blood  circulating 
through  the  apparatus  and  a  quantitjr  of  gas  whose  composition  was  known  at 
the  beginning  of  the  experiment  and  inclosed  in  the  apparatus.  The  mixture 
of  gases  was  analyzed  after  an  equalization  of  the  gases  by  diffusion.  In  this 
way  the  tension  of  the  oxygen  and  carbon  dioxide  in  the  circulating  arterial  blood 
was  determined.  During  the  experiment  the  composition  of  the  inspired  and 
expired  air  was  also  determined,  the  number  of  inspirations  noted,  and  the  extern 
of  respiratory  exchange  of  gas  measured.  To  be  able  to  make  a  comparison 
between  the  gas  tension  in  the  blood  and  in  an  expired  air  whose  composition  was 
closer  to  the  unknown  composition  of  the  alveolar  air  than  the  ordinary  expired 
air,  the  composition  of  the  expired  air  at  the  moment  it  passed  the  bifurcation  of 
the  trachea  was  ascertained  by  special  calculation.  The  tension  of  the  gases  ;; 
this  "  bifurcated  air  "  could  be  compared  with  the  tension  of  the  gases  of  the  blood 
and  in  such  a  way  that  the  comparison  took  place  simultaneously.     Recentlv 


1  Pert,  La  pression  barometrique,  Paris,  1878;  Herter,  Zeitschr.  f.  phvsiol.  Chem. 
3;  Hufner,  1.  c;  Fredericq,  Centralbl.  f.  Physiol.,  7,  and  Travaux  der  laborat.  de 
l'inst.  de  phvsiol.  de  Liege,  5,  1896. 

-  StrasBburg,  Pfluger's  Arch.,  6;  Falloisi:,  Bull.  Acad.  Roy.  Belg.,  1902.  Wolfberg, 
Pfliiger's  Arch.,  4  and  6;  Xussbauni,  ibid.,  1;  Loewy  and  v.  Schrotter,  cited  by  Loewy 
in  Gppenheimer'a  Elandb.,  4,  76. 

*  Skand.  Arch.  f.  Physiol.,  2,  and  Nagel's  Handbuch  der  Physiologic 


862  RESPIRATION  AND  OXIDATION. 

Krogh  •    constructed  an  apparatus,  called    by  him  microtonometer,  to  be  used 
for  the  same  purpose. 

Bohr  found  remarkably  high  results  for  the  oxygen  tension  in  arterial 
blood  in  this  series  of  experiments.  They  varied  between  101  and  144 
mm.  Hg  pressure.  In  eight  out  of  nine  experiments  on  the  breathing 
of  atmospheric  air,  and  in  four  out  of  five  experiments  on  breathing  air 
containing  carbon  dioxide,  the  oxygen  tension  in  the  arterial  blood  was 
higher  than  the  "  bifurcated  air."  The  greatest  difference,  where  the 
oxygen  tension  was  higher  in  the  blood  than  in  the  air  of  the  lungs,  was 
38  mm.  Hg. 

Hufner  and  Fredericq  2  have  made  the  objection  to  Bohr's  experi- 
ments and  views  that  a  perfect  equilibrium  had  probably  not  been 
attained  between  the  air  in  the  apparatus  and  the  gases  of  the  blood. 
Fredericq,  by  new  experiments,  presents  strong  objections  to  the 
acceptance  of  Bohr's  findings,  while  on  the  other  hand  Bohr  not  only 
defends  his  experiments,  but  also  finds  errors  in  the  experiments  of  his 
opponents,  while  Haldane  and  Smith's3  experiments,  making  use  of 
an  entirely  different  principle,  tend  to  corroborate  the  high  results  attained 
by  Bohr. 

Haldane's  method  is  as  follows :  The  individual  experimented  upon  is  allowed 
to  inspire  air  containing  an  exactly  known  but  small  quantity  of  carbon  monoxide 
(0.045-0.06  per  cent),  until  no  further  absorption  of  carbon  monoxide  takes  place 
and  the  percentage  saturation  of  the  haemoglobin  in  the  arterial  blood  with  carbon 
monoxide  has  become  constant,  as  shown  by  a  special  titration  method.  This 
percentage  saturation  is  dependent  upon  the  relation  between  the  tension  of  the 
oxygen  in  the  blood  and  the  tension  of  the  carbon  monoxide,  as  known  from  the 
composition  of  the  inspired  air.  When  this  last  and  the  percentage  saturation 
with  carbon  monoxide  and  oxygen  are  known  the  oxygen  tension  in  the  blood 
can  be  easily  calculated. 

According  to  this  method  Haldane  and  Smith  found  still  higher 
figures  than  Bohr  for  the  oxygen  tension  in  the  blood,  and  they  calculated 
the  average  tension  of  the  oxygen  in  human  arterial  blood  to  be  equal 
to  293  mm.  Hg. 

Based  upon  the  experiments  of  A.  and  M.  Krogh,  which  will  be  dis- 
cussed below  (page  864)  A.  Krogh  4  has  presented  objections  to  the 
experiments  of  Haldane  and  Smith. 

Let  us  now  compare  the  figures  for  the  oxygen  tension  of  the  arterial 
blood  as  found  by  various  investigators  with  the  tension  of  the  oxygen 
in  the  air  of  the  lungs. 

1  Skand.  Arch.  f.  Physiol.,  20. 

2  Hufner,  Arch.  f.  (Anat.  u.)  Physiol.,  1890;  Fredericq,  Centralbl.  f.  Physiol.,  7, 
and  Traveaux  du  laboratorie  de  l'institute  de  physiologie  de  Liege,  5,  1896. 

3  Haldane,  Journ.  of  Physiol.,  18;  Haldane  and  Smith,  ibid.,  20. 
♦Skand.  Arch.  f.  Physiol.,  23,  217,  253. 


THE  ALVEOLAR  AIR.  8G3 

Numerous  investigations  as  to  the  composition  of  the  inspired  atmos- 
pheric air  as  well  as  the  expired  air  are  at  hand,  and  it  can  be  said  that 
these  two  kinds  of  air  at  0°  C.  and  a  pressure  of  700  mm.  Hg  have  the 
following  average  composition  in  volume  per  cent. 

Oxvizen  Nitrogen  Carbon 

vxygen  (aQ(,  argonj  Dioxide 

Atmospheric  air 20.90  79.02  0.03 

Expired  air 16.03  79.59  4.38 

The  partial  pressure  of  the  oxygen  of  the  atmospheric  air  corresponds 
at  a  normal  barometric  pressure  of  760  mm.  to  a  pressure  of  150  mm. 
Hg.  The  loss  of  oxygen  which  the  inspired  air  suffers  in  respiration 
amounts  to  about  4.93  per  cent,  while  the  expired  air  contains  about 
one  hundred  times  as  much  carbon  dioxide  as  the  inspired  air. 

The  expired  air  is  therefore  a  mixture  of  alveolar  air  with  the  residue 
of  inspired  air  remaining  in  the  air-passages;  hence  in  the  study  of 
the  gaseous  exchange  in  the  lungs  the  alveolar  air  must  first  be  con- 
sidered. There  exists  no  direct  determination  of  the  composition  of  the 
alveolar  air  in  man,  but  only  approximate  calculations.  From  the 
average  results  found  by  Vierordt  in  normal  respiration  for  the  carbon 
dioxide  in  the  expired  air,  4.63  per  cent,  Zuntz  l  has  calculated  the 
probable  quantity  of  carbon  dioxide  in  the  alveolar  air  as  equal  to  5.44 
per  cent.  If  we  start  from  this  value,  with  the  assumption  that  the 
quantity  of  nitrogen  in  the  alveolar  air  does  not  essentially  differ  from 
the  expired  air,  and  admit  that  the  quantity  of  oxygen  in  the  alveolar 
air  is  6  per  cent  less  than  the  inspired  air,  it  will  be  seen  that  the  alveolar 
air  contains  15  per  cent  oxygen.  As  the  total  pressure  of  the  air  of  the 
lungs  after  deducting  the  aqueous  tension  of  about  50  mm.  can  be  cal- 
culated as  about  710  mm.  the  partial  pressure  of  the  oxgyen  in  man 
can  be  put  at  about  106  mm.  and  that  of  the  carbon  dioxide  as  about 
45  mm. 

Based  upon  several  respiration  experiments  upon  different  persons, 
Loewy  has  been  able  to  calculate  the  composition  of  the  alveolar  air 
of  human  beings  almost  at  the  atmospheric  pressure,  from  the  com- 
position of  the  expired  air  and  the  depth  of  inspiration  and  expiration, 
taking  into  consideration  the  air  in  the  upper  air-passages.  He  obtained 
results  which  varied  between  101  and  105  mm.  Hg  for  the  oxygen  tension 
and  between  32-42  mm.  for  the  carbon  dioxide  tension. 

The  alveolar  oxygen  tension  in  dogs  can  be  calculated  from  the  car- 
bon dioxide  content  of  the  alveolar  air  and  is  also  found  to  be  above 
100  mm.  Hg. 

If  the  oxygen  partial  pressure  in  the  alveoli  is  put  at  about   105 

1  See  Zuntz,  1.  c,  Hermann's  Handbuch,  105  and  106. 


S64  RESPIRATION  AND  OXIDATION. 

i 
mm.  Hg  and  we  compare  this  with  the  highest  results  obtained  for  the 
oxygen  tension  of  the  arterial  blood  as  determined  by  tonometric  means, 
we  find  that  the  taking  up  of  oxygen  in  the  lungs  can  be  simply  explained 
according  to  physical  laws  as  a  diffusion  process.  The  conditions  are 
quite  different  if  we  start  with  the  high-tension  results  of  Bohr,  101-144 
mm.  Hg,  or  the  still  higher  results  of  Haldane  and  Smith.  The  oxygen 
tension  in  the  blood  is,  in  many  cases,  according  to  these  latter  authors, 
always  higher  than  the  tension  in  the  lungs,  as  average  for  various  races 
of  animals.  In  these  cases  the  passage  of  oxygen  from  the  lungs  to  the 
blood  cannot  be  explained  simply  by  a  diffusion.  We  must  therefore 
with  Bohr,  accept  a  special  activity  cf  the  lungs,  and  according  to, 
him  a  secretory  activity  of  the  lungs  also  exists  besides  diffusion.  In  his 
most  recent  work  Bohr  x  presents  the  view  that  the  specific  action  of  the 
lungs  essentially  consists  in  maintaining  a  necessary  difference  in  pressure 
for  the  diffusion.  Nevertheless  besides  this  a  secretory  process  is  nec- 
essary, especially  for  the  taking  up  of  oxygen.  Based  upon  newer 
measurements  Douglas  and  Haldane  2  also  advocate  the  view  that  the 
taking  up  of  oxygen  can  be  brought  about  by  diffusion  alone,  but  that 
with  the  existing  lack  of  oxygen  in  the  tissues  an  active  secretion  of 
oxygen  takes  place  in  the  lungs. 

By  means  of  a  tonometer  described  by  A.  Krogh,  he  and  M.  Krogh 
have  compared  the  oxygen  tension  in  the  arterial  blood  with  that  in  the 
alveolar  air.  In  these  experiments  the  tension  in  the  blood  was  always 
found  lower  than  in  the  alveolar  air.  From  this  A.  Krogh  3  concludes 
that  the  exchange  of  gas  in  the  lungs  is  chiefly  brought  about  by  diffusion. 
Fredericq4  has  recently  arrived  at  the  same  view  by  his  experiments 
on  the  respiratory  exchange  of  gas  in  aquatic  animals. 

As  reports  on  the  taking  up  of  oxygen  are  conflicting  so  also  are  those 
on  the  giving  up  of  carbon  dioxide. 

The  tension  of  the  carbon  dioxide  in  the  blood  has  been  determined 
in  different  ways  by  Pfluger  and  his  pupils  Wolffberg,  Strassbtjrg, 
and  Nussbaum.5 

According  to  the  aero  tonometric  method  the  blood  is  allowed  to  flow  directly 
from  the  artery  or  vein  through  a  glass  tube  which  contains  a  gas  mixture  of  a 
known  composition.  If  the  tension  of  the  carbon  dioxide  in  the  blood  is  greater 
than  the  gas  mixture,  then  the  blood  gives  up  carbon  dioxide,  while  in  the  reverse 
case  it  takes  up  carbon  dioxide  from  the  gas  mixture.     The  analysis  of  the  gas 

»Centralbl.  f.  Physiol.  23,  274;  Skand.  Arch.  f.  Physiol.,  22,  221  (1909). 
*Skand.  Arch.  f.  Physiol.,  25,  169  (1911);    Proc.  Roy.  Soc,  1911;  see  also  Journ. 
of  Physiol.,  44,  305  (1912). 

'Skand.  Arch.  f.  Physiol.,  20,  203;  23,  179,  200,  213,  (1910). 

fAfdh.  intern,  de  Physiol.,  10,  391  (1911). 

*  Wolffbonr,  Pfltigef's  Arch.,  fi;  Strassburg,  ibid.;  Nussbaum,  ibid.,  7. 


CARBON  DIOXIDE  TENSION.  865 

mixture  after  passing  the  blood  through  it  will  also  decide  if  the  tension  of  the 
Carbon  dioxide  in  the  blood  is  greater  or  less  than  in  the  gas  mixture;  and  by  a 
sufficiently  great  number  of  determinations,  especially  when  the  quantity  of  car- 
bon dioxide  of  the  gas  mixture  corresponds  as  closely  as  possible,  in  the  beginning, 
to  the  probable  tension  of  this  gas  in  the  blood,  we  may  learn  the  tension  of  the 
carbon  dioxide  in  the  blood.  As  above  mentioned  the  oxygen  tension  can  be 
determined  by  the  same  method. 

According  to  this  method  the  carbon-dioxide  tension  of  the  arterial 
blood  is  on  an  average  2.8  per  cent  of  an  atmosphere,1  corresponding  to 
a  pressure  of  20  mm.  mercury  (Strassburg).  In  the  blood  from  the 
pulmonary  aveoli  NlJSSBAUM  found  a  carbon-dioxide  tension  of  3.81 
per  cent  of  an  atmosphere,  corresponding  to  a  pressure  of  27  mm.  mer- 
cury. Strassburg,  who  experimented  in  non-tracheotomized  dogs 
in  which  the  ventilation  of  the  lungs  was  less  active  and  therefore  the 
carbon  dioxide  was  removed  from  the  blood  with  less  readiness,  found 
in  the  venous  blood  of  the  heart,  a  carbon-dioxide  tension  of  5.4  per 
cent  of  an  atmosphere,  corresponding  to  a  partial  pressure  of  38.3  mm. 
mercury. 

Another  method,  which  was  first  used  by  Pfluger  and  his  pupils 
Wolffberg  and  Nussbaum,  depends  upon  excluding  a  part  of  the  lungs 
be  means  of  the  lung  catheter 

The  principle  of  this  method  is  as  follows:  By  the  introduction  of  a  catheter, 
of  a  special  construction,  into  a  branch  of  a  bronchus  the  corresponding  lobe  of 
the  lung  may  be  hermetically  sealed,  while  in  the  other  lobes  of  the  same  lung, 
and  in  the  other  lung,  the  ventilation  remains  unchanged,  so  that  no  accumulation 
of  carbon  dioxide  takes  place  in  the  blood.  When  the  cutting  off  lasts  so  long  that 
a  complete  equalization  between  the  gases  of  the  blood  and  the  retained  air  of 
the  lungs  is  assumed,  a  sample  of  this  air  of  the  lungs  is  removed  by  means  of 
the  catheter  and  analyzed. 

When  a  complete  exchange  between  the  gases  of  the  inclosed  part  of 
the  lungs  and  the  gases  of  the  circulating  venous  blood  has  taken  place, 
the  tension  of  the  gases  in  this  part  of  the  lungs  can  be  considered  as  a 
measure  for  the  gas  tension  in  the  venous  blood,  if  we  admit  that  the 
gas  exchange  is  due  only  to  physical  forces.  In  their  experiments 
Wolffberg  and  Nussbaum  found  only  3.6  per  cent  CO2  in  the  air  taken 
out  with  the  catheter.  Nussbaum  also  determined  the  carbon-dioxide 
tension  in  the  blood  from  the  right  heart  in  a  case  simultaneous  with 
the  catheterization  of  the  lungs.  He  found  almost  identical  results, 
namely,  a  carbon-dioxide  tensioD  of  3.84  per  cent  and  3.81  per  cent 
of  an  atmosphere,  which  also  shows  that  complete  equalization  between 
the  gases  of  the  blood  and  lungs  in  the  inclosed  parts  of  the  lungs  had  taken 

1  Here  and  in  the  following  discussion  we  mean  by  atmospheric  pressure  the  pressure 
in  the  lungs  after  subtracting  the  aqueous  vapor  tension  (about  50  mm.),  namely, 
760  —  50  =  710  mm.  mercury  pressure. 


866  RESPIRATION  AND  OXIDATION. 

place.  The  method  of  catheterizing  the  lungs  is,  as  shown  by  Loewy 
and  v.  Schrotter,1  also  applicable  to  man,  and  they  found  that  the 
carbon  dioxide  tension  of  human  venous  blood  was  equal  to  6  per  cent 
of  the  atmospheric  pressure  in  the  lungs  =  42.6  mm.  Hg,  while  according 
to  Loewy's  calculations  the  carbon  dioxide  tension  in  the  respired  lung 
aveoli  varied  between  31.8  and  41.8  mm.  Hg  with  an  average  of  37.3 
mm.  Hg  for  eleven  cases. 

According  to  these  investigations  the  giving  up  of  carbon  dioxide 
may  also  be  explained  by  physical  laws;  but  Bohr,  in  his  experiments 
above  mentioned  (page  861),  has  arrived  at  other  results  in  regard  to 
the  carbon-dioxide  tension.  In  eleven  experiments  with  inhalation  of 
atmospheric  air  the  carbon-dioxide  tension  in  the  arterial  blood  varied 
from  0  to  38  mm.  Hg,  and  in  five  experiments  with  inhalation  of  air  con- 
taining carbon  dioxide,  from  0.9  to  57.8  mm.  Hg.  A  comparison  of  the 
carbon-dioxide  tension  in  the  blood  with  the  bifurcated  air  gave  in  several 
cases  a  greater  carbon-dioxide  pressure  in  the  air  of  the  lungs  than  in 
the  blood,  and  as  maximum  this  difference  amounted  to  17.2  mm.  in 
favor  of  the  air  of  the  lungs  in  the  experiments  with  inhalation  of  atmos- 
pheric air.  As  the  aveolar  air  is  richer  in  carbon  dioxide  than  the  bifur- 
cated air  this  experiment  unquestionably  proves,  according  to  Bohr, 
that  the  carbon  dioxide  has  migrated  against  the  high  pressure. 

In  opposition  to  these  investigations,  Fredericq,2  in  his  above-men- 
tioned experiments,  obtained  the  same  figures  for  the  carbon-dioxide 
tension  in  arterial  peptone  blood  as  Pfluger  and  his  pupils  found  for 
normal  blood.  Weisgerber,3  in  Fredericq's  laboratory,  has  made 
experiments  with  animals  which  respired  air  rich  in  carbon  dioxide,  and 
these  experiments  confirm  Pfluger's  theory  of  respiration.  Recently 
Falloise  has  made  determinations  of  the  carbon-dioxide  tension  of 
venous  blood  by  means  of  Fredericq's  aerotonometer.  The  carbon- 
dioxide  tension  was  found  to  equal  6  per  cent  of  an  atmosphere,  hence 
somewhat  higher  than  the  results  found  by  Pfluger's  pupils.  To  these 
investigations  Bohr  has  presented  strong  objections;  he  has  demon- 
strated the  principles  for  the  construction  of  the  tonometer,  and  claims  that 
the  earlier  experiments  with  the  tonometer  are  not  conclusive,  as  a 
complete  equilibrium  of  the  gas  tension  was  not  attained. 

A  certain  importance  has  been  ascribed  to  oxygen  in  regard  to  the 
elimination  of  carbon  dioxide  in  the  lungs,  in  that  it  has  an  expelling 
action  on  the  carbon  dioxide  from  its  combinations  in  the  blood.  This 
theory,  first  advanced  by  Holmgren,  has  recently  found  an  advocate 


1 1.  c,  footnote  2,  page  861. 

2  See  footnote  1,  page  861. 

•Centralbl.  f.  Physiol.,  10,  482;  Falloise,  see  Maly's  Jahresber.,  32. 


INTERNAL  RESPIRATION.  867 

in  Werigo.  Still  Zuntz  has  presented  weighty  objections  to  Werigo's 
experiments,  and  Bohr1  has  later  also  shown  that  we  have  no  positive 
basis  for  the  above  assumption. 

The  conditions  as  to  the  elimination  of  carbon  dioxide  in  the  lungs 
are  not  quite  clear.  On  the  one  hand  we  have  advocates  of  the  view 
that  the  gas  exchange  takes  place  simply  according  to  physical  laws 
and  is  chiefly  considered  as  a  diffusion  process.  According  to  the  former 
views  of  Bohr,2  which  he  has  supported  by  recent  experiments,  a  diffusion 
does  take  place,  but  the  lung  is  a  gland  which  has  the  power  of  secreting 
gases,  and  the  gas  exchange  in  the  lungs  is  essentially  a  secretory  proc- 
ess. In  his  most  recent  work  Bohr,3  after  a  thorough  criticism  of  the 
methods  used  in  the  measurement  of  the  lung  diffusion  and  based  upon  a 
new  calculation  of  the  extent  of  diffusion,  has  come  to  the  conclusion 
that  the  specific  activity  of  the  lungs  consists  in  that  a  difference  in 
pressure  necessary  for  the  diffusion  is  brought  about. 

That  a  true  secretion  of  gases  occurs  in  animals  follows  from  the  composition 
and  behavior  of  the  gases  in  the  swimming-bladder  of  fishes.  These  gases  con- 
sist of  oxygen  and  nitrogen  with  only  small  quantities  of  carbon  dioxide.  In 
fishes  which  do  not  live  at  any  great  depth  the  quantity  of  oxygen  is  ordinarily 
as  high  as  in  the  atmosphere,  while  fishes  which  live  at  great  depths  may,  accord- 
ing to  Biot  and  others,  contain  considerably  more  oxygen  and  even  above  80  per 
cent.  AIoreau  has  also  found  that  after  emptying  the  swimming-bladder  by 
means  of  a  trocar,  new  air  collected  after  a  time,  and  this  air  was  richer  in  oxygen 
than  the  atmospheric  air,  and  contained  even  85  per  cent  oxygen.  Bohr,  "who 
has  proven  and  confirmed  these  statements,  also  found  that  this  accumulation 
is  under  the  influence  of  the  nervous  system,  because  on  the  section  of  certain 
branches  of  the  pneumogastric  nerve  it  is  discontinued.  It  is  beyond  dispute  that 
there  is  here  a  secretion  and  not  a  diffusion  of  oxygen.  Recently  Jaeger  *  has 
given  a  further  explanation  as  to  the  secretory  activity  of  the  swimming-bladder. 

From  what  has  been  said  above  (page  858)  in  regard  to  the  internal 
respiration,  one  can  conclude  that  it  consists  chiefly  that  in  the  capil- 
laries the  oxygen  passes  from  the  blood  into  the  tissues,  while  the  car- 
bon dioxide  passes  from  the  tissues  into  the  blood. 

The  assertion  of  Estor  and  Saint  Pierre  that  the  quantity  of  oxygen 
in  the  blood  of  the  arteries  decreases  with  the  remoteness  from  the  heart 
has  been  shown  to  be  incorrect  by  Pfluger,5  and  the  oxygen  tension  in 
blood  on  entering  the  capillaries  must  be  higher.      The  oxygen  tension 


1  Holmgren,  Wien.  Sitzungsber.,  48;    Werigo,  Pfliiger's  Arch.,  51  and  52;    Zuntz, 
ibid.,  52;  Bohr,  see  Nagel's  Handbuch  der  Physiologic 

2  Centralbl.  f.  Physiol.,  21,  367. 

3  Ibid.,  23,  374;  Skand.  Arch.  f.  Physiol.,  22,  221  (1909). 

4  Biot,  see  Hermann's  Handbuch  d.  Physiol.,  4,  Thl.  2,  151;  Moreau,  Compt. 
Rend.,  57;  Bohr,  Journ.  of  Physiol.,  15.  See  also  Hiifner,  Arch.  f.  (Anat.  u.)  Physiol. 
1892;  Jaeger,  Pfluger 's  Arch.,  94. 

5  Estor  and  Saint  Pierre  with  Pfluger  in  Pfliiger's  Arch.,  1. 


868  RESPIRATION  AND  OXIDATION. 

of  the  plasma  is  of  importance  in  the  giving  up  of  oxygen  to  the  tissues, 
as  the  blood  corpuscles  contain  a  supply  of  oxygen  only  sufficient  to 
replace  that  removed  from  the  plasma  by  the  tissue.  This  quantity 
of  oxygen,  which  is  dissolved  in  the  plasma  and  at  the  disposal  of  the 
tissues,  is  dependent  upon  the  oxygen  tension  in  the  blood  and  only 
indirectly  dependent  upon  the  total  quantity  of  oxygen  in  the  blood. 
As  this  tissue  is  almost  or  entirely  free  from  oxygen,  a  considerable  dif- 
ference in  regard  to  the  oxygen  pressure  must  exist  between  the  blood 
and  the  tissues.  The  possibility  that  this  difference  in  pressure  is  suf- 
ficient to  supply  the  tissues  with  the  necessary  quantity  of  oxygen  is 
hardly  to  be  doubted. 

The  animal  body,  it  seems,  also  has  the  command  over  means  of  regu- 
lating and  varying  the  oxygen  tension,  and  such  a  means  is  the  carbon 
dioxide  produced  in  the  tissue  which,  according  to  Bohr,  Hasselbalch, 
and  Krogh,1  raises  the  oxygen  tension.  This  is  of  special  importance 
when  the  tension  of  the  oxygen  in  the  blood  of  the  capillaries  is  very  low; 
then  the  ability  of  the  carbon  dioxide  to  raise  the  dissociation  tension 
of  the  ox3'hsemoglobin  comes  into  play,  especially  with  low  oxygen  tension." 
Another  regulating  moment  is,  Bohr  claims,  the  specific  oxygen  capacity 
of  the  blood,  which  means  the  relation  of  the  maximum  oxygen  combina- 
tion to  the  quantity  of  iron  of  the  blood  or  the  haemoglobin  solution. 

In  regard  to  the  carbon-dioxide  tension  in  the  tissue  it  must  be 
assumed  a  priori  that  it  is  higher  than  in  the  blood.  This  is  found  to 
be  true.  Strassburg  2  found  in  the  urine  of  dogs  and  in  the  bile  a  car- 
bon-dioxide tension  of  9  per  cent  and  7  per  cent  of  an  atmosphere, 
respectively.  The  same  experimenter  has,  further,  injected  atmospheric 
air  into  a  ligatured  portion  of  the  intestine  of  a  living  dog  and  analyzed 
the  air  taken  out  after  some  time.  He  found  a  carbon-dioxide  tension 
of  7.7  per  cent  of  an  atmosphere.  The  carbon-dioxide  tension  in  the 
tissues  is  considerably  greater  than  in  the  venous  blood,  and  there  is 
no  opposition  to  the  view  that  the  carbon  dioxide  simply  diffuses  from 
the  tissues  into  the  blood  according  to  the  law  of  diffusion. 

Several  methods  have  been  suggested  for  the  study  of  the  quantitative 
relation  of  the  respiratory  exchange  of  gas.  The  reader  must  be  referred 
to  other  text-books  for  details  as  to  these  methods,  and  we  will  here 
mention  only  the  chief  features  of  the  most  important  methods.  It  must 
also  be  remarked,  in  regard  to  these  methods,  that  those  of  Regnault 
and  Reiset  and  of  Pettenkofer,  determine  the  total  gas  exchange, 
and  indeed  for  a  long  time,  while  the  other  three  methods  determine  the 
respiratory  gas  exchange  alone,  and  this  only  for  a  short  time. 

1  Centralbl.  f.  Physiol.,  17,  and  Skand.  Arch.  f.  Physiol.,  16. 

2  Pfiuger's  Arch.,  6. 


METHODS  FOR  DETERMINING  RESPIRATORY  EXCHANGE.     £69 

Regnault  and  Reiset's  Method.  According  to  this  method  the  animal 
•or  person  experimented  upon  is  allowed  to  respire  in  an  inclosed  Bpace.  The 
carbon  dioxide  is  removed  from  the  air,  as  it  forms,  by  Strong  caustic  alkali,  from 
which  the  quantity  may  be  determined,  while  the  oxygen  is  replaced  continually 
in  exactly  measured  quantities.  This  method,  which  also  makes  possible  a  direct 
determination  of  the  oxygen  used  as  well  as  the  carbon  dioxide  produced,  has 
since  been  modified  by  other  investigators,  such  as  Pfluger  and  his  pupils,  Seegen 
and  Nkwak,  and  Hoppe-Seyler,  Rosenthal  and  Oppenheimer  and  especially 
by  Atwater  and  Benedict.1 

Pettenkofer's  Method.  According  to  this  method  the  individual  to  be 
experimented  upon  breathes  in  a  room  through  which  a  current  of  atmospheric 
air  is  passed.  The  quantity  of  air  passed  through  is  carefully  measured.  As  it 
is  impossible  to  analyze  all  the  air  made  to  pass  through  the  chamber,  a  small 
fraction  of  this  air  is  diverted  into  a  branch  line  during  the  entire  experiment, 
carefully  measured,  and  the  quantity  of  carbon  dioxide  and  water  determined. 
From  the  composition  of  this  air  the  quantity  of  water  and  carbon  dioxide  con- 
tained in  the  large  quantity  of  air  made  to  pass  through  the  chamber  can  be 
calculated.  The  consumption  of  oxygen  cannot  be  directly  determined  in  this 
method,  but  may  be  calculated  indirectly  by  difference,  which  is  a  defect  in  this 
method.  The  large  respiration  apparatus  of  Sonden  and  Tigerstedt  as  well 
as  of  Atwater  and  Rosa  2  are  based  upon  this  principle. 

Speck's  Method.3  For  briefer  experiments  on  man  Speck  used  the  follow- 
ing: He  breathes  through  a  mouthpiece  with  two  valves,  closing  the  nose  with  a 
clamp,  into  two  spirometer-receivers,  where  the  gas-volume  can  be  read  off  very 
accurately.  The  air  from  one  of  the  spirometers  is  inhaled  through  one  valve 
and  the  expired  air  passes  through  the  other  into  the  other  spirometer.  By  means 
of  a  rubber  tube  connected  with  the  expiration-tube  an  accurately  measured  part 
of  the  expired  air  may  be  passed  into  an  absorption-tube  and  analyzed. 

Zuntz  and  Geppert's  Method.*  This  method,  which  has  been  improved  by 
Zuntz  and  his  pupils  from  time  to  time,  consists  in  the  following:  The  individual 
being  experimented  upon  inspires  pure  atmospheric  air  through  a  very  wide  feed- 
pipe leading  from  the  open  air,  the  inspired  and  the  expired  air  being  separated  by 
two  valves  (human  subjects  breathe  with  closed  nose  by  means  of  a  soft-rubber 
mouthpiece,  animals  through  an  air-tight  tracheal  canula).  The  volume  of  the 
expired  air  is  measured  by  a  gas-meter  and  an  aliquot  part  of  this  air  collected  and 
the  quantity  of  carbon  dioxide  and  oxygen  determined.  As  the  composition  of 
the  atmospheric  air  can  be  considered  as  constant  within  a  certain  limit,  the 
production  of  carbon  dioxide  as  well  as  the  consumption  of  oxygen  may  be  readily 
calculated  (see  the  works  of  Zuntz  and  his  pupils). 

Hanriot  and  Richet's  Method6  is  characterized  by  its  "simplicity.  These 
investigators  allow  the  total  air  to  pass  through  three  gasometers,  one  after  the 
other.  The  first  measures  the  inspired  air,  whose  composition  is  known.  The 
second  gasometer  measures  the  expired  air,  and  the  third  the  quantity  of  the 


xSee  Zuntz  in  Hermann's  Handbuch,  4,  Thl.  2,  and  Hoppe-Seyler,  Zeitschr.  f. 
physiol.  Chem.,  19;  Rosenthal,  Arch.  f.  (Anat.  u.)  Physiol.,  1902;  Zuntz  and  Oppen- 
heimer, Arch.'  f.  (Anat.  u.)  Physiol.,  1905,  and  Bioch.  Zeitschr.,  14;  Atwater  and 
Benedict,  Bull.  Dept.  of  Agriculture,  Washington,  69,  109,  and  136.  See  also  Krogh, 
Wien.  Sitz.  Ber.,  115,  III.,  and  Skand.  Arch.  f.  Physiol.,  18. 

2  Pettenkofer's  method;  see  Zuntz,  1.  c;  Sond6n  and  Tigerstedt,  Skand.  Arch, 
f.  Physiol.,  6;  Atwater  and  Rosa,  Bull,  of  Dept.  of  Agriculture,  63.     Washington. 

1  Speck,  Physiologie  des  menschlichen  Atmens.     Leipzig,  1892. 

4  Pfluger's  Arch.,  42.  See  also  Magnus-Levy  in  Pfluger's  Arch.,  55,  10,  in  which 
the  work  of  Zuntz  and  his  pupils  is  cited. 

5  Compt.  Rend.,  104. 


870  RESPIRATION  AND  OXIDATION. 

expired  air  after  the  carbon  dioxide  has  been  removed  by  a  suitable  apparatus. 
The  quantity  of  carbon  dioxide  produced  and  the  oxygen  consumed  can  be_readily 
calculated  from  these  data. 


Appendix 

THE   LUNGS   AND   THEIR  EXPECTORATIONS 

Besides  'proteins  and  the  albuminoids  of  the  connective-substance 
group,  lecithin,  taurine  (especially  in  ox-lungs),  uric  acid,  and  inosite 
have  been  found  in  the  lungs.  Poulet  1  claims  to  have  found  a  special 
acid  in  the  lung-tissue,  which  he  has  called  -pulmotartaric  acid.  Glyco- 
gen occurs  abundantly  in  the  embryonic  lung,  but  is  absent  in  the  adult 
organ.  The  proteolytic  enzymes  also  belong  to  the  physiological  con- 
stituents of  the  lungs.  They  are  active  in  the  autolysis  of  the 
lungs  (Jacoby)  as  well  as  in  the  solution  of  pneumonic  infiltrations  (Fr. 
Muller)  ? 

The  lungs  have  a  strong  reducing  property,  which  Bohr  explains  by 
the  extensive  oxidation  processes  in  the  lungs.  According  to  N.  Sieber 
they  also  have  the  ability  to  decompose  neutral  fats,  while  Riehl3 
says  they  do  not  have  the  ability  to  invert  milk  sugar. 

The  black  or  dark-brown  pigment  in  the  lungs  of  human  beings  and  domestic 
animals  consists  chiefly  of  carbon,  which  originates  from  the  soot  in  the  air.  The 
pigment  may  in  part  also  consist  of  melanin.  Besides  carbon,  other  bodies,  such 
as  iron  oxide,  silicic  acid,  and  clay,  may  be  deposited  in  the  lungs,  being  inhaled 
as  dust. 

Among  the  bodies  found  in  the  lungs  under  pathological  conditions 
must  be  specially  mentioned,  proteoses  (and  peptones?)  in  pneumonia 
and  suppuration,  glycogen,  a  slightly  dextrorotatory  carbohydrate 
differing  from  glycogen,  found  by  Potjchet  in  consumptives,  and  finally 
also  cellulose,  which,  according  to  Freund,4  occurs  in  the  lungs,  blood, 
and  pus  of  persons  with  tuberculosis. 

C.  \V.  Schmidt  found  in  1000  grams  of  miners1  bodies  from  the  normal 
human  lung  the  following:  NaCl  130,  K20  13,  Na20  195,  CaO  19,  MgO 
19,  Fe203  32,  P205  485,  S03  8,  and  sand  134  grams.  According  to 
Oidtmann,5  the  lungs  of  a  14-day  old  child  contained  796.05  p.  m.  water, 
198.19  p.  m.  organic  bodies,  and  5.76  p.  m.  inorganic  bodies. 

1  Cited  from  Maly's  Jahresber.,  18,  248. 

2  Jacoby,  Zeitschr.  f.  physiol.  Chem.,  33;  Muller,  Verhandl.  d.  Kongress.  f.  inn. 
Medizin,  1002. 

1  X.  Sieber,  Zeitschr.  f.  physiol.  Chem.,  55;  Riehl,  Zeitschr.  f.  Biol.,  48. 
4  Pouchet,  Compt.  Rend.,  96;  Freund,  cited  from  Maly's  Jahresber.,  16,  471. 
'Schmidt,   cited   from   v.   Gorup-Besanez,   Lehrbuch,   4.   Aufi.,   727;    Oidtmann, 
ibid.,  732. 


PHYSIOLOGICAL  OXIDATION  PROCESSES.  871 

The  sputum  is  a  mixture  of  the  mucous  secretion  of  the  respiratory- 
passages,  of  saliva  and  buccal  mucus.  Because  of  this  its  composition 
is  variable,  especially  under  pathological  conditions  when  various  prod- 
ucts mix  with  it.  The  chemical  constituents  are,  besides  the  mineral 
substances,  chiefly  mucin  with  a  little  proteid  and  nuclein  substance. 
Inilcr  pathological  conditions  proteoses  and  peptones  (?),  which  are 
probably  produced  by  bacterial  action  cr  by  autolysis  (Wanner,  Simon1), 
volatile  fatty  acids,  glycogen,  Charcot's  crystals,  and  also  crystals  of 
cholesterin,  hsematoidin,  tyrosine,  fat  and  fatty  acids,  triple  phosphates, 
etc.,  have  been  found. 

The  form  constituents  are,  under  physiological  circumstances,  epithe- 
lium-cells of  various  kinds,  leucocytes,  sometimes  also  red  blood-cor- 
puscles and  various  kinds  of  fungi.  In  pathological  conditions  elastic 
fibers,  spiral  formations  consisting  of  a  mucin-like  substance,  fibrin 
ceagulum,  pus,  pathogenic  microbes  of  various  kinds  and  the  above- 
mentioned  crystals  occur. 

The  lung  concretions  contain  chiefly  calcium  and  phosphoric  acid  as  inorganic 
constituents.  Silicic  acid  is,  in  Zickgraf's  opinion;  an  essential  and  constant 
constituent,  but  according  to  Gerhartz  and  Strigel  2  is  not  always  constant. 

in.     HOW  ARE  THE  PHYSIOLOGICAL  OXIDATION  PROCESSES  BROUGHT 

ABOUT? 

After  the  oxygen  passes  from  the  blood  to  the  tissues  a  very  extensive 
oxidation  is  there  carried  out,  which  in  conjunction  with  cleavage  processes 
yields  finally  the  products  carbon  dioxide,  water,  urea  and  other  bodies. 
Little  is  known  as  to  the  manner  in  which  the  organism  carries  out  such 
complete  oxidations.  Attempts  have  been  made  for  a  long  time  to 
explain  the  mechanism  of  the  oxidation  processes.  Thus  Schonbein3 
believed  in  the  presence  in  the  organism  of  oxygen  in  a  peculiar  form, 
suited  for  the  oxidation.  Hoppe-Seyler4  connects  the  oxidation  with  a 
simultaneous  reduction;  reducible  or  readily  oxidizable  substances  first  rup- 
ture the  oxygen  molecule  (  =  02)  into  atoms  and  take  one  up;  the  other 
at  the  moment  it  is  set  free  is  especially  able  to  oxidize.  M.  Traube  5 
believes  that  in  the  case  that  a  readily  oxidizable  (auto-oxidizable)  substance 
is  present,  the  oxidation  is  produced  by  means  of  the  entire  oxygen 
molecule,  and  indeed  in  the  manner  that  water  is  transformed  at  the  same 
time  to  hydrogen  peroxide,  for  example  A-r-2H20+Oo  =  A(OH)2  +  H202. 

1  Wanner,  Deutsch.  Arch.  f.  klin.  Med.,  75;  Simon,  Arch.  f.  exp.  Path.  u.  Pharm.,  49. 

*  Gerhartz  and  Strigel,  Beitr.  z.  klin.  d.  Tuberkulose,  10,  which  also  cites  Zickgraf. 
'Baseler,  Verh.,  Bd.  1,  339  (1853);    Sitzungsber.   Bayer.  Akad.  Wiss.,    1863,   BA. 

1,  274. 

4  Zeitschr  f.  physiol.  Chem.,  2,  1  (1878). 

*  Ber.  d.  d.  chem.  Gesellsch.,  Bd.  15  to  26  (1882  to  1893). 


872  RESPIRATION  AND  OXIDATION. 

With  these  views  as  basis  and  at  the  same  time  although  independently 
of  each  other  Engler  1  and  Bach  2  have  developed  a  theory  which  for 
the  present  is  the  one  generally  accepted.  According  to  this  theory, 
peroxides  of  the  hydrogen  peroxide  type  are  always  formed  as  primary 
oxidation  products.  The  peroxides  are  either  formed  by  a  direct  attach- 
ment of  oxygen  with  readily  oxidizable  substances  or  in  consequence  of  a 
simultaneous  oxidation  with  other  substances — in  the  last  way,  for 
example,  the  formation  of  H2O2  in  the  oxidation  of  indigo-white  to  indigo 
according  to  the  formula: 


Indigo^      +02  =  Indigo + H2O2 


Indigo-white 

Only  certain  substances  have  the  ability  either  directly  or  indirectly  of 
forming  peroxides.  Certain  protein-like  substances  occurring  especially 
in  the  plants  which  have  this  ability,  have  been  called  oxygenases  by 
Bach  and  Chodat.3  Most  of  the  substances  which  are  oxidized  within 
the  organism  lose  their  ability  to  be  directly  oxidized.  The  oxidation 
of  such  substances  can,  according  to  Bach,4  be  accomplished  in  that  the 
oxygen  is  transported  to  the  substance  to  be  oxidized  from  the  peroxide 
simultaneously  present  by  means  of  special  enzymes,  the  peroxidases. 
These  latter  were  first  prepared  from  pumpkins  and  from  horse-radish 
roots.  In  the  absence  of  peroxides  or  oxygenases  the  peroxidases  are 
without  action.  Chodat  and  Bach  5  have  also  found  that  certain 
preparations,  which  have  previously  been  called  oxidases,  can  be  decom- 
posed into  oxygenases  and  peroxidases.  According  to  Bach's  theory  the 
formation  of  peroxides  is  a  constant  process  going  on  in  the  organism, 
to  which  the  organism  accommodates  itself,  in  that  the  cells  by  means  of 
the  peroxidases  can  make  use  of  the  peroxides  for  the  oxidation  processes. 
Besides  this  the  organism  also  forms  other  enzymes,  the  so-called 
catalases,  which  have  the  ability  of  decomposing  the  peroxides  with  the 
formation  of  molecular  oxygen  (O2)  and  in  this  way  making  a  possible 
excess  of  peroxides  harmless.6     In  reference  to  the  behavior  of  the  perox- 


1  Verh.  naturw.  Verein,  Karlsruhe,  20,  XI  (1896),  Bd.  13,  72;  see  also  Zeitschr. 
f.  physiol.  Chem.,  59,  327  (1909). 

2Compt.  Rend.,  124,  951  (1897);  see  also  Bioch.  Centralbl.,  1,  417,  and  457  (1903); 
9,  1  (1909). 

a  Ber.  d.  d.  chem.  Gesellsch.,  36,  600  (1903). 

*Ibid.,  36,  600  (1903). 

5  Ibid.,  36,  606  (1903). 

«  Bioch.  Centralbl.,  1,  460. 


PHYSIOLOGICAL  OXIDATION  PROCESSES.  873 

idases  to  heat  the  views  are  contradictory.  Czyhlauz  and  v.  Fiuni 
found  that  the  peroxidases  from  animal  tissues  were  remarkably  resistant 
to  high  temperatures,  while  Batelli  and  Stern  x  find  that  animal  peroxi- 
dases arc  destroyed  even  at  66°  C. 

According  to  Bach's  theory  on  the  one  hand,  substances  are  nec- 
essary for  the  oxidation,  which  take  up  oxygen  with  the  formation  of 
peroxides  (oxygenases)  and  on  the  other  hand,  substances  which  are 
able  to  transport  the  oxygen  from  the  peroxides  to  the  oxidizable  bodies 
(peroxidases).  In  certain  oxidations,  for  example  in  the  phenols,  the 
peroxidases  can  be  replaced  by  certain  metallic  combinations.2  The 
iron,  of  the  blood  pigments,  acts  in  this  way  in  the  guaiacum  reaction 
(see  Chapter  XIV).  The  oil  of  turpentine  here  represents  the  peroxide 
and  can  be  replaced  by  hydrogen  peroxide.  The  oxidizable  substance, 
which  becomes  blue  in  the  reaction,  is  the  guaiaconic  acid  in  the  guaiac 
gum.3 

Irrespective  of  whether  the  division  of  the  oxidation  enzymes  into 
oxygenases  and  peroxidases  can  be  carried  out  in  all  cases,  there  are 
various  oxidation  processes,  whose  occurrence  by  a  combination  of  oxy- 
genase (or  peroxide)  with  peroxidase  (or  metallic  salt)  can  be  explained  only 
with  difficulty.  According  to  Bertrand's4  view  the  action  of  plant 
oxidation  enzymes  is  connected  with  their  manganese  content.  Never- 
theless Bach  5  has  been  able  to  prepare  enzymes  from  plants  which  were 
entirely  free  from  iron  as  well  as  manganese  salts.  Starting  from  Ber- 
trand's view,  Trillat6  has  prepared  solutions  of  manganese  salts,  alkali 
and  colloidal  substances,  which  acted  like  oxidizing  enzymes.  Dony- 
Henault7  has  prepared  artificial  "oxidases"  from  a  faintly  alkaline 
solution  of  gum  treated  with  a  solution  of  manganese  salt.  According  to 
Euler  and  Bolin  8  the  salts  of  certain  organic  acids  have  the  ability  of 
setting  the  oxidation  power  of  manganese  salts  free.  Similar  observations 
have  been  made  by  Wolff.9  In  the  oxidation  of  auto-oxidizable  substances 
the  presence  of  extremely  small  amounts  of  iron  salts  may  be  of  advantage, 


1  Czyhlarz  and  v.  Ftirth,  Hofmeister's  Beitrage,  10,  358  (1907);    Batelli  and   Stern, 
Bioch.  Zeitschr.,  13,  44  (1908). 

2  Ber.  d.  d.  chem.  Gesellsch.,  43,  366  (1910). 

3C.   E.  Carlson,  Zeitschr.   f.   physiol.  Chem.,   48,   69   (1906).     P.  Richter,  Arch. 
d.  Pharm.,  244,  90  (1906). 

<Compt.  Rend.,  124,  1032,  1355  (1897). 

6  Ber.  d.  d.  ehem.  Gesellsch.,  43,  364  (1910). 
8Compt.  Rend.,  138,  274  (1904). 

7  Bull.  acad.  roy.  de  Belgique,  1908,  105. 

8  Zeitschr.  f.  physiol.  Chem.,  57,  80  (1908). 

9  Ann.  inst.  Past.,  24  (1910). 


87-4  RESPIRATION  AND  OXIDATION. 

for  example  with  the  lecithins  (Thunberg,  Warberg  and  Meyerhof  l 
as  well  as  in  the  oxidation  of  certain  thio-compounds.2 

Batelli  and  Stern  3  have  made  careful  investigations  as  to  the 
occurrence  of  peroxidases  in  the  animal  organism.  In  order  to  eliminate 
the  action  of  catalases  which  are  present  in  the  tissues  and  which, 
as  shown  by  earlier  investigators,  decompose  the  hydrogen  peroxide, 
these  experimenters  used  ethyl  hydrogen  peroxide,  C2H5.O.O.H,  on 
which  the  catalases  do  not  act.  With  ethyl  hydrogen  peroxide  and 
hydroidic  acid  nearly  all  animal  tissues  gave  the  peroxidase  reaction, 
wherein  free  iodine  was  formed.  Scheunert,  Grimmer  and  Andryewski  4 
make  use  of  the  following  solution  as  a  reagent  for  peroxidases:  100  cc. 
fresh  tincture  of  guaiacum  and  0.1  to  0.2  cc.  3  per  cent  H2O2  solution. 
Blood  does  not  give  any  blue  coloration  with  this  reagent,  but  in  the  pres- 
ence of  large  quantities  of  H2O2  or  other  superoxide  solutions  (ethylhy- 
drogen  peroxide,  oil  of  turpentine)  it  does  give  a  blue  coloration.  With 
this  active  tincture  of  guaiacum  these  experimenters  were  able  to  detect 
peroxidases  in  the  salivary  glands,  as  well  as  the  mucous  membrane 
of  the  stomach  and  intestine  of  certain  varieties  of  animals.  The  liver 
was  always  free  from  peroxidases.  On  the  other  hand,  Batelli  and  Stern5 
also  tested  the  ability  of  various  tissues  of  acting  upon  formic  acid  in  the 
presence  of  H2O2  with  the  evolution  of  carbon  dioxide.  In  later  works 
these  experimenters  claim  that  in  all  animal  tissues  there  exists  a  substance 
of  an  unknown  nature,  the  pnein,  which  has  the  ability  of  bringing  about 
the  respiration  in  all  animal  tissues.  Pnein,  which  is  soluble  in  water, 
dializable  and  resistant  to  temperature,  increases  the  so-called  chief  respira- 
tion, which  is  connected  with  the  life  of  the  cells  and  which  stops  more 
or  less  rapidly  after  the  death  of  the  animal.  The  so-called  accessory 
respiration  continues  quite  a  long  time,  after  death,  and  this  can  continue 
in  the  absence  of  cell  elements  and  is  of  an  enzymotic  character.  Thun- 
berg 6,  who  has  constructed  an  apparatus  for  measuring  the  respiratory 
exchange  of  gas  in  small  organs  and  organisms  (microrespirometer)  finds 
that  the  salts  of  certain  organic  acids  (succinic  acid,  citric  acid,  malic 
acid,  fumaric  acid)  accelerate  more  or  less  the  gas  exchange  in  surviv- 
ing frog's  muscles.  In  their  last  communication  Batelli  and  Stern  7 
differentiate  between  two  kinds  of  oxidation  catalysts:  the  oxidases 
and  the  oxidones.    The  first  to  which,  among  others  the  tyrosinase,  alcohol- 

1  Skand.  Arch.  f.  Physiol.,  24,  90  (1911);  Zeitschr.  f.  physiol.  Chem.,  85,  412  (1913). 

2  Thunberg,  Lunds  Univ.  Arsskr.,  N.  F.,  2,  Bd.  9  (1913). 
«  Bioch.  Zeitschr.,  13,  44  (1908). 

* Ibid.,  53,  300  (1913). 

*Ibid.,  21,  487  (1910);  30,  172  (1910);  33,  315  (1911). 

■  Skand.  Arch.  f.  Physiol.,  17,  23,  24,  25  (1911). 

7  Bioch.  Zeitschr.,  46,  317,  343  (1912);  Compt.  rend.,  soc.  biol.,  74,  212  (1913). 


PHYSIOLOCAL  OXIDATION  PROCESSES.  875 

oxidase,  xanthin  oxidase  (see  below)  belong,  are  soluble  in  water,  re- 
sistant to  alcohol  and  acetone  and  can  be  heated  to  55-60°  C.  The 
oxodones,  which  for  example  oxidize  succinic  acid  to  malic  acid  and 
act  upon  p-phenyldiamine,  cannot  be  extracted  from  the  tissues  by  water; 
they  are  injured  or  destroyed  by  alcohol,  acetone  or  by  being  heated 
to  55-60°  C. 

Warburg  !  has  carried  on  extensive  experiments  on  the  influence  of 
foreign  bodies  upon  the  respiration  in  the  cells  and  has  conformed  the 
results  to  Overton's  theory  of  lipoid  membrane. 

No  deep  oxidation  processes  have  been  produced  under  the  influence 
of  the  mentioned  oxidizing  substances  outside  of  the  organism.  The 
various  divergent  views  on  the  nature  of  the  oxidizing  substances  strik- 
ingly indicates  how  little  exact  knowledge  we  have  of  this  subject. 
Perhaps  the  oxidation  within  the  body  takes  place  step  by  step,  and  it 
seems  possible  that  the  consecutive  stages  of  the  reaction  can  be  brought 
about  by  different  agents.  A  positive  division  of  the  so-called  oxidizing 
enzymes  cannot  be  made  at  the  present  time.  For  in  many  cases  it 
is  undecided  whether  we  are  dealing  with  enzymotic  processes  or  with 
non-enzymotic  catalytic  action  of  metallic  salts,  especially  as  the 
reports  on  the  heat-resistance  of  the  active  substances  are  contra- 
dictory. In  the  enzymotic  oxidations  we  are  in  many  cases  in  doubt 
whether  the  oxygen  is  transported  directly  to  the  oxidizing  substance  or 
whether  the  oxidation  is  brought  about  by  the  system  peroxide  plus 
peroxidase.  When  the  oxidation  cannot  be  shown  as  due  to  the  just- 
mentioned  system,  then  the  active  enzyme  is  called  simply  oxidase. 

In  consideration  of  the  substances  upon  which  the  oxidation  enzymes  act, 
we  can  divide  them  for  the  present  into  the  following  groups,  according  to 
Oppexheimer:2 

1.  Alcoholases,  which  transform  alcohols  into  acids,  for  example  the  acetic- 
acid-forming  enzyme  of  certain  varieties  of  bacteria. 

2.  Aldehydases,  which  oxidize  aldehydes  into  acids,  for  example,  salicylase. 

3.  Purine-oxidases,  which  oxidize  hypoxanthine  and  xanthine  into  uric  acid  and 
which  act  upon  uric  acid  with  the  formation  of  allantoin.  This  last  reaction  is 
produced  by  the  action  of  the  so-called  uriease. 

4.  Phenol-oxidases,  which  oxidize  various  phenols  and  related  bodies  with  the 
formation  of  pigments.     The  guaiac  reaction  is  of  this  kind. 

5.  Tyrosinases,  which  oxidize  tyrosine  and  closely  related  bodies  into  dark 
pigments. 

The  system  peroxide -f  peroxidase  has  been  shown  only  in  connection  with 
enzymes  of  group  4. 

Besides  the  above-mentioned  bodies,  upon  which  the  different  classes  of  oxidiz- 
ing enzymes  act  more  specifically,  we  can  mention  the  following  as  oxidase  reagents. 


xZeitschr.  f.  physiol.  Chem.,  67.  69,  70,  (1910);  71,  76  (1911). 

1  Die  Fermente  und  ihre  Wirkungen,  3.  Aufl.,  Spec.  Teil,  351  (1909). 


876  RESPIRATION  AND  OXIDATION. 

1.  Iodides  in  acid  solution  in  the  presence  of  H202.  According  to  Bach  and 
Chodat  >  this  reaction  is  completely  parallel  with  the  guaiac  reaction. 

2.  Formic  acid  with  H202  (see  above). 

3.  Amines  (especially  p-phenyldiamine)  which  forms  colored  products  on 
t  aking  up  oxygen. 2 

4.  Leucobases  or  mixtures  of  their  formers,  which  by  oxidation  are  converted 
into  pigments.  A  solution  of  a  mixture  of  a-naphthol  and  p-phenylen-diamine 
made  alkaline  with  soda  gives  indophenol  on  taking  up  oxygen  (Rohmann  and 
Spitzer3). 

5.  Certain  benzene  derivatives  which  on  oxidation  and  loss  of  water  are 
transformed  into  diphenol  derivatives,  for  example  vanillin  into  dehydrodivanillin.4 

For  quantitative  estimation  of  the  extent  of  oxidation  Bach  and  Chodat  5 
use  the  transformation  of  pyrogallol  into  purpurogallin,  which  latter  can  be  weighed. 
Bach  6  determines  the  amount  of  iodine  set  free  in  the  reaction  between  hydrogen 
peroxide  and  hydriodic  acid  and  Batelli  and  Stern  7  determine  the  quantity  of 
C02  formed  in  the  oxidation  of  formic  acid. 

There  is  no  doubt  that  reductions  occur  to  a  great  extent  in  the  animal 
body  and  often  go  hand  in  hand  with  oxidations.  The  question  as  to 
the  extent  in  which  special  reduction  enzymes  are  concerned,  is  still 
undecided.  As  the  oxidations  are  explained  by  the  action  of  special 
enzymes,  so  also  we  can  admit  of  special  reduction  enzymes,  so-called 
reductases  or  hydrogenases.  To  this  group  belongs  the  so-called  "  philo- 
thion" (De  Rey-Pailhade),  which  in  the  presence  of  sulphur  and 
water  develops  sulphuretted  hydrogen,  while  others  on  the  contrary  do 
not  accept  this  view,  and  deny  the  enzyme  nature  of  philothion.8  The 
investigations  of  Nasse  and  Rosing  9  on  the  oxidation  of  protein  in  the 
presence  of  sulphur  contradict  the  enzymotic  nature  of  this  formation 
of  sulphuretted  hydrogen,  and  the  recent  investigations  of  Heffter  10 
have  shown  that  certain  reductions  occurring  in  the  tissues  are  not  pro- 
duced by  enzymes.  He  has  also  shown  that  those  reductions,  which  are 
not  influenced  by  HCN,  like  the  reduction  of  pigments  (methylene  blue),. 

1  Ber.  d.  d.  chem.  Gesellsch.,  35,  2466  (1902). 

2  Bioch.  Zeitschr.,  46,  317  (1912). 

» Ber.  d.  d.  chem.  Gesellsch.,  28,  567  (1894). 

*  In  regard  to  this  and  other  reagents,  see  Zeitschr.  f.  physiol.  Chem.,  59,  359 
(Engler  and  Herzog). 

5  Ber.  d.  d.  chem.  Gesellsch.,  37,  1342  (1904). 
*IUd.,  37,  3785  (1904). 

7  Bioch.  Zeitschr.,  31,  443;  33,  282  (1911). 

8  De  Rey-Pailhade,  Recherches  exp6r.  sur  le  Philothion,  etc.,  Paris  (G.  Masson), 
1891,  and  Nouvelles  recherches  sur  le  Philothion,  Paris  (Masson),  1892;  Bull.  soc. 
chim.  (4),  1;  Pozzi-Escot,  Bull.  soc.  chim.  (3),  27,  and  Chem.  Centralbl.,  1904,  1, 
1645;  Chodat  and  Bach,  Ber.  d.  d.  chem.  Gesellsch.,  36;  Abelous  and  Ribaut,  Compt. 
Rend.,  137,  and  Bull.  soc.  chim.,  (3),  31. 

»E.  Rosing,  Unters.  liber  die  Oxydation  von  Eiweiss  in  Gegenwart  von  Schwefel, 
I norg. -Dissert,  Rostock,  1891. 

10  Med.-naturw.  Arch.,  1,  81-104;  Marburg,  cited  in  Chem.  Centralbl.,  2,  1907,, 
822;  Thunberg,  Ergebn.  d.  Physiol.,  11. 


REDUCTION   PROCESSES.  877 

of  sulphur  to  H2S  and  others,  can  be  brought  about  by  the  labile  H  of  the 
Bulphydryl-compounds.  In  this  manner  for  example  the  cysteine 
(see  Chapter  II)  arts  and  quickly  reacts  with  sulphur  with  the  formation 
of  H2S  and  similarly  acting  substances  have  been  detected  by  IIkffter 
in  various  organs  and  organ  extracts.  We  have  here  a  group  of  reductions 
which  are  not  of  an  enzyme  nature. 

From  the  investigations  of  Abelous  and  Aloy  l  it  follows  that 
plants  as  well  as  animal  organs  have  the  ability  at  the  same  time  of  oxidiz- 
ing salicylaldehyde  and  of  reducing  nitrates  to  nitrites.  On  the  other 
hand  ScHARDiNGEit,  Trommsdorff  and  Bach2  have  shown  that  fresh 
cow's  milk,  which  alone  is  without  action  upon  methylene  blue  and  on 
nitrates,  reduces  these  bodies  in  the  presence  of  aldehydes  into  leucobases 
or  nitrites.  Boiled  milk  does  not  have  this  power  and  the  action  is 
explained  by  the  presence  of  a  reductase,  the  so-called  Schardinger 
enzyme.  The  optimum  of  action  lies  at  about  70°  C.  Bach  found  the  same 
action  in  various  animal  organs.  The  process  may  to  be  just  as  well 
considered  as  an  oxidation  under  the  influence  of  an  aldehydase  whereby 
the  methylene  blue  or  the  nitrate  gives  up  the  oxygen  for  the  oxidation 
of  the  aldehyde.  On  the  other  hand  Strassner3  ascribes  the  reduction 
of  the  methylene  blue  to  the  above-mentioned  reducing  action  of  the 
sulphydryl  groups. 

1  Compt.  Rend.,  138,  382  (1904);  see  also  Pozzi-Escot,  ibid.,  13S,  511. 

2  Schardinger,  Zeitschr.  f.  Unters.  d.  Nahrune;s-  und  Genussmittel,  5,  22  (1902); 
Trommsdorff,  Centralbl.  f.  Bakter.,  49,  291  (1909);  Bach,  Ber.  d.  d.  chem.  Gesellsch., 
42.  4463  (1909);  Bioch.  Zeitschr.,  31,  443;  33,  2S2;  38,  154  (1911). 

» Ibid.,  29,  295  (1910). 


CHAPTER  XVII. 

METABOLISM  WITH  VARIOUS  FOODS,  AND  THEIR  NECESSITY 

TO   MAN. 

I.     GENERAL  DISCUSSION  AND  METHODS  USED  IN  THE  STUDY  OF  MATTER 
AND   FORCE   METABOLISM. 

The  conversion  of  chemical  energy  into  heat  and  mechanical  work 
which  characterizes  animal  life,  leads  to  the  formation  of  relatively 
simple  compounds — carbon  dioxide,  urea,  etc. — which  leave  the  organism, 
and  which,  moreover,  being  very  poor  in  energy,  are  for  this  reason  of 
little  or  no  value  to  the  body.  It  is  therefore  absolutely  necessary  for 
the  continuance  of  life  and  the  normal  course  of  the  functions  of  the  body 
that  the  organism  and  its  different  tissues  should  be  supplied  with  new 
material  to  replace  that  which  has  been  exhausted.  This  is  accom- 
plished by  means  of  food.  Those  bodies  are  designated  as  food  which 
have  no  injurious  action  upon  the  organism  and  which  serve  as  a  source 
of  energy  and  can  replace  those  constituents  of  the  body  that  have  been 
consumed  in  metabolism  or  that  can  prevent  or  diminish  the  consumption 
of  such  constituents. 

Among  the  numerous  dissimilar  substances  which  man  and  animals 
take  with  the  food  all  cannot  be  equally  necessary  or  have  the  same  value. 
Some  perhaps  are  unnecessary,  while  others  may  be  indispensable.  We 
have  learned  by  direct  observation  and  a  wide  experience  that  besides  the 
oxygen,  which  is  necessary  for  oxidation,  the  essential  foods  for  animals 
in  general,  and  for  man  especially,  are  water,  mineral  bodies,  proteins, 
carbohydrates,  and  fats. 

It  is  also  apparent  that  the  various  groups  of  food-stuffs  necessary  for 
the  tissues  and  organs  must  be  of  varying  importance;  thus,  for  instance, 
water  and  the  mineral  bodies  have  another  value  than  the  organic  foods, 
and  these  again  must  differ  in  importance  among  themselves.  The 
knowledge  of  the  action  of  various  nutritive  bodies  on  the  exchange  of 
material  from  a  qualitative  as  well  as  a  quantitative  point  of  view  must 
be  of  fundamental  importance  in  determining  the  value  of  different 
nutritive  substances  relative  to  the  demands  of  the  body  for  food  under 
various  conditions,  and  also  in  deciding  many  other  questions — for  instance, 
the  proper  nutrition  for  an  individual  in  health  and  in  disease. 

878 


EXCRETA  OF   THE  ORGANISM.  879 

Such  knowledge  can  be  attained  only  by  a  series  of  systematic  and 
thorough  observations,  in  which  the  quantity  of  nutritive  material,  rela- 
tive to  the  weight  of  the  body,  taken  and  absorbed  in  a  given  time  is 
compared  with  the  quantity  of  final  metabolic  products  which  leave  the 
organism  at  the  same  time.  Researches  of  this  kind  have  been  made  by 
investigators,  but  above  nil  should  be  mentioned  those  made  by  BlBCHOFF 
and  Voir,  by  Pettenkofer  and  Voit,  and  by  Voit  and  his  pupils,  by 
Rubner,  Zintz  and  by  Atwater. 

It  is  absolutely  necessary  in  researches  on  the  exchange  of  material 
to  be  able  to  collect,  analyze,  and  quantitatively  estimate  the  excreta 
of  the  organism,  so  that  they  may  be  compared  with  the  quantity  and 
composition  of  the  nutritive  bodies  ingested.  In  the  first  place,  one  must 
know  what  the  habitual  excreta  of  the  body  are  and  in  what  way  these 
bodies  leave  the  organism.  One  must  also  have  trustworthy  methods 
for  their  quantitative  estimation. 

The  organism  may,  under  physiological  conditions,  be  exposed  to 
accidental  or  periodic  losses  of  valuable  material — such  losses  as  occur 
only  in  certain  individuals,  or  in  the  same  individual  only  at  a  certain 
period;  for  instance,  the  secretion  of  milk,  the  production  of  eggs,  the 
ejection  of  semen  or  menstrual  blood.  It  is  therefore  apparent  that  these 
losses  can  be  the  subject  of  investigation  and  estimation  only  in  special 
cases. 

The  regular  and  constant  excreta  of  the  organism  are  of  the  very 
greatest  importance  in  the  study  of  metabolism.  To  these  belong,  in 
the  first  place,  the  true  final  metabolic  products — carbon  dioxide,  urea 
(uric  acid,  hippuric  acid,  creatinine,  and  other  urinary  constituents), 
and  a  part  of  the  water.  The  remainder  of  the  water,  the  mineral  bodies, 
and  those  secretions  or  tissue  constituents — mucus,  digestive  fluids,  sebum, 
perspiration,  and  epidermal  formations — which  are  either  poured  into 
the  intestinal  tract,  or  secreted  from  the  surface  of  the  body,  or  broken 
off  and  thereby  lost  to  the  body,  also  belong  to  the  constant  excreta. 

The  remains  of  food,  sometimes  indigestible,  sometimes  digestible  but  not 
acted  upon,  which  are  contained  in  the  feces,  and  which  vary  considerably  in 
quantity  and  composition  with  the  nature  of  the  food,  also  belong  to  the  excreta 
of  the  organism.  Even  though  these  remains,  which  are  never  absorbed  and 
therefore  are  never  constituents  of  the  animal  fluids  or  tissues,  cannot  be  con- 
sidered as  excreta  of  the  body  in  a  strict  sense,  still  their  quantitative  estimation 
is  absolutely  necessary  in  certain  experiments  on  the  exchange  of  material. 

The  determination  of  the  constant  loss  is  in  some  cases  accompanied  with  the 
greatest  difficulties.  The  loss  from  the  detached  epidermis,  from  the  secretion 
of  the  sebaceous  glands,  etc.,  cannot  be  determined  with  exactness  without  dif- 
ficulty, and  therefore— as  they  do  not  occasion  any  appreciable  loss  because  of 
their  small  quantity — they  need  not  he  considered  in  quantitative  experiments 
on  metabolism.  This  also  applies  to  the  constituents  of  the  mucus,  bile,  pancreatic 
and  intestinal  juices,  etc.,  occurring  in  the  contents  of  the  intestine,  and  which, 
leaving  the  body  with  the  feces,  cannot  be  separated  from  the  other  contents  of 


880  METABOLISM. 

the  intestine  and  therefore  cannot  be  quantitatively  determined  separately.  The 
uncertainty  which,  because  of  the  intimated  difficulties,  attaches  itself  to  the 
results  of  the  experiment,  is  very  small  as  compared  to  the  variation  which  is  caused 
by  different  individualities,  different  modes  of  living,  different  foods,  etc.  Only 
approximate  values  can  therefore  be  given  for  the  constant  excreta  of  the  human 
body. 

The  following  figures  represent  the  quantity  of  excreta  for  twenty- 
iour  hours  from  a  grown  man,  weighing  60-70  kilos,  on  a  mixed  diet. 
The  figures  are  compiled  from  the  results  of  different  investigators: 

Grams. 

Water 2500-3500 

Salts  (with  the  urine) 20-30 

Carbon  dioxide 750-900 

Urea 20-40 

Other  nitrogenous  urinary  constituents 2-5 

Solids  in  the  excrement 20-50 

These  total  excreta  are  approximately  divided  among  the  various 
excretions  in  the  following  way;  but  still  it  must  not  be  forgotten  that 
this  division  may  vary  to  a  great  extent  under  different  external  circum- 
stances: By  respiration  about  32  per  cent,  by  the  evaporation  from  the 
skin  17  per  cent,  with  the  urine  46-47  per  cent,  and  with  the  excrement 
5-9  per  cent.  The  elimination  by  the  skin  and  lungs,  which  is  sometimes 
differentiated  by  the  name  "  per spir alio  insensiblis  "  from  the  visible 
elimination  by  the  kidneys  and  intestine,  is  on  an  average  about  50  per 
cent  of  the  total  elimination.  This  proportion,  quoted  only  relatively, 
is  subject  to  considerable  variation,  because  of  the  great  difference  in 
the  loss  of  water  through  the  skin  and  kidneys  under  varying  circum- 
stances. 

The  nitrogenous  constituents  of  the  excretions  consist  cniefly  of  urea, 
or  uric  acid  in  certain  animals,  and  the  other  nitrogenous  urinary  con- 
stituents. A  disproportionately  large  part  of  the  nitrogen  leaves  the  body 
with  the  urine,  and,  as  the  nitrogenous  constituents  of  this  excretion  are 
final  products  of  the  metabolism  of  proteins  in  the  organism,  the  quantity 
of  proteins  catabolized  in  the  body  may  be  easily  calculated  by  multiply- 
ing-the  quantity  of  nitrogen  in  the  urine  by  the  coefficient  6.25  (:L1Q6(l  =  6.25), 
if  it  is  admitted  that  the  proteins  contain  in  round  numbers  16  per  cent 
of  nitrogen. 

Still  another  question  is  whether  the  nitrogen  leaves  the  body  only 
with  the  urine  or  by  other  channels.  The  latter  is  habitually  the  case. 
The  discharges  from  the  intestine  always  contain  some  nitrogen,  which 
consists  in  part  of  non-absorbed  remnants  of  the  food,  but  in  chief  part 
and  sometimes  entirely  of  constituents  of  the  epithelium  and  the  secre- 
tions. Under  these  circumstances  it  is  apparent  that  one  cannot  give 
any  exact  figures  which  are  valid  for  all  cases  for  that  part  of  the  nitrogen 
of  the  excrement  which  originates  in  the  digestive  tract  and  in  the  digestive 


NITROGEN  ELIMINATION.     NITROGEN  DEFICIT.  881 

fluids.  It  may  not  vary  in  different  individuals  only,  but  also  in  the  same 
individual  after  more  or  less  active  secretion  and  absorption.  In  the 
attempts  made  to  determine  this  part  of  the  nitrogen  of  the  excrement 
it  has  been  found  that  in  man,  on  non-nitrogenous  or  nearly  nitrogen- 
free  food,  it  amounts  in  round  numbers  to  somewhat  less  than  1  gram 
per  twenty-four  hours  (Rieder,  Rubner).  Even  with  such  food  the 
.absolute  quantity  of  nitrogen  eliminated  by  the  feces  increases  with 
the  quantity  of  food  because  of  the  accelerated  digestion  (Tsuboi  1), 
and  is  greater  than  in  starvation.  Muller2  found  in  his  observations 
on  the  faster  C'etti  that  only  0.2  gram  nitrogen  was  derived  from  the 
intestinal  canal. 

The  quantity  of  nitrogen  which  leaves  the  body  under  normal  circum- 
stances by  means  of  the  hair  and  nails,  with  the  scaling  off  of  the  skin, 
and  with  the  perspiration  cannot  be  accurately  determined.  It  is 
nevertheless  so  small  that  it  may  be  ignored.  Only  in  profuse  sweating 
need  the  elimination  by  this  channel  be  taken  into  consideration. 

The  view  was  formerly  held  that  in  man  and  carnivora  an  elimination 
of  gaseous  nitrogen  took  place  through  the  skin  and  lungs,  and  because  of 
this,  on  comparing  the  nitrogen  of  the  food  with  that  of  the  urine  and 
feces,  a  nitrogen  deficit  occurred  in  the  visible  elimination. 

This  question  has  been  the  subject  of  much  discussion  and  of  numerous 
investigations,  the  most  recent  by  Krogh  and  Oppenheimer.3  These 
researches  have  shown  that  the  above  assumption  is  unfounded,  and 
moreover  several  authorities,  especially  Pettenkofer  and  Voit,  and 
Gruber,4  have  shown  by  experiments  on  man  and  animals  that  with 
the  proper  quantity  and  quality  of  food  the  body  can  be  brought  into 
nitrogenous  equilibrium,  in  which  the  quantity  of  nitrogen  voided  with 
the  urine  and  feces  is  equal  or  nearly  equal  to  the  quantity  contained  in 
the  food.  Undoubtedly  we  must  admit,  with  Voit,  that  a  deficit  of  nitro- 
gen does  not  exist,  or  it  is  so  insignificant  that  in  experiments  upon 
metabolism  it  need  not  be  considered.  Ordinarily,  in  investigations  on 
the  catabolism  of  proteins  in  the  body,  it  is  only  necessary  to  consider  the 
nitrogen  of  the  urine  and  feces,  but  it  must  be  remarked  that  the  nitrogen 
of  the  urine  is  a  measure  of  the  extent  of  the  catabolism  of  the  proteins 


1  Rieder,  Zeitschr.  f.  Biologie,  20;  Rubner,  ibid.,  15;  Tsuboi,  ibid.,  35. 

2  Berlin,  klin.  Wochenschr.,  1887. 

3  See  Regnault  and  Reiset,  Annal.  d.  chem.  et  phys.  (3),  26,  and  Annal.  d.  Chem. 
U.  Pharm.,  73;  Seegen  and  Nowak,  Wien.  Sitzungsber.,  71,  and  Pfliiger's  Arch.,  25; 
Pettenkofer  and  Voit,  Zeitschr.  f.  Biologie,  16;  Leo,  Pfliiger's  Arch.,  26;  Krogh, 
Skand.  Arch.  f.  Physiol.,  18,  and  Wien.  Sitz.  Ber.,  115,  III;  Oppenheimer,  Bioch. 
Zeitschr.,  4. 

4  Pettenkofer  and  Voit,  in  Hernnan's  Handbuch,  6,  Thl.  1;  Gruber,  Zeitschr.  f. 
Biologie,  16  and  19. 


882  METABOLISM. 

in  the  body,  while  the  nitrogen  of  the  feces  (after  deducting  about  1  gram 
on  a  mixed  diet)  is  a  measure  of  the  non-absorbed  part  of  the  nitrogen  of 
the  food.  The  nitrogen  of  the  food,  as  well  as  of  the  excreta,  is  generally- 
determined  by  Kjeldahl's  method. 

In  the  oxidation  of  the  proteins  in  the  organism, their  sulphur  is  oxidized 
into  sulphuric  acid,  and  on  this  depends  the  fact  that  the  elimination  of 
sulphuric  acid  by  the  urine,  which  in  man  is  but  to  a  small  extent  derived 
from  the  sulphates  of  the  food,  nearly  makes  equal  variations  with  the 
elimination  of  nitrogen  by  the  urine.  If  the  amount  of  nitrogen  and  sul- 
phur in  the  proteins  is  considered  as  16  per  cent  and  1  per  cent  respectively, 
then  the  proportion  between  the  nitrogen  of  the  proteins  and  the  sulphuric 
acid,  H2SO4,  produced  by  their  combustion  is  in  the  ratio  5.2  : 1,  or  about 
the  same  as  in  the  urine  (see  page  765).  The  determination  of  the  quantity 
of  sulphuric  acid  eliminated  in  the  urine  gives  us  an  important  means  of 
controlling  the  extent  of  the  transformation  of  proteins,  and  such  a  con- 
trol is  especially  important  in  cases  in  which  it  is  expected  to  study  the 
action  of  certain  nitrogenous  non-albuminous  bodies  on  the  metabolism 
of  proteins,  or  to  decide  the  question  whether  a  true  protein  combustion 
and  not  only  a  washing  out  of  the  nitrogenous  products  of  metabolism 
from  the  tissues  is  taking  place.  A  determination  of  the  nitrogen  alone 
is  naturally  not  sufficient  in  such  cases.  A  perfectly  positive  measure 
of  the  protein  catabolism  cannot  be  made  from  the  sulphuric  acid  of  the 
urine,  as  the  various  protein  substances  have  a  rather  variable  sulphur 
content,  and  on  the  other  hand  also  a  variable  quantity  of  the  sulphur 
in  the  urine  exists  as  so-called  neutral  sulphur. 

In  metabolism  experiments  the  total  sulphur  of  the  urine  as  well  as  the 
feces  must  be  determined,  and  it  may  also  be  of  importance  to  determine 
the  relation  between  the  sulphuric-acid  sulphur  and  the  neutral  sulphur 
of  the  urine.  The  elimination  of  the  sulphur  originating  from  the  proteins 
does  not,  according  to  v.  Wendt,  Hamalainen  and  Helme  and  Ch. 
Wolff  x  always  run  parallel  with  the  protein  nitrogen,  and  for  the 
white  of  egg  the  maxima  of  the  elimination  curves  may  indeed  be 
separated  during  a  period  of  twenty-four  hours  (Wolff).  The  sulphur 
is  more  quickly  eliminated  than  the  nitrogen,  and  this  behavior  of  sul- 
phur gives  in  certain  cases  a  more  positive  picture  of  the  temporal 
catabolism  of  protein  than  the  nitrogen.  This  is  of  importance,  as  the 
elimination  of  the  nitrogen  corresponding  to  a  certain  amount  of  protein 
requires  several  days  for  completion.  Falta  has  also  observed  that  the 
chief  amount  of  nitrogen  in  man  on  taking  different  proteins  is  secreted 
with  varying  rapidity,   and  the  same  is  true,  according  to  Hamalainen 


1  Wendt,  Skand.  Arch.  f.  Physiol.,  17;    Hamalainen  and  Helme,  ibid.,  19;    Falta, 
Deutsch.  Arch.  f.  klin.  Med.,  86;  Ch.  G.  Wolff,  Bioch.  Zeitschr.,  40. 


CALCULATION  OF  METABOLISM.  883 

and  Helme,  for  the  elimination  of  sulphur,  as  in  their  experiments  the 
sulphur  elimination  from  white  of  egg  required  about  six  days  and  from 
casein  only  two  days.  These  conditions  must  be  considered  in  metab- 
olism experiments. 

Besides  lecithins  and  other  phosphatides  the  body  takes  with  its  food 
pseudonucleins  as  well  as  true  nucleins,  and  these  are  absorbed  more  or 
less  completely  from  the  intestinal  tract  and  then  assimilated.  On  the 
other  hand,  the  phosphorized  protein  substances,  lecithins  and  phos- 
phatides, are  also  decomposed  within  the  body,  and  their  phosphorus  is 
chiefly  eliminated  as  phosphoric  acid  and  also  in  part  as  organic  phos- 
phorus (see  page  757).  For  these  reasons  the  phosphorus  is  of  great 
importance  in  certain  investigations  on  metabolism. 

It  is  found,  on  comparing  the  nitrogen  of  the  food  with  that  of  the 
urine  and  feces,  that  there  is  an  excess  of  the  first ;  this  means  that  the 
body  has  increased  its  stock  of  nitrogenous  substances — proteins.  If, 
on  the  contrary,  the  urine  and  feces  contain  more  nitrogen  than  the  food 
taken  at  the  same  time,  this  denotes  that  the  body  is  giving  up  part  of  its 
nitrogen — that  is,  part  of  its  own  proteins  has  been  decomposed. 

We  can,  from  the  quantity  of  nitrogen,  as  above  stated,  calculate  the  corre- 
sponding quantity  of  proteins  by  multiplying  by  6.25. x  Usually,  according  to 
Voit's  proposition,  the  nitrogen  of  the  urine  is  not  calculated  as  decomposed 
proteins,  but  as  decomposed  muscle-substance  or  flesh.  Lean  meat  contains  on 
an  average  about  3.4  per  cent  nitrogen;  hence  each  gram  of  nitrogen  of  the  urine 
corresponds  in  round  numbers  to  about  30  grams  of  flesh.  The  assumption  that 
lean  meat  contains  3.4  per  cent  nitrogen  is  arbitrary,  and  the  relation  of  N  :  C 
in  the  proteins  of  dried  meat,  which  is  of  great  importance  in  certain  experiments 
on  metabolism,  is  given  differently  by  various  experimenters,  namely,  1  :  3.22- 
1  :  3.68.  Argutinsky  found  in  beef,  after  complete  removal  of  fat  and  subtrac- 
tion of  glycogen,  that  the  relation  was  1  :  3.24  (see  Chapter  X). 

The  carbon  leaves  the  body  chiefly  as  carbon  dioxide,  which  is  elimi- 
nated by  the  lungs  and  skin.  The  remainder  of  the  carbon  is  excreted  in 
the  urine  and  feces  in  the  form  of  organic  compounds,  in  which  the  quan- 
tity of  carbon  can  be  determined  by  elementary  analysis.  It  was  for- 
merly considered  sufficient  to  calculate  the  quantity  of  carbon  in  the  urine 
from  the  quantity  of  nitrogen  according  to  the  relation  N:C  =  1:0.67  to 
0.72.  This  does  not  seem  to  be  trustworthy,  as  this  relation  varies  and 
depends,  according  to  Tangl,  Pfluger,  Langstein,  and  Steinitz,2  upon 
the  kind  of  food.  Tangl  has  shown  that  the  richer  the  food  is  in  car- 
bohydrates the  more  carbon  and  hence  the  more  heat  of  combustion  per 

1  In  calculating  the  protein  catabolism  from  the  nitrogen  of  the  urine  it  must  not 
be  forgotten  that  the  food  often  contains  nitrogenous  extractives  whose  nitrogen  cannot 
be  calculated  as  protein  and  for  which  a  special  correction  must  be  made,  if  necessary. 

2  Tangl,  Arch.  f.  (Anat.  u.)  Physiol.,  1899,  Suppl.  Bd.;  Pfluger  in  Pfliiger's  Arch., 
79;  Langstein  and  Steinitz,  Centralbl.  f.  Physiol.,  19. 


884  METABOLISM. 

gram  of  nitrogen  does  the  urine  contain.  He  found  the  following  for  1 
gram  of  nitrogen  in  the  urine:  With  diet  rich  in  fat  0.747  gram  C  and  9.22 
calories;  for  carbohydrate-rich  diet  he  found  0.936  gram  C  and  11.67 
calories.     The  quantity  of  carbon  in  the  feces  can  be  calculated  from  the 

C 

quantity  of  nitrogen  in  the  feces  by  using  the  quotient  —  =  9.2  (average 

with  mixed  diet,  according  to  Atwater  and  Benedict.1) 

The  extent  of  the  gas  exchange  can  be  determined  by  any  of  the 
methods  given  on  page  869.  By  multiplying  the  quantity  of  carbon  dioxide 
found  by  0.273  one  obtains  the  quantity  of  carbon  eliminated  as  CO2. 
If  the  total  quantity  of  carbon  eliminated  in  various  ways  is  compared 
with  the  carbon  contained  in  the  food,  some  idea  can  be  obtained  as  to 
the  transformation  of  the  carbon  compounds.  If  the  quantity  of  carbon 
in  the  food  is  greater  than  in  the  excreta,  then  the  excess  is  deposited; 
while  if  the  reverse  be  the  case,  it  shows  a  corresponding  loss  of  body 
substance. 

The  nature  of  the  substances  here  deposited  or  lost,  whether  they  consist 
of  proteins,  fats,  or  carbohydrates,  is  learned  from  the  total  quantity  of  the 
nitrogen  of  the  excretions.  The  corresponding  quantity  of  proteins  may  be  cal- 
culated from  the  quantity  of  nitrogen,  and,  as  the  average  quantity  of  carbon 
in  the  proteins  is  known,  the  quantity  of  carbon  which  corresponds  to  the  decom- 
posed proteins  may  be  easily  ascertained.  If  the  quantity  of  carbon  thus  found 
is  smaller  than  the  quantity  of  the  total  carbon  in  the  excreta,  it  is  then  obvious 
that  some  other  nitrogen-free  substance  has  been  consumed  besides  the  proteins. 
If  the  quantity  of  carbon  in  the  proteins  is  considered  in  round  numbers  as  52.5 
per  cent,  then  the  relation  between  carbon  (52.5)  and  nitrogen  (16)  is  3.28,  or  in 
round  numbers  3.3  :  1.  If  the  total  quantity  of  nitrogen  eliminated  is  multiplied 
by  3.3,  the  excess  of  carbon  in  the  eliminations  over  the  product  found  represents 
the  carbon  of  the  decomposed  non-nitrogenous  compounds.  For  instance,  in 
the  case  of  a  person  experimented  upon,  10  grams  of  nitrogen  and  200  grams  of 
carbon  were  eliminated  in  the  course  of  twenty-four  hours;  then  these  62.5  grams  of 
protein  correspond  to  33  grams  of  carbon,  and  the  difference,  200  — (3.3X10)  =167, 
represents  the  quantity  of  carbon  in  the  decomposed  non-nitrogenous  compounds. 
If  we  start  from  the  simplest  case,  starvation,  where  the  body  lives  at  the  expense 
of  its  own  substance,  then,  since  the  quantity  of  carbohydrates  as  compared  with 
the  fats  of  the  body  is  extremely  small,  in  such  cases  in  order  to  avoid  mistakes 
the  assumption  must  be  made  that  the  person  experimented  upon  has  used  only 
fat  and  proteins.  As  animal  fat  contains  on  an  average  76.5  per  cent  carbon, 
the  quantity  of  transformed  fat  may  be  calculated  by  multiplying  the  carbon  by 

~-v  =  1.3.     In  the  case  of  the  above  example,  the  person  experimented  upon 

would  have  used  62.5  grams  of  proteins  and  167X1.3=217  grams  of  fat,  of  his 
own  body,  in  the  course  of  the  twenty-four  hours. 

Starting  from  the  nitrogen  balance,  it  can  be  calculated  in  the  same  way 
whether  an  excess  of  carbon  in  the  food  as  compared  with  the  quantity  of  carbon 
in  the  excreta  is  retained  by  the  body  as  proteins  or  fat  or  as  both.  On  the  other 
hand,  with  an  excess  of  carbon  in  the  excreta  one  can  determine  how  much  of  the 
loss  of  the  substance  of  the  body  is  due  to  a  consumption  of  the  proteins  on  the 
one  side  and  of  non-nitrogenous  bodies  on  the  other  side.     How  to  especially 

1  Bull,  of  Dept.  of  Agric,  U.  S.,  Washington,  No.  136. 


CALORIC  VALUES  OF  FOOD-STUFFS.  885 

calculate  the  part  taken  by  the  fats  and  carbohydrate  will  be  shown  in  connection 
with  the  calculation  of  the  energy  metabolism. 

The  quantity  of  water  and  mineral  bodies  voided  with  the  urine  and 
feces  can  easily  be  determined.  The  quantity  of  wrater  eliminated  by 
the  skin  and  lungs  may  be  directly  estimated  by  means  of  the  large 
respiration  apparatus. 

The  organic  constituents  of  the  body  as  well  as  the  foodstuffs  intro- 
duced, represent  a  sum  of  chemical'  energy  which  the  body  can  use 
for  force.  The  exchange  of  material  is  also  an  exchange  of  force, 
and  the  first  stands  in  such  close  relation  to  the  second  that  the  study 
of  one  cannot  be  separated  from  the  other.  The  energy  theory  of 
metabolism  has  exercised  an  extraordinarily  fruitful  influence  upon 
the  entire  study  of  metabolism  and  nutrition,  and  this  is  due  in  great 
measure  to  the  work  of  Rubner. 

This  energy  of  the  various  foods  may  be  represented  by  the  amount 
of  heat  which  is  set  free  in  their  combustion.  This  quantity  of  heat  is 
expressed  as  calories,  and  a  small  calorie  is  the  quantity  of  heat  necessary 
to  warm  1  gram  of  water  from  0°  to  1°  C.  A  large  calorie  is  the  quantity 
of  heat  necessary  to  warm  1  kilo  of  water  1°  C.  Here  and  in  the  follow- 
ing pages  large  calories  are  to  be  understood.  There  are  numerous 
investigations  by  different  experimenters,  such  as  Frankland,  Dan- 
ilewski,  Rubner,  Berthelot,  Stohmann,  Benedict  and  Osborne, 
and  others,  on  the  calorific  value  of  different  foodstuffs.  The  following 
results,  which  represent  the  calorific  value  of  a  few  nutritive  bodies  on 
complete  combustion  outside  of  the  body  to  the  highest  oxidation  prod- 
ucts, are  taken  from  Stohmann's  1  work. 

Calories. 

Casein 5 .  86 

Ovalbumin 5 .  74 

Conglut  in 5 .  48 

Protein   (average) 5.71 

Animal  tissue-fat 9 .  50 

Butter-fat 9 .  23 

Cane-sugar 3 .  96 

Milk-sugar 3 . 95 

Glucose 3 .  74 

Starch 4 .  19 

Fats  and  carbohydrates  are  completely  burnt  in  the  body,  and  one  can 
therefore  consider  their  combustion  equivalent  as  a  measure  of  the  living 
force  developed  by  them  within  the  organism.  We  generally  designate 
9.3  and  4.1  calories  for  each  gram  of  substance  as  the  average  for  the 
physiological  calorific  value  of  fats  and  carbohydrates  respectively. 

1  See  Rubner,  Zeitschr.  f.  Biologic,  21,  which  also  cites  the  works  of  Frankland 
and  Danilewski;  see  also  Berthelot,  Compt.  Rend.,  102,  104,  and  110;  Stohmann, 
Zritschr.  f.  Biologie,  31;  Benedict  and  Osborne  (vegetable  proteins),  Journ.  of  biol. 
Chem.,  3. 


886  METABOLISM. 

The  proteins  act  differently  from  the  fats  and  carbohydrates.  They 
are  only  incompletely  burnt,  and  they  yield  certain  decomposition  prod- 
ucts, which,  leaving  the  body  with  the  excreta,  still  represent  a  certain 
quantity  of  energy  which  is  lost  to  the  body.  The  heat  of  combustion 
of  the  proteins  is  smaller  within  the  organism  than  outside  of  it,  and  they 
must  therefore  be  specially  determined.  For  this  purpose  Rubner1 
fed  a  dog  on  washed  meat,  and  he  subtracted  from  the  heat  of  combustion 
of  the  food  the  heat  of  combustion  of  the  urine  and  feces,  which  cor- 
responded to  the  food  taken  plus  the  quantity  of  heat  necessary  for  the 
swelling  up  of  the  proteins  and  the  solution  of  the  urea.  Rubner  has 
also  tried  to  determine  the  heat  of  combustion  of  the  proteins  (muscle- 
proteins)  decomposed  in  the  body  of  rabbits  in  starvation.  According 
to  these  investigations,  the  physiological  heat  of  combustion  in  calories 
for  each  gram  of  substance  is  as  follows: 

1  gram  of  the  dry  substance  Calories. 

Protein  from  meat 4.4 

Muscle 4.0 

Protein  in  starvation 3.8 

Fat  (average  for  various  fats) 9.3 

Carbohydrates  (calculated  average) 4.1 

The  physiological  combustion  value  of  the  various  foods  belonging  to 
the  same  group  is  not  quite  the  same.  It  is,  for  instance,  3.97  calories 
for  a  vegetable  protein,  conglutin,  and  4.42  calories  for  an  animal  protein 
body,  syntonin.  According  to  Rubner  the  normal  heat  value  per  1 
gram  of  animal  protein  may  be  considered  as  4.23  calories,  and  of  vegetable 
protein  as  3.96  calories.  When  a  person  on  a  mixed  diet  takes  about 
60  per  cent  of  the  proteins  from  animal  foods  and  about  40  per  cent  from 
vegetable  foods,  the  value  of  1  gram  of  the  protein  of  the  food  is  equivalent 
to  about  4.1  calories.  The  physiological  value  of  each  of  the  three 
chief  groups  of  organic  foods,  by  their  decomposition  in  the  body,  is  in 
round  numbers  as  follows: 

Calories. 

1  gram  protein 4.1 

1  gram  fat 9.3 

1  gram  carbohydrate 4.1 

1  gram  alcohol 7.1 

These  figures  are  generally  used  in  the  calculation  of  the  energy  con- 
tent of  various  foodstuffs  and  diets. 

The  extent  of  gas  exchange  and  the  so-called  respiratory  quotient 
is,  besides  the  extent  of  nitrogen  elimination,  of  the  greatest  importance 
in  the  calculation  of  the  extent  of  energy  metabolism  and  the  division 
of  the  energy  between  the  protein,  fat  and  carbohydrate. 

<  >n  comparing  the  inspired  and  expired  air  we  learn,  on  measuring 
them  when  dry  and  at  the  same  temperature  and  pressure,  that  the  volume 

1  Zeitschr.  f .  Biologie,  21. 


RESPIRATORY  QUOTIENT.  Ss7 

of  the  expired  air  is  less  than  that  of  the  inspired  air.  This  depends 
upon  the  fact  that  not  all  of  the  oxygen  appears  again  in  the  expired 
air  as  carbon  dioxide,  because  it  is  not  only  used  in  the  oxidation  of  car- 
bon, hut  also  in  part  in  the  formation  of  water,  sulphuric  acid,  and  other 
bodies.     The  volume  of  expired  carbon  dioxide  is  regularly  less  than  the 

CO 
volume  of  the  inspired  oxygen,  and  the  relation  -rr11,  which  is  called  the 

respiratory  quotient,  is  generally  less  than  1. 

The  magnitude  of  the  respiratory  quotient  is  dependent  upon  the  kind 
of  substances  destroyed  in  the  body.  In  the  combustion  of  pure  carbon 
one  volume  of  oxygen  j'ields  one  volume  of  carbon  dioxide,  and  the 
quotient  is  therefore  equal  to  1.  The  same  is  true  in  the  burning  of 
carbohydrates,  and  in  the  exclusive  decomposition  of  carbohydrates  in 
the  animal  body  the  respiratory  quotient  must  be  approximately  1.  In 
the  exclusive  metabolism  of  proteins  it  is  close  to  0.80,  and  with  the  decom- 
position of  fat  it  is  0.7.  In  starvation,  as  the  animal  draws  on  its  own 
flesh  and  fat,  the  respiratory  quotient  must  be  a  close  approach  to  the 
latter  figure.  The  respiratory  quotient,  which  is  calculated  with  exclusive 
combustion  of  carbohydrate,  fat  and  protein,  as  respectively,  1,  0.707  and 
0.809  and  with  alcohol  is  0.667,  also  gives  important  information  as  to 
the  quality  of  material  decomposed  in  the  body,  especially  with  the 
supposition  that  the  carbon  dioxide  elimination  is  not  influenced  by  some 
special  condition  such  as  a  change  in  the  respirator}'  mechanism.  Another 
supposition  is  that  no  incomplete  oxidation  step  in  combustion  is  elimi- 
nated. 

The  respiratory  quotient  can  also  be  strongly  influenced  by  inter- 
mediary processes  in  the  animal  bod}',  as  by  the  formation  of  glycogen 
from  protein,  or  from  fat  or  by  the  formation  of  fat  from  carbohydrates. 
In  the  first  case  the  quotient  may  be  lower  than  0.7  and  in  the  last  case 
it  can  be  higher  than  1. 

Knowledge  as  to  the  extent  of  oxygen  consumption  is  of  special 
importance  in  the  calculation  of  the  energy  metabolism  from  the  extent 
of  gas  exchange,  and  one  can  under  some  circumstances  approximately 
calculate  the  energy  exchange  from  the  calorific  value  of  the  oxygen 
alone — with  regard  to  the  respiratory  quotient  (Zuntz  and  co-workers). 
The  calorific  value  of  oxygen  must  be  different  for  each  of  the  three  men- 
tioned foodstuffs,  as  they  require  different  quantities  of  oxygen  for  their 
combustion.  For  fat  and  carbohydrate  this  calorific  value  can  be  readily 
calculated,  as  these  bodies  are  completely  burnt  into  carbon  dioxide  and 
water.  One  gram  of  starch  uses  828.8  cc.  oxygen  in  its  combustion 
and  produces  828.8  cc.  carbon  dioxide,  and  4183  calories  of  heat  are 
developed.  For  one  liter  (  =  1.43  gram)  oxygen,  5047  calories  are  pro- 
duced, therefore  for  every  liter  (  =  1.966  gram)  carbon  dioxide  formed, 


888  METABOLISM. 

the  same  number,  5047  calories,  are  produced.  In  an  analogous  manner 
the  average  calorific  value  of  fat  for  1  liter  of  oxygen,  4686  calories,  and 
for  1  liter  carbon  dioxide,  6629  calories,  can  be  calculated. 

These  figures,  which  represent  the  physiological  combustion  values 
per  1  gram  of  food-stuffs,  derived  from  the  carbon  dioxide  output  or  the 
oxygen  in-take  in  (grams  or)  liters  which  are  represented  by  the  quotients 

'    or      „',  have  been  called  the  calorific  coefficients. 
L.CO2       L.O2 

With  proteins,  because  of  the  unequal  composition  of  the  different 
proteins,  the  results  are  uncertain  and  variable,  and  the  calculation  is 
much  more  complicated.  As  example  we  will  give  the  following  calcula- 
tion of  Zuntz  J  for  the  fat-free  dry  substance  of  meat. 

This  substance  consisted  in  100  parts 

52.38g.C.;  7.27g.H.;  22.68g.O.;  16.65g.N.;  1.02g.S. 
Of  which  were  found  in  the  urine.    9.406         2.663  14.099         16.28  1.02 

Of  which  were  found  in  the  feces .    1.471         0.212  0.889  0.37 

Retained 41.50C;     4.40H;         7.690;         0.0N;         0.0S. 

From  this  residue,  with  the  taking  up  of  96.63  liters  of  oxygen,  besides  39.6 
grams  water,  77.39  liters  carbon  dioxide  were  formed  and  the  respiratory  quotient 
is  therefore  0.801.  Now  100  grams  of  such  dry  meat  substance  on  complete 
combustion  yields  563.09  calories,  and  if  we  subtract  the  calorific  value  of  the 
corresponding  urine  (=113.70  calories)  and  feces  (  =  17.76  calories),  the  sum, 
131.46  calories,  then  431.63  calories  were  set  free  in  the  body.     For  every  gram 

of  nitrogen  eliminated  in  the  urine  (16.28  gram)  there  is  produced  -  „  ~q  =26.51. 

calories;    the  corresponding  quantity  of  oxygen  is       'n 0  =5.91  liter  O  and  the 

77.39 
corresponding  quantity  of  C02  produced  is      '„»  =4.75  liters  C02.     The  calorific 

cyo    r  -I 

value  for  1  liter  of  oxygen  consumed  is  therefore    _  Q.   =  4.485  calories,  and  for 

1  liter  of  carbon  dioxide  produced    ,  7C.  =  5.579  calories. 

For  milk  protein  Zuntz  has  calculated  for  1  gram  urea  nitrogen  5.8  liters 
oxygen,  4.6  liters  carbon  dioxide  and  27  calories.  The  calorific  value  can  be  cal- 
culated from  this  for  1  liter  0=4.66  and  for  1  liter  C02  =  5.87  calories.     If  we 

Cal. 
take  the  average  of  these  calculations  we  obtain  the  calorific  coefficients  T  rt'  =4.57 

L/.U2 

Cal. 
and  >  r<n  =5.73  for  protein. 

For  the  three  foodstuffs  we  have  the  following  calorific  values: 

Per  1  liter  Relative                Per  1  liter  Relative 

Oxygen.                value.  Carbon  dioxide.  value. 

Protein 4.57               100                   5.73  113.4 

Fat 4.69              102.6              6.63  131.3 

Carbohydrate 5 .  05              110. 5              5 .05  100 . 0 

1  Zuntz,  Loewy,  Miiller  and  Caspari,  Hohenklima  und  Bergwanderungen,  Berlin, 
1906,  pages  102,  103. 


CALCULATION  OF  THE  CALORIC  VALUE.         889 

The  figures  for  the  oxygen  vary  less  than  those  for  the  carbon  dioxide, 
and  this  is  a  reason  why  the  oxygen  values  are  better  suited  than  the 
CO2  values  for  calculating  the  energy  production  from  the  extent  of  gas 
exchange.  Other  investigators  have  obtained  results  which  correspond 
more  or  less  with  the  above  values  for  the  heat  value  of  oxygen,  and  E. 
Voit  and  Kummaciiek,1  who  have  made  calculations  in  another  way, 
have  obtained  still  smaller  differences  for  the  relative  oxygen  value. 

From  what  was  said  above  we  can  calculate  the  extent  of  protein 
metabolism,  the  corresponding  development  of  energy  and  the  correspond- 
ing absorption  of  oxygen  and  carbon  dioxide  formation,  from  the  quantity 
of  nitrogen  in  the  urine.  If  we  subtract  the  oxygen  and  carbon  dioxide 
values  from  the  total,  directly  determined  gas  exchange,  the  result  repre- 
sents the  fats  and  carbohydrates  used.  According  to  Zuntz  from  this 
residue  we  can  calculate  the  heat  value  of  the  oxygen  used  as  well  as  the 
division  of  the  decomposition  of  the  fat  and  carbohydrate  by  consider- 
ing the  respiratory  quotient.  For  this  purpose  Zuntz  and  Schumburg 
have  constructed  a  table,  an  abstract  of  which  we  give  below,  taken 
from  the  work  of  Magnus-Levy.2 

Division  in  per  cent. 

Carbohydrate.  Fat. 

100  0 

83  17 

66  34 

49  51 

32  68 

15  85 

0  100 

As  the  calorific  oxygen  values  in  the  combustion  of  protein,  fat 
and  carbohydrate  show  no  great  differences  among  themselves,  in  those 
cases  where,  as  in  starvation,  the  part  taken  by  the  proteins  in  the  total 
metabolism  is  relatively  small,  one  can  calculate  the  total  energy  exchange, 
without  any  striking  error,  from  the  respiratory  quotient  and  the  oxygen 
used.  This  is  especially  important  in  experiments  of  short  duration 
where  the  protein  metabolism  cannot  be  directly  determined,  but  is 
calculated  from  the  nitrogen  elimination  occurring  during  a  longer  time. 
The  method  of  Zuntz  and  Geppert,  mentioned  on  page  869,  has  shown 
itself  especially  useful  in  the  study  of  the  material  and  force  exchange 
in  these  experiments  of  short  duration,  while  the  respiration  apparatus 
constructed  on  Pettenkofer's  or  the  Regnault-Reiset  principle  are 
only  useful  in  experiments  over  a  longer  period. 

Kaufmann  '  incloses  the  individual  to  be  experimented  upon  in  a  capacious 
sheet-iron  room,  which  serves  both  as  a  respiration-chamber  and  a  calorimeter, 

1  Voit,  Zeitschr.  f.  Biol.,  44;  Kummacher,  ibid. 

■  A.  Magnus-Levy  in  v.  Noorden's  Handb.  d.  Pathol,  des  Stoffwechsels,  Bd.  1.  (1906). 

1  Arch.  d.  Physiologie  (5),  8. 


R.  Q. 

Calories  value 

per  1  liter  O. 

1.000 

5.047 

0.950 

4.986 

0.900 

4.924 

0.S50 

4.803 

0.800 

4.801 

0.750 

4.740 

0.707 

4.086 

890  METABOLISM. 

and  which  permits  the  estimation  of  the  nitrogen  of  the  urine  and  the  carbon 
dioxide  expired,  as  well  as  the  inspired  oxygen  and  the  quantity  of  heat  produced. 
If  we  start  from  the  theoretically  calculated  formulae  for  the  various  possible 
transformations  of  the  proteins,  fats,  and  carbohydrates  in  the  body,  it  is  clear 
that  other  values  must  be  obtained  for  the  heat,  carbon  dioxide,  oxygen,  and 
nitrogen  of  the  urine,  when  one,  for  example,  admits  of  a  complete  combustion 
of  proteins  to  urea,  carbon  dioxide,  and  water,  or  of  a  partial  splitting  off  of  fat. 
Another  relation  between  heat,  carbon  dioxide,  and  oxygen  is  also  to  be  expected 
when  the  fat  is  completely  burnt  or  when  it  is  decomposed  into  sugar,  carbon 
dioxide,  and  water.  In  this  way,  by  a  comparison  of  the  values  found  in  special 
cases  with  the  figures  calculated  for  the  various  transformations,  Kaufmann 
attempts  to  explain  the  various  decomposition  processes  in  the  body  under  dif- 
ferent nutritive  conditions. 

The  organic  foodstuffs  serve  in  part  to  replace  the  necessary  losses 
of  the  organs  and  in  part  as  sources  of  energy.  Under  all  circumstances 
a  restitution  of  the  protein-like  constituents  of  the  organs  is  necessary. 
This  replacement  is,  according  to  Rubner,  represented  by  the  so-called 
wear-and-tear  quota  (see  below)  which  amounts  to  about  4-6  per  cent  of 
the  total  energy  transformed  and  which  can  be  supplied  by  proteins  only. 
For  the  supply  of  the  remaining  exchange,  which  according  to  Rubner 
serves  as  source  of  energy,  all  three  groups  of  organic  foodstuffs  can  be 
used,  and  investigations  carried  out  by  Rubner  have  taught  that  these 
foodstuffs  can  act  as  sources  of  energy  in  the  animal  body  in  a  proportion 
which  corresponds  with  the  respective  figures  of  their  heat  value.  This 
is  apparent  from  the  following  table.  In  this  is  found  the  weight  of 
the  various  foods  equal  to  100  grams  of  fat,  a  part  determined  from 
experiments  on  animals  and  a  part  calculated  from  figures  of  the  heat 
values: 

From  Experiments  From  the  Difference, 

on  Animals.  Heat  Value.  per  cent. 

Syntonin 225  213  +5.6 

Muscle-flesh  (dried) ....  243  235  +4.3 

Starch 232  229  +1.3 

Cane-sugar 234  235  -0 

Glucose 256  255  -0 

From  the  given  isodynamic  value  of  the  various  foods  it  follows  that 
these  substances  replace  one  another  in  the  body  almost  in  exact  ratio 
to  the  energy  contained  in  them.  Thus  in  round  numbers  227  grams  of 
protein  and  carbohydrate  are  equal  to  or  isodynamic  with  100  grams  of 
fat  in  regard  to  source  of  energy,  because  each  yields  930  calories  on  com- 
bustion in  the  oody. 

By  means  of  recent  very  important  calorimetric  investigations,  Rub- 
ner !  has  shown  that  the  heat  produced  in  an  animal  in  several  series  of 
experiments  extending  over  forty-five  days  corresponded  to  within  0.47 
per  cent  of  the  physiological  heat  of  combustion  calculated  from  the  decom- 


Zeitschr.  f.  Biologie,  30. 


ISODYNAMICS  OF  FOOD-STUFFS.  891 

posed  body  and  foods.  Atwater  and  his  collaborators  x  have  made  some 
very  thorough  investigations  on  this  subject  on  men.  In  their  experi- 
ments they  made  use  of  a  large  respiration  calorimeter,  which  not  only 
exactly  determined  the  excreta,  but  also  made  a  calorimetric  determina- 
tion of  the  heat  given  out  by  the  person  experimented  upon,  i.e.,  the  work 
performed.  From  the  results  of  these  experiments  they  found  an  almost 
absolutely  complete  agreement  between  the  calories  found  directly  and 
those  calculated. 

This  isodynamic  law  is  of  fundamental  value  in  the  study  of  metabo- 
lism and  nutrition.  The  quantity  of  energy  in  the  transformed  foods 
or  the  constituents  of  the  body  may  be  used  as  a  measure  for  the  total 
consumption  of  energy,  and  the  knowledge  of  the  quantity  of  energy 
in  the  foods  must  also  be  the  basis  for  the  calculation  of  dietaries  for 
human  beings  under  various  conditions. 

The  isodynamic  theory  has  been  accepted  by  a  large  number  of  inves- 
tigators, but  not  by  all.  Certain  of  them,  especially  the  French,  accept 
an  isoglucosic  instead  of  the  isodynamic.  According  to  this  theory  the 
organism  for  its  physiological  functions  can  use  glucose  only,  and  as  a 
formation  of  glucose  is  possible  from  proteins  as  well  as  fats,  those  quan- 
tities of  food-stuffs  are  to  be  considered  as  equivalent  which  yield  an 
equal  amount  of  glucose. 

The  heat  value  of  a  foodstuff  can  be  directly  determined  in  a  calorim- 
eter, but  may  also  be  calculated  from  its  composition.  If  one  subtracts 
from  the  gross  heat  value  of  the  food  obtained  in  one  way  or  another 
the  combustion  heat  of  the  feces  and  urine  with  the  same  diet,  there  is 
obtained  the  net  calorific  value  of  the  diet.  This  value,  calculated  in 
percentage  of  the  total  energy  content  of  the  food,  is  called  the  physio- 
logical availability  by  Rubner.2  In  order  to  elucidate  this  we  will  give 
a  few  of  Rubner's  values.  The  loss  in  calories,  as  well  as  the  physio- 
logical availability,  is  calculated  in  percentages  of  the  total  energy 
content  of  the  food. 

p00(j  r  Loss  in  per  cent.  Total  loss       Availability 

In   Urine.        In  the  Feces,     in  per  cent,     in  per  cent. 

Cow's  milk 5.13  5.07  10.20  89.8 

Mixed  diet  (rich  in  fat) 3.87  5.73  9.60  90.4 

Mixed  diet  (poor  in  fat) 4.70  6.00  10.70  89.3 

Potatoes 2.00  5.60  7.60  92.4 

Graham  bread 2.40  15.50  17.90  82.1 

Rve  bread 2.20  24.30  26.50  73.5 

Meat 16.30  6.90  23.20  76.8 

In  order  to  simplify  the  calculation  of  the  energy  exchange  there  exist  other 
standard  factors,  besides  the  above-mentioned  standard  figures  for  the  physiological 

1  Bull,  of  Dept.  of  Agric,  Washington,  44,  63,  69,  and  109,  and  Ergebnisse  der 
Physiologie,  3. 

2  Zeitschr.  f.  Biologie,  42. 


892  METABOLISM. 

calorific  value  of  the  organic  foodstuffs,  also  for  the  carbon  of  the  carbon  dioxide, 
and  for  the  oxygen.  Thus  for  1  gram  of  meat  (dry  substance)  free  from  fat 
and  extractives  we  have  the  calculated  value  of  5.44-5.77  calories.  Kohler  x 
found  5.678  calories  for  1  gram  of  ash  and  fat-free  dried-meat  substance  of  the 
ox  and  5.599  calories  for  horse  meat.  According  to  Frentzel  and  Schreuer  2 
45.4  calories  is  calculated  for  1  gram  of  nitrogen  in  fat  and  ash-free  dried-meat 
feces  (dog),  while  6.97  to  7.45  calories  is  calculated  for  1  gram  of  nitrogen  in  meat- 

urine.     The  calorific  urine  quotient  — ^ —  seems  still,  as  above  given,  not  to  be 

constant  for  human  beings,  but  is  dependent  upon  the  variety  of  food. 


H.     METABOLISM  IN  STARVATION  AND  WITH  INSUFFICIENT  NUTRITION. 

In  starvation  the  decomposition  in  the  body  continues  uninterruptedly, 
though  with  decreased  intensity;  but,  as  it  takes  place  at  the  expense  of 
the  substance  of  the  body,  it  can  continue  for  a  limited  time  only.  When 
an  animal  has  lost  a  certain  fraction  of  the  mass  of  the  body,  death  is  the 
result.  This  fraction  varies  with  the  condition  of  the  body  at  the  begin- 
ning of  the  starvation  period.  Fat  animals  succumb  when  the  weight  of 
the  body  has  sunk  to  one-half  of  the  original  weight.  Otherwise,  accord- 
ing to  Chossat,3  animals  die  as  a  rule  when  the  weight  of  the  body  has 
sunk  to  two-fifths  of  the  original  weight.  The  period  when  death  occurs 
from  starvation  not  only  varies  with  the  varied  nutritive  condition  at  the 
beginning, of  starvation,  but  also  with  the  more  or  less  active  exchange 
of  material.  This  is  more  active  in  small  and  young  animals  than  in  large 
and  older  ones,  but  different  classes  of  animals  show  an  unequal  activity. 
Children  succumb  in  starvation  in  3-5  days  after  having  lost  one-fourth 
of  their  body  mass.  Grown  persons  may,  as  observed  upon  Succi,4  and 
other  professional  fasters,  starve  for  twenty  days  or  more  without  lasting 
injury;  and  there  are  reports  of  cases  of  starvation  extending  over  a 
period  of  even  more  than  forty  to  fifty  days.  Dogs  may  starve,  accord- 
ing to  several  observers,  50-60  days.  Hawk5  and  co-workers  have 
recorded  a  case  where  a  dog  was  starved  for  117  days  and  lost  about 
63  per  cent  of  its  original  weight.  Snakes  and  frogs  can  starve  for  one- 
half  a  year  or  even  a  whole  year. 

In  starvation  the  weight  of  the  body  decreases.  The  loss  of  weight  is 
greatest  in  the  first  few  days,  and  then  decreases  rather  uniformly.  In 
small  animals  the  absolute  loss  of  weight  per  day  is  naturally  less  than 
in  larger  animals.     The  relative  loss  of  weight — that  is,  the  loss  of  weight 

1  Zeitschr.  f.  physiol.  Chem.,  31. 

2  The  works  of  Frentzel  and  Schreuer  may  be  found  in  Arch.  f.  (Anat.  u.)  Physiol., 
1901,  1002,  and  L903. 

3  Cited  from  \'<>it  in  Hermann's  Handbuch,  6,  Thl.  1,  100. 
*  See  Luciani,  Das  Hungern.     Hamburg  u.  Leipzig,  1890. 

6  P.  B.  Hawk,  P.  E.  Howe,  and  H.  A.  Mattil,  Journ.  of  biol.  Chem.,  11. 


METABOLISM  IN  STARVATION.  893 

of  the  unit  of  the  weight  of  the  body,  namely,  1  kilo — is,  on  the  contrary, 
greater  in  small  animals  than  in  larger  ones.  The  reason  for  this  is  that 
the  smaller  animals  have  a  greater  surface  of  body  in  proportion  to  their 
mass  than  larger  animals,  and  the  greater  loss  of  heat  caused  thereby 
must  be  replaced  by  a  more  active  consumption  of  material. 

It  follows  from  the  decrease  in  the  weight  of  the  body  that  the  absolute 
extent  of  metabolism  must  diminish  in  starvation.  If,  on  the  contrary, 
the  extent  of  metabolism  is  referred  to  the  unit  of  weight  of  the  body, 
namely,  1  kilo,  it  appears  that  this  quantity  remains  almost  un- 
changed during  starvation.  The  investigations  of  Zuntz,  Lehmann, 
and  others,1  on  the  professional  faster  Cetti,  showed  on  the  third  and 
sixth  days  of  starvation  an  average  consumption  of  4.65  cc.  oxygen  per 
kilo  in  one  minute,  and  on  the  ninth  to  eleventh  day  an  average  of  4.73 
cc.  The  calories,  as  a  measure  of  the  metabolism,  fell  on  the  first  to 
fifth  day  of  starvation  from  1850  to  1G00  calories,  or  from  32.4  to  30  per 
kilo,  and  it  remained  nearly  unchanged,  if  referred  to  the  unit  of  body 
weight.2  In  man  the  average  daily  energy  consumption  in  starvation 
amounts  to  about  30-32  calories  per  kilo. 

The  extent  of  the  metabolism  of  proteins,  or  the  elimination  of  nitrogen 
by  the  urine,  which  is  a  measure  of  the  same,  diminishes  as  the  weight 
of  the  body  diminishes.  This  decrease  is  not  regular  or  the  same  during 
the  entire  period  of  starvation,  and  the  extent  depends,  as  the  experi- 
ments made  upon  carnivora  have  shown,  upon  several  circumstances. 
During  the  first  few  days  of  starvation  the  excretion  of  nitrogen  is  greatest, 
and  the  richer  the  body  is  in  protein,  due  to  the  food  previously  taken, 
the  greater  is  the  protein  catabolism  or  the  nitrogen  elimination,  accord- 
ing to  Voit.  The  nitrogen  elimination  diminishes  the  more  rapidly — 
that  is,  the  curve  of  the  decrease  is  more  sudden — the  richer  in  proteins 
the  food  was  which  was  taken  before  starvation.  This  condition  is 
apparent  from  the  following  table  of  data  of  three  different  starvation 
experiments  made  by  Voit3  on  the  same  dog.  This  dog  received  2500 
grams  of  meat  daily  before  the  first  series  of  experiments,  1500  grams  of 
meat  daily  before  the  second  series,  and  a  mixed  diet  relatively  poor  in 
nitrogen  before  the  third  series. 

Day  of  Starvation.  grams  of  Urea  Eliminated  in  Twenty-four  Hgurs.^ 

First 60.1  '  26.5   '  13.8 

Second 24.9  18.6  11.5 

Third 19.1  15.7  10.2 

Fourth 17.3  14.9  12.2 

Fifth 12.3  14.8  12.1 

Sixth 13.3  12.8  12.6 

Seventh 12.5  12.9  11.3 

Eighth 10.1  12.1  10.7 

1  Berlin,  klin.  Wochenschr.,  1887. 

2  See  also  Tigerstedt  and  collaborators  in  Skand.  Arch.  f.  Physiol.,  7. 
1  See  Hermann's  Handbuch,  6,  Thl.  1,  89. 


894  METABOLISM. 

In  man  and  also  in  animals  sometimes  a  rise  in  the  nitrogen  excretion 
is  observed  about  the  second  or  third  starvation  day,  which  is  then  fol- 
lowed by  a  regular  diminution.  This  rise  is  explained  by  Prausnitz, 
Tigerstedt,  Landergren,1  as  follows:  At  the  commencement  of  star- 
vation the  protein  metabolism  is  reduced  by  the  glycogen  still  present 
in  the  body.  After  the  consumption  of  the  glycogen,  which  takes  place 
in  great  part  during  the  first  days  of  starvation,  the  destruction  of  pro- 
teins increases  as  the  glycogen  action  decreases,  and  then  decreases  again 
when  the  body  has  become  poorer  in  available  proteins. 

Other  conditions,  such  as  varying  quantities  of  fat  in  the  body,  have 
an  influence  on  the  rapidity  with  which  the  nitrogen  is  eliminated  during 
the  first  days  of  starvation.  After  the  first  few  days  of  starvation  the 
elimination  of  nitrogen  is  more  uniform.  It  may  diminish  gradually 
and  regularly  until  the  death  of  the  animals  or  there  may  be  a  rise  in  the 
last  days,  a  so-called  premortal  increase.  Whether  the  one  or  the  other 
occurs  depends  upon  the  relation  between  the  protein  and  fat  content 
of  the  body. 

Like  the  destruction  of  proteins  during  starvation,  the  decomposi- 
tion of  fat  proceeds  uninterruptedly,  and  the  greatest  part  of  the'  calories 
needed  during  starvation  are  supplied  by  the  fats.  According  to  Rubner 
and  Voit  the  protein  catabolism  varies  only  slightly  in  starving  animals 
at  rest  and  at  an  average  temperature,  and  forms  a  constant  portion 
of  the  total  exchange  of  energy;  of  the  total  calories  in  dogs  10-16  per 
cent  comes  from  the  protein  decomposition  and  84-90  per  cent  from  the 
fats.  This  is  at  least  true  for  starving  animals  which  had  a  sufficiently 
great  original  fat  content.  If  on  account  of  starvation  the  animal  has 
become  relatively  poorer  in  fat  and  the  fat  content  of  the  body  has  fallen 
below  a  certain  limit,  then  in  order  to  supply  the  calories  necessary,  a 
larger  quantity  of  protein  is  destroyed  and  the  premortal  increase  now 
occurs  (E.  Voit).  The  reason  for  this  premortal  rise  in  protein  catabol- 
ism is  still  not  completely  understood   (Schulz   and   collaborators2). 

Since  the  fat  has  a  diminishing  influence  on  the  destruction  of  pro- 
teins corresponding  to  what  was  said  above,  the  elimination  of  nitrogen 
in  starvation  is  less  in  fat  than  in  lean  individuals.  For  instance,  only 
9  grams  of  urea  were  voided  in  twenty-four  hours  during  the  later  stages 
of  starvation  by  a  well-nourished  and  fat  person  suffering  from  disease 
of  the  brain,  while  I.  Munk  found  that  20-29  grams  urea  were  voided 
daily  by  Cetti,3  who  had  been  poorly  nourished. 

'Prausnitz,  Zeitsohr.  f.  Biologie,  29;  Tigerstedt  and  collaborators,  1.  c;  Landergren, 
Undersokningar  ofver  menniskans  agghviteomsattning,  Inaug.-Diss.  Stockholm,. 
1902. 

2  Voit,  Zeitschr.  f.  Biologie,  41;  167  and  502.  See  also  Kaufmann,  ibid.,  and  N_ 
Schulz,  ibid.,  and  Pflu^-r's  Arch.,  76,  with  Mangold,  Stubel  and  Hempel,  ibid.,  114. 

»  Berl.  klin.  Wochenschr.,  J887. 


METABOLISM   IN  STARVATION.  895 

The  investigations  on  the  exchange  of  gas  in  starvation  have  shown, 
as  previously  mentioned,  that  its  absolute  extent  is  diminished,  hut 
that  when  the  consumption  of  oxygen  and  elimination  of  carbon  dioxide 
are  calculated  on  the  unit  weight  of  the  body,  1  kilo,  this  quantity 
quickly  sinks  to  a  minimum  and  then  remains  unchanged,  or,  on  the 
continuation  of  the  starvation,  may  actually  rise.  It  is  a  well-known 
fact  that  the  body  temperature  of  starving  animals  remains  almost  con- 
stant, without  showing  any  appreciable  decrease,  during  the  greater 
part  of  the  starvation  period.  The  temperature  of  the  animal  first  sinks 
a  few  days  before  death,  which  occurs  at  about  33-30°  C. 

From  what  has  been  said  about  the  respiratory  quotient  it  follows 
that  in  starvation  it  is  about  the  same  as  with  fat  and  meat  exclusively 
as  food,  i.e.,  approximately  0.7.  This  is  often  the  case,  but  it  may  occa- 
sionally be  lower,  0.65-0.50,  as  observed  in  the  cases  of  Cetti  and  Succi. 
This  can  be  explained  by  an  elimination  of  acetone  bodies  by  the  urine; 
a  part  can  be  accounted  for  perhaps  by  a  formation  and  deposition  of 
glycogen  from  protein. 

Water  passes  uninterruptedly  from  the  body  in  starvation  even  when 
none  is  taken.  If  the  quantity  of  water  in  the  tissues  rich  in  proteins 
is  considered  as  70-80  per  cent,  and  the  quantity  of  proteins  in  them 
20  per  cent,  then  for  each  gram  of  protein  destroyed  about  4  grams  of 
water  are  set  free.  This  liberation  of  water  from  the  tissues  is  generally 
sufficient  to  supply  the  loss  of  water,  and  starvation  is  ordinarily  not 
accompanied  with  thirst. 

The  loss  of  water  calculated  on  the  percentage  of  the  total  organism  must 
naturally  be  essentially  dependent  upon  the  previous  amount  of  fatty  tissue  in  the 
body.  In  certain  cases  the  starving  animal  body  has  indeed  been  found  richer 
in  water;  but  if  we  bear  these  conditions  in  mind,  then,  it  seems,  according  to 
Bohtlingk,1  that,  from  experiments  upon  white  mice,  the  animal  body  is  poorer 
in  water  during  inanition.  The  body  loses  more  water  than  is  set  free  by  the 
destruction  of  the  tissues. 

The  mineral  substances  leave  the  body  uninterruptedly  in  starvation 
until  death,  and  the  influence  of  the  destruction  of  tissues  is  plainly 
perceptible  by  their  elimination.  Because  of  the  destruction  of  tissues 
rich  in  potassium  the  proportion  between  potassium  and  sodium  in 
the  urine  changes  in  starvation,  so  that,  contrary  to  the  normal  condi- 
tions, the  potassium  is  eliminated  in  proportionately  greater  quantities. 

Contrary  to  the  above  Bohtlingk  with  starving  white  mice,  and  Katsuyama  ! 
with  starving  rabbits  found  a  greater  excretion  of  sodium  than  potassium. 


1  Arch,  des  sciences  biol.  de  St.  Petersbourg,  5. 

*  Bohtlingk,  1.  c;  Katsuyama,  Zeitschr.  f.  physiol.  Chem.,  26. 


896 


METABOLISM. 


Munk  observed,  in  Cetti's  case,  an  increase  in  the  elimination  of 
phosphoric  acid  in  relation  to  the  iV-elimination,  which  indicates  an 
increased  decomposition  of  bone-substance,  and  this  explanation  is 
supported  by  the  fact  that  a  simultaneous  increase  in  the  elimination  of 
lime  and  magnesia  occurs.  Recently  Wellmann  *  showed  that  in  rabbits, 
the  increase  in  the  elimination  of  phosphorus,  calcium  and  magnesium 
in  starvation  corresponds  to  the  loss  in  the  bones  of  these  constituents. 

The  question  as  to  the  participation  of  the  different  organs  in  the  loss 
of  weight  of  the  body  during  starvation  is  of  special  interest.  In  elucida- 
tion of  this  point  we  give  the  following  results  of  Chossat's  experiments 
on  pigeons,  and  those  of  Voit  2  on  a  male  cat.  The  results  are  percentages 
of  weight  lost  from  the  original  weight  of  the  organ. 


Pigeon  (Chossat). 

Adipose  tissue 93  per  cent. 

Spleen 71 

Pancreas 64 

Liver 52 

Heart 45 

Intestine 42 

Muscles 42 

Testicles — 

Skin 33 

Kidneys 32 

Lungs 22 

Bones 17 

Nervous  system 2 


Male  Cat  (Voit). 
97  per  cent. 
67 
17 
54 

3 
18 
31 
40 
21 
26 
18 

4 

3 


The  total  quantity  of  blood,  as  well  as  the  quantity  of  solids  contained 
therein,  decreases,  as  Panum  and  others3  have  shown,  in  the  same  pro- 
portion as  the  weight  of  the  body.  Concerning  the  loss  of  water  by 
different  organs  authorities  disagree,  Lukjanow4  claiming  that  the 
various  organs  differ  from  each  other  in  this  respect. 

The  above-tabulated  results  cannot  serve  as  a  measure  of  the  metabol- 
ism in  the  various  organs  during  starvation.  For  instance,  the  nervous 
system  shows  only  a  small  loss  of  weight  as  compared  with  the  other 
organs,  but  from  this  it  must  not  be  concluded  that  the  exchange  of 
material  in  this  system  of  organs  is  least  active.  The  conditions  may  be 
quite  different;  for  one  organ  may  derive  its  nutriment  during  starva- 
tion from  some  other  organ  and  exist  at  its  expense.  A  positive  con- 
clusion cannot  be  drawn  in  regard  to  the  activity  of  the  metabolism  in 
an  organ  from  the  loss  of  weight  of  that  organ  in  starvation.  Death 
by  starvation  is  not  the  result  of  the  death  of  all  the  organs  of  the  body, 


1  Munk,  Berl.  klin.  Wbchenschr.,  1887;  Wellmann,  Pfliiger's  Arch.,  121. 

2  Cited  from  Voit  in  Hermann's  Handbwh,  fi.  Part  I,  96  and  97. 

5  Panum,  Virchow's  Arch.,  29;  London,  Arch.  d.   scienc.  biol.  de  St.  P£tersbourg,  4.. 
•  Zeitechr.  f.  physiol.  Chem.,  13. 


METABOLISM  IN  STARVATION.  897 

but  it  depends  more  likely  upon  the  disturbance  in  the  nutrition  of  a  few 
less  vitally  important  organs  (E.  Voit  1). 

In  calculating  or  determining  the  loss  of  weight  of  the  organs  in 
starvation  the  original  fat  content  of  the  organs  must  be  considered. 
With  the  consideration  of  the  fat  content  of  the  organs,  determined  or 
estimated  in  a  special  way  before  the  starvation  period  and  at  the  end, 
E.  Voit2  found  the  following  loss  of  weight  in  the  supposed  fat-free 
organs  in  starvation,  namely,  muscles  41  per  cent,  viscera  42  per  cent, 
skin  28  per  cent,  and  skeleton  5  per  cent. 

The  quantity  of  urine  nitrogen  sinks  in  starvation  corresponding  to 
the  protein  catabolism,  but  to  a  varying  degree  in  different  individuals. 
The  lowest  value  observed  thus  far  in  man  was  2.82  grams  per  diem  as 
found  by  E.  and  0.  Freund  on  the  twenty-first  day  in  the  faster  Succi. 
Calculated  on  1  kilogram  of  body  weight,  the  urine  nitrogen,  as  is  to  be 
expected,  shows  striking  differences  in  different  persons;  in  Cetti  and 
Succi  it  was  0.150-0.200  gram  on  the  fifth  to  tenth  day  of  starvation. 
The  division  of  the  nitrogen  in  the  urine  in  starvation  is  unl'.ke  that  in 
the  normal  condition.  The  relative  amount  cf  urea  diminishes,  as 
shown  by  E.  and  ().  Freund,  Brugsch  and  Cathcart,3  so  that  instead 
of  being  about  85  per  cent  of  the  total  nitrogen  under  normal  conditions 
it  can  sink  to  54  per  cent  (Brugsch).  At  the  same  time  because  of  the 
abundant  formation  of  acetone  bodies  (starvation  acidosis)  the  quantity 
of  ammonia  increases  considerably  (Brugsch,  Cathcart).  A  relative 
increase  in  the  neutral  sulphur  of  the  urine  also  takes  place  (Benedict, 
Cathcart4).  Creatine  also  occurs  in  starvation  urine  and  according 
to  Hawk  5  and  co-workers  the  elimination  of  creatine  is  much  greater  than 
the   creatinine   a  few   days  before  the  premortal  nitrogen  elimination. 

One  must  differentiate  between  the  real  starvation  metabolism  and  the 
metabolism  in  the  inanition  condition,  the  basal  requirement  (Magnus- 
Levy)  or  the  maintenance  value  (Loewy  6).  With  this  we  understand  the 
metabolism  in  uniform,  medium  temperature,  with  absolute  bodily  rest 
and  inactivity  of  the  intestinal  canal.  As  a  measure  of  this  we  deter- 
mine the  gas  exchange  in  a  person  lying  down  with  as  perfect  com- 
plete muscular  rest  as  possible,  or  sleeping  in  the  early  morning  and 
at  least  twelve  hours  after  a  light  meal  not  rich  in  carbohydrates.     This 


1  Zeitschr.  f.  Biologie,  41. 

2  Ibid.,  46. 

J  E.  and  O.  Freund,  Wien.  klin.  Rundschau,  1901,  Nos.  5  and  G;  Brugsch,  Zeitschr. 
f.  exp.  Path.  u.  Therap.,  1  and  3;  Cathcart,  Bioch.  Zeitschr.,  6. 

4  Zeitschr.  f.  klin.  Med.,  36;  Cathcart,  1.  c. 

5  Journ.  of  biol.  Chem.,  11. 

6  Magnus-Levy  in  v.  Noorden's  Handbuch,  and  Loewy  in  Oppenheimer's  Handbuch 
d.  Biochemie,  Bd.  4. 


898  METABOLISM. 

basal  requirement  is  the  measure  of  the  energy  necessary  for  the  per- 
formance of  all  the  functions  necessary  to  maintain  life  during  rest;  and 
all  work  above  this  minimum  activity  is  called  productive  increase  by 
Magnus-Levy.  The  basal  requirement  is  almost  constant  for  the  same 
individual  and  serves  as  the  starting  point  in  the  study  of  the  action  of 
different  influences  such  as  work,  food,  diseased  conditions,  etc.,  upon 
metabolism.  The  extent  of  this  basal  requirement,  as  determined  by 
the  gas  exchange  according  to  the  Zuntz-Geppert  method,  and  by 
Johansson  l  and  collaborators  amounts  in  men  of  60-70  kilos  body 
weight  to  about  220-250  cc.  oxygen  and  160-200  cc.  carbon  dioxide  per 
minute,  which  equals  20-24  grams  carbon  dioxide  per  hour.  Johansson 
found  in  forced  complete  muscular  rest  20.7  grams  CO2  per  hour  and  24.8 
grams  CO2  in  the  ordinary  resting.  Gigon  2  found  about  23.4  grams 
CO2  and  21  grams  oxygen  for  the  basal  requirement.  According  to  Mag- 
nus-Levy the  total  daily  metabolism  can  be  calculated  for  the  basal 
requirement  as  1625  calories,  or  including  the  rise  due  to  the  partaking 
of  food  as  1800  calories.  According  to  Gigon  the  basal  requirement 
consists  of  15.22  per  cent  protein,  15-35.2  per  cent  carbohydrates  and 
44.5-70  per  cent  fat. 

The  food  may  be  quantitatively  insufficient,  and  the  final  result  of 
this  is  absolute  inanition.  The  food  may  also  be  qualitatively  insufficient 
or,  as  we  say,  inadequate.  This  occurs  when  any  of  the  necessary 
nutritive  bodies  are  absent  in  the  food,  while  the  others  occur  in  sufficient 
or  perhaps  even  in  excessive  amounts. 

Lack  of  Water  in  the  Food.  The  quantity  of  water  in  the  organism  is 
greatest  during  fcetal  life  and  then  decreases  with  increasing  age.  Nat- 
urally, the  quantity  differs  essentially  in  different  organs.  The  enamel, 
with  only  2  p.  m.  water,  is  the  tissue  poorest  in  water,  while  the  teeth 
contain  about  100  p.  m.  and  the  fatty  tissue  60-120  p.  m.  water.  The 
bones,  with  140-440  p.  m.,  and  the  cartilage  with  540-740  p.  m.  are 
somewhat  richer  in  water,  while  the  muscles,  blood  and  glands,  with  750 
to  more  than  800  p.  m.,  are  still  richer.  The  quantity  of  water  is  even 
greater  in  the  animal  fluids  (see  preceding  chapter),  and  the  adult  body 
contains  in  all  about  630  p.  m.  water.3  It  .follows  from  what  has  been 
given  in  Chapter  I  in  regard  to  the  great  importance  of  water  for  living 
processes,  that  if  the  loss  of  water  is  not  replaced  by  fresh  supply,  the 
organism  must  succumb  sooner  or  later.  Death  occurs  indeed  sooner 
from  lack  of  water  than  from  complete  inanition  (Landauer,  Nothwang). 


1  The  literature  can  be  found  in  the  works  of  Magnus-Levy  and  Loewy. 
1  Johansson,   Skand.  Arch.  f.  Physiol.,  7,  8,  21,  and   Nord.    Med.  Arch.  Festband, 
1897;  see  also  Magnus-Levy;  Gigon,  Pfluger's  Arch.,  140. 
*  See  Voit,  in  Hermann's  Handbuch,  6,  part  1,  345. 


LACK  OF  MINERAL  SUBSTANCES.  899 

If  water  is  withdrawn  for  a  certain  time,  as  Landauer  and  espe- 
cially Straub  have  shown,  it  has  an  accelerating  influence  upon  the 
decomposition  of  protein.  This  increased  destruction  has,  according  to 
Landauer,  the  purpose  of  replacing  a  part  of  the  water  removed,  by  the 
production  of  water  by  means  of  the  increased  metabolism.  The  depriva- 
tion of  water  for  a  short  time  may,  according  to  Spiegler,1  especially  in 
man,  cause  a  diminution  in  the  protein  metabolism  by  means  of  a  reduced 
protein  absorption. 

Lack  of  Mineral  Substances  in  the  Food.  In  the  previous  chapters 
attention  has  repeatedly  been  called  to  the  importance  of  the  mineral 
bodies  and  also  to  the  occurrence  of  certain  mineral  substances  in  certain 
amounts  in  the  various  organs.  The  mineral  content  of  the  tissues  and 
fluids  is  not  very  great  as  a  rule.  With  the  exception  of  the  skeleton, 
which  contains  as  average  about  220  p.  m.  mineral  bodies  (Volkmann2), 
the  animal  fluids  or  tissues  are  poor  in  inorganic  constituents,  and  the 
quantity  of  these  amounts  as  a  rule,  only  to  about  10  p.  m.  Of  the 
total  quantity  of  mineral  substances  in  the  organism,  the  greatest  part 
occurs  in  the  skeleton,  830  p.  m.,  and  the  next  greatest  in  the  muscles, 
about  100  p.  m.  (Volkmann). 

The  mineral  bodies  seem  to  be  partly  dissolved  in  the  fluids  and  partly 
combined  with  organic  substances,  but  nothing  definite  can  be  given  as 
to  the  kind  of  combination,  or  whether  they  occur  in  stoichiometric 
proportions,  or  whether  they  are  simply  adsorption  combinations.  In 
accordance  with  this  the  organism  persistently  retains,  with  food  poor 
in  salts,  a  part  of  the  mineral  substances,  also  such  as  are  soluble,  as  the 
chlorides.  On  the  burning  of  the  organic  substances  the  mineral  bodies 
combined  therewith  are  set  free  and  may  be  eliminated.  It  is  also 
admitted  that  they  in  part  combine  with  the  new  products  of  the  com- 
bustion, and  in  part  with  organic  nutritive  bodies  poor  in  salts  or  nearly 
salt-free,  which  are  absorbed  from  the  intestinal  canal  and  are  thus  retained 
(Voit,  Forster3). 

If  this  statement  is  correct,  it  is  possible  that  a  constant  supply  of 
mineral  substances  with  the  food  is  not  absolutely  necessary,  and  that  the 
amount  of  inorganic  bodies  which  must  be  administered  is  insignificant. 
The  question  whether  this  is  so  or  not  has  not,  especially  in  man,  been 
sufficiently  investigated;    but  generally  we  consider  the  need  of  mineral 

1  Landauer,  Maly's  Jahresber.,  24;  Nothwang,  Arch.  f.  Hyg.,  1892;  Straub,  Zeitschr. 
f.  Biol.,  37  and  38;  Spiegler,  ibid.,  41. 

2  See  Hermann's  Handbuch.,  6,  pt.  1,  353. 

3  Forster,  Zeitschr.  f.  Biologie,  9.  See  also  Voit,  in  Hermann's  Handbuch,  6, 
Part  1,  354.  In  regard  to  the  occurrence  and  the  behavior  of  the  various  mineral 
constituents  of  the  animal  body  see  the  work  of  Albu  and  Neuberg,  Physiologie  und 
Pathologie  des  Mineralstoffwechsel,  Berlin,  1906. 


900  METABOLISM. 

substances  by  man  as  very  small.  It  may,  however,  be  assumed  that 
man  usually  takes  with  his  food  a  considerable  excess  of  mineral  sub- 
stances. 

Experiments  to  determine  the  results  of  an  insufficient  supply  of 
mineral  substances  with  the  food  in  animals  have  been  made  by  several 
investigators,  especially  Fcrster.  He  observed,  on  experimenting  with 
dogs  and  pigeons  with  food  as  poor  as  possible  in  mineral  substances, 
that  a  very  suggestive  disturbance  of  the  functions  of  the  organs,  par- 
ticularly the  muscles  and  the  nervous  system,  appeared,  and  that  death 
resulted  in  a  short  time,  earlier  in  fact  than  in  complete  starvation.  On 
observations  made  upon  himself,  Taylor  l  found  on  partaking  less  than 
0.1  gram  salts  per  diem  that  the  chief  disturbance  occurred  in  the  mus- 
cular system. 

Bunge  in  opposition  to  these  observations  of  Forster's  has  suggested 
that  the  early  death  of  these  cases  was  not  caused  by  the  lack  of  mineral 
salts,  but  more  likely  by  the  lack  of  bases  necessary  to  neutralize  the  sul- 
phuric acid  formed  in  the  combustion  of  the  proteins  in  the  organism; 
these  bases  must  then  be  taken  from  the  tissues.  In  accordance  with 
this  view,  Bunge  and  Lunin2  also  found,  in  experimenting  with  mice, 
that  animals  which  received  nearly  ash-free  food  with  the  addition  of 
sodium  carbonate  were  kept  alive  twice  as  long  as  those  which  had  the 
same  food  without  the  sodium  carbonate.  Special  experiments  also 
show  that  the  carbonate  cannot  be  replaced  by  an  equivalent  amount  of 
sodium  chloride,  and  that  to  all  appearances  it  acts  by  combining  with 
the  acids  formed  in  the  body.  The  addition  of  alkali  carbonate  to  the 
otherwise  nearly  ash-free  food  may  indeed  delay  death,  but  cannot  pre- 
vent it,  and  even  in  the  presence  of  the  necessary  amount  of.  bases  death 
results  from  lack  of  mineral  substances  in  the  food. 

With  an  insufficient  supply  of  chlorides  with  the  food  the  elimination 
of  chlorine  by  the  urine  decreases  constantly,  and  at  last  it  may  stop 
entirely,  while  the  tissues  still  persistently  retain  the  chlorides.  It  has 
already  been  stated  (Chapter  VIII)  how  chloride  starvation  influences 
other  functions,  especially  the  secretion  of  gastric  juice.  If  there  be  a 
lack  of  sodium  as  compared  with  potassium,  or  if  there  be  an  excess  of 
potassium  compounds  in  any  other  form  than  KC1,  the  potassium  com- 
binations are  replaced  in  the  organism  by  NaCl,  so  that  new  potassium 
and  sodium  compounds  are  produced  which  are  voided  with  the  urine. 
The  organism  becomes  poorer  in  NaCl,  which  therefore  must  be  taken 
in  greater  amounts  from  the  outside  (Bunge).     This  occurs  continuously 


1  University  of  California  Publications,  Pathol.,  1. 

2  Bunge,  Lehrbuch  der  physiol.  Chem.,  4.  Aufl.,  97;  Lunin,  Zeitschr.  f.  physiol. 
Chem.,  5. 


ALKALI  CARBONATES.  PHOSPHATES  AND  EARTHS.    (J01 

in  herbivora,  and  in  man  with  vegetable  food  rich  in  potash.  For  human 
beings,  and  especially  for  the  poorer  classes  of  people  who  live  chiefly 
on  potatoes  and  foods  rich  in  potash,  common  salt  is  not  only  a  condi- 
ment, but  a  necessary  addition  to  the  food  (Bunge  l).  On  the  behavior 
of  chlorides,  especially  sodium  chloride,  in  the  animal  body  as  well  as  the 
elimination  or  the  retention  of  NaCl  in  diseases,  we  have  an  abundance 
of  investigations,  which  may  be  found  in  Albu  and  Neuberg's2  work, 
previously  cited. 

Lack  of  Alkali  Carbonates  or  Bases  in  the  Food.  The  chemical  processes 
in  the  organism  are  dependent  upon  the  presence  in  the  tissues  and  tissue- 
fluids  of  a  certain  reaction,  and  this  reaction,  which  is  habitually  alkaline 
toward  litmus  and  neutral  toward  phenolphthalein,  is  chiefly  due  to  the 
presence  of  alkali  carbonates  and  carbon  dioxide  and  in  a  lesser  degree  to 
alkali  phosphates.  The  alkali  carbonates  are  also  cf  great  importance, 
not  only  as  a  solvent  for  certain  protein  bodies  and  as  constituents  of 
certain  secretions,  such  as  the  pancreatic  and  intestinal  juices,  but  they 
are  also  a  means  of  transportation  of  the  carbon  dioxide  in  the  blood. 
It  is  therefore  easy  to  understand  that  a  decrease  below  a  certain  point 
in  the  quantity  of  alkali  carbonate  must  endanger  life.  Such  a  decrease 
not  only  occurs  with  lack  of  bases  in  the  food  which  brings  about  various 
disturbances  and  death  by  a  relatively  great  production  of  acids  through 
the  burning  of  the  proteins,  but  it  also  occurs  when  an  animal  is  given 
dilute  mineral  acids  for  a  period.  The  importance  of  ammonia  as  a 
means  of  neutralizing  the  acids  produced  or  introduced  into  the  body 
as  well  as  the  unequal  resistance  of  man  and  other  animals  toward  this 
action  of  acids  has  already  been  discussed  in  Chapter  XIV. 

Lack  of  Phosphates  and  Earths.  With  the  exception  of  the  value  of 
the  alkaline  earths  as  carbonates  and  more  especially  as  phosphates  in 
the  physical  composition  of  certain  structures,  such  as  the  bones  and 
teeth,  their  physiological  importance  is  almost  unknown.  The  importance 
of  calcium  for  certain  enzymotic  processes  and  of  calcium  ions  for  the 
functions  of  the  muscles,  and  especially  for  cell  life,  gives  an  indication  of 
the  necessity  of  the  alkaline  earths  to  the  animal  organism.  Little  is 
known  of  the  need  of  these  earth  in  adults,  and  no  average  results  can 
be  given.  According  to  Kochmann  and  Petzsch  3  we  cannot  conceive 
of  a  certain  calcium  minimum  (in  dogs)  as  the  Ca  needs  vary  with  dif- 
ferent foods.  With  a  Ca  equilibrium  we  can  cause  an  increased  elimi- 
nation of  calcium  by  increasing  the  quantity  of  protein,  of  fat,  or  of 
carbohydrate  in  the  food  and  this  probably  depends  upon  a  giving  up 


1  Zeitschr.  f .  Biologie,  9. 

*  See  footnote  3,  page  899. 

*  Kochmann,  Bioch.  Zeitschr.,  31,  with  Petzsch,  ibid.,  32. 


902  METABOLISM. 

of  calcium  phosphate  by  the  skeleton.  It  is  impossible  to  give 
positive  figures  for  the  need  of  phosphates  or  phosphoric  acid,  whose 
value  is  recognized  not  only  in  the  construction  of  the  bones,  but  also' 
in  the  functions  of  the  muscles,  the  nervous  system,  the  glands,  the 
organs  of  generation,  etc.  The  extent  of  this  need  is  most  difficult  to 
determine,  as  the  body  shows  a  strong  tendency,  when  increased  amounts 
of  phosphorus  are  introduced,  to  retain  more  than  is  necessary.  The 
need  of  phosphates,  which,  according  to  Ehrstrom,1  corresponds  in  adults 
to  a  minimum  of  1  to  2  grams  phosphorus,  is  relatively  smaller  in  adults 
than  in  young,  developing  animals,  and  in  these  latter  the  question  of 
the  result  of  an  insufficient  supply  of  earthy  phosphates  and  alkaline 
earths  upon  the  bone  tissue  is  of  special  interest.  For  details  we  refer 
to  Chapter  IX  and  to  the  cited  work  of  Albu-Neuberg. 

Another  important  question  is,  How  far  do  the  phosphates  take  part 
in  the  construction  of  the  phosphorized  constituents  of  the  body  or  to 
what  extent  are  they  necessary?  The  experiments  of  Rohmann  and  his 
pupils 2  with  phosphorized  (casein,  vitellin)  and  non-phosphorized  pro- 
teins (edestin)  and  phosphates  show  that  with  the  introduction  of  casein 
and  vitellin  a  deposition  of  nitrogen  and  phosphorus  takes  place,  while 
with  non-phosphorized  protein  and  phosphates  this  does  not  seem  to 
occur.  The  body  apparently  does  not  have  the  power  of  building  up 
the  phosphorized  cell  constituents  necessary  for  cell  life  from  non-phos- 
phorized proteins  and  phosphates.  On  the  contrary,  according  to  the 
observations  of  several  investigators,  the  lecithins  seem  to  possess  this 
power.  As  known  from  the  investigations  of  Meischer,  the  develop- 
ment of  the  generative  organs  of  the  salmon,  which  are  very  rich  in  nuclein 
substances  and  phosphatides,  from  the  muscles  which  are  relatively  poor 
in  organic-combined  phosphorus,  seem  to  indicate  a  synthesis  of  phos- 
phorized organic  substance  from  the  phosphates.  The  investigations 
of  Hart,  McCollum  and  Fuller,3  who  found  that  pigs  with  food 
poor  in  phosphorus  develop  just  as  well  with  inorganic  phosphates  as, 
with  organic  phosphorus  compounds,  also  indicate  such  a  formation. 
The  recent  investigations  of  McCollum  4  on  rats  show  that  these  animals 
can  take  up  the  entire  need  of  phosphorus  for  the  skeleton  as  well  as  for 
the  reformation  of  nucleins  and  phosphatides  in  the  form  of  inorganic 


1  Skand.  Arch.  f.  Physiol.,  14. 

2  The  literature  on  feeding  experiments  with  phosphorized  and  non-phosphorized 
food  can  be  found  in  McCollum,  Amer.  Journ.  of  Physiol.,  25. 

1  Hart,  McCollum  and  Fuller,  Amer.  Journ.,  of  Physiol.,  23.  See  also  Lipschiitz, 
Pfliiger's  Arch.,  143.  The  literature  on  the  phosphorus  metabolism  can  also  be 
found  in  Albu  and  Neuberg,  Physiologie  und  Pathologie  des  Mineralstoffwechsel,. 
Berlin,  1906. 

4  Amer.  Journ.  of  Physiol.,  25. 


LACK  OF  IRON.  903 

phosphorus.  Also  the  investigations  of  v.  Wendt  and  Holsti  l  show 
that  a  synthesis  of  organic  phosphorized  substances  from  phosphates  is 
very  probable.  The  feeding  experiments  of  Osborne  and  collabora- 
tors, which  we  will  soon  discuss,  and  which  extend  over  a  long  period 
where  the  animals  were  fed  with  proteins,  fat,  carbohydrates  and  min- 
eral substances  free  from  phosphorus,  give  especially  strong  proof  of 
the  ability  of  the  animal  to  construct  phosphatides  and  nucleins  from  only 
inorganic  phosphorus. 

Lack  of  Iron.  As  iron  is  an  integral  constituent  of  haemoglobin, 
absolutely  necessary  for  the  supply  of  oxygen,  it  is  an  indispensable 
constituent  of  food.  Iron  is  a  never-failing  constituent  of  the  nucleins 
and  nucleoproteins,  and  herein  lies  another  reason  for  the  necessity 
of  the  introduction  of  iron.  Iron  is  also  of  great  importance  in  the 
action  of  certain  enzymes,  the  oxidases.  In  iron  starvation,  iron  is 
continually  eliminated,  even  though  in  diminished  amounts;  and  with 
an  insufficient  supply  of  iron  with  the  food  the  formation  of  haemoglobin 
decreases.  The  formation  of  haemoglobin  is  not  only  enhanced  by  the 
supply  of  organic  iron,  but  also,  according  to  the  general  view,  by  inor- 
ganic iron  preparations.  The  various  divergent  reports  of  this  question 
have  already  been  given  in  a  previous  chapter  (on  the  blood). 

In  the  absence  of  protein  bodies  in  the  food. the  organism  must  nourish 
itself  by  its  own  protein  substances,  and  with  such  nutrition  it  must  sooner 
or  later  succumb.  By  the  exclusive  administration  of  fat  and  carbohy- 
drates the  consumption  of  proteins  in  these  cases  is  very  considerably 
reduced.  For  a  long  time  we  believed  in  the  view  suggested  by  C.  and  E. 
Voit2  that  with  a  nitrogen- free  diet  the  protein  metabolism  could  never 
be  reduced  to  as  small  a  value  as  in  starvation,  but  now,  due  to  the  investi- 
gations Of    HlRSCHFELD,    IvUMAGAWA,    KLEMPERER,    SlVEN,    LaNDERGREN 

and  recently  those  of  Thomas,3  we  learn  that  the  protein  metabolism 
with  such  a  diet  can  be  smaller  than  in  complete  starvation.  With  exclusive 
feeding  of  sugar,  according  to  Thomas,  the  nitrogen  elimination  can  be 
reduced  in  a  few  days  to  the  wear  and  tear  quota,  and  he  has  observed 
an  elimination  of  only  30  milligrams  nitrogen  per  day  and  per  kilo  of 
body  weight. 

The  absence  of  fats  and  carbohydrates  in  the  food  affects  carnivora  and 
herbivora   somewhat   differently.     It   is   not   known    whether   carnivora 


1  v.  Wendt,  Skand.  Arch.  f.  Physiol.,  17;  Holsti;  ibid.,  23.  See  also  Gregersen, 
Zeitschr.  f.  physiol.  Chem.,  71. 

2  Zeitschr.  f.  Biologie,  32. 

'  Hirschfeld,  Yirchow's  Arch.,  114;  Kumagawa,  ibid.,  116;  Klemperer,  Zeitschr. 
f.  klin.  Med.,  16;  Siven,  Skand.  Arch.  f.  Physiol.,  10  and  11;  Landergren,  1.  c,  11; 
footnote  1,  page  894,  also  Maly's  Jahresber.,  32;  Karl  Thomas,  Arch.  f.  (Anat.  u.) 
Physiol.,  1909  and  1910,  Suppl.  Bd. 


904  METABOLISM. 

can  be  kept  alive  for  any  length  of  time  by  food  entirely  free  from  fat 
and  carbohydrates.1  But  it  has  been  positively  demonstrated  that  they 
can  be  kept  alive  a  long  time  by  feeding  exclusively  with  meat  freed  as 
much  as  possible  from  visible  fat  (Pfluger  2) .  Human  beings  and 
herbivora,  on  the  contrary,  cannot  live  for  any  length  of  time  on  such 
food.  On  the  one  hand  they  lose  the  property  of  digesting  and  assimilating 
the  necessarily  large  amounts  of  meat,  and  on  the  other  a  distaste  for 
large  quantities  of  meat  or  proteins  soon  appears.  The  elimination  of 
acetone  bodies  with  an  exclusion  of  carbohydrates  from  the  food  of  man 
is  of  interest  (see  Chapter  XIV) . 

A  question  of  greater  importance  is  whether  it  is  possible  to  maintain 
life  in  an  animal  for  anjr  length  of  time  with  a  mixture  of  simple  organic 
and  inorganic  foodstuffs.  The  earlier  experiments  carried  out  by  many 
investigators  to  decide  this  question  have  not  yielded  satisfactory  results, 
and  Rohmann  3  was  first  able,  by  feeding  a  mixture  of  several  proteins 
with  fat,  starch,  glucose  and  salts,  to  keep  mice  alive  for  a  long  time,  and 
was  also  able  to  raise  young  mice  by  artificial  feeding  of  the  mother  and 
then  the  small  animals.  Rohmann  concludes  from  his  experiments 
that  for  the  continuous  maintenance  or  for  development  of  the  animal 
a  mixture  of  different  proteins  is  necessery,  but  more  recently  he4  has 
found  that  this  can  be  accomplished  by  a  single  protein,  and  the  results 
of  his  experiment  coincide  well  in  this  regard  with  the  investigations  of 
Osborne  and  Mendel  (and  E.  Ferry5). 

In  experiments  with  white  mice  these  investigators  have  found  that 
on  feeding  with  a  mixture  of  only  one  protein  with  cane-sugar,  starch, 
fat,  agar-agar  and  mineral  substances,  adult  mice  could  be  kept  for  169- 
259  days  without  changing  their  body  weight.  The  reason  why  the 
adult  mice  could  not  be  maintained  for  a  still  longer  time  and  why  young 
mice  did  not  grow  was  that  certain  substances  of  unknown  kind  were 
lacking.  Such  substances  occur  in  milk,  and  by  adding  to  the  food,  milk 
from  which  the  proteins  have  been  removed,  although  the  food  contained 
only  one  protein,  the  animals  can  be  kept  alive  for  a  longer  time— 500-600 
days,  and  the  normal  growth  accomplished  as  well.  These  proteins  were, 
especially,  casein,  lactalbumin,  ovalbumin,  hemp-seed  edestin,  wheat 
glutenin  and  excelsin,  while   on  the  contrary  they  were  not  able  to  pro- 

1  See  Horbaczewski,  Maly's  Jahresber.,  31,  715. 

2  Pfliiger's  Arch.,  50. 

*  F.  Rohmann,  Klin,  therap.  Wochenschr.,  No.  40,  1902,  and  Allg.  med.  Centralbl. 
Zeitung,  1908,  No.  9. 

*  Rohmann,  Bioch.  Zeitschr.,  39. 

*  Th.  B.  Osborne  and  L.  B.  Mendell,  Science,  34.  The  Carnegie  Institution,  Wash- 
ington, Parts  1  and  2,  1911;  with  Edna  Ferry,  Journ.  of  biol.  Chem.,  12  and  13,  and 
Zeitschr.  f.  physiol.  Chem.,  80. 


FEEDING  WITH  FOOD-STUFFS   AND   LIPOIDS.  S05 

duce  a  sufficient  growth  with  pea-legumin,  zein,  gliadin  and  hordein  when 
added  to  the  other  foodstuffs  and  protein-free  milk.  These  experiments 
showed  that  animals  fed  with  gliadin  as  the  only  protein  had  the  normal 
ability  to  produce  offspring  and  had  the  ability  to  produce  milk  necessary 
for  their  food. 

In  another  series  of  experiments  it  was  shown  that  the  protein-free 
milk  could  be  replaced  by  a  proper  mixture  of  salts  and  that  the  organic 
constituents  of  such  milk  were  not  necessary.  On  feeding  with  fat,  car- 
bohydrates, casein  and  such  a  salt  mixture  they  were  able  to  attain 
normal  growth  in  a  series  of  experiments  of  more  than  80  or  100  days. 
Growth  was  produced  in  the  animals  also  in  the  absence  of  substances 
soluble  in  ether  (lipoids).  This  is  remarkable,  as  according  to  the 
observations  and  experiments  of  Stepp,  lipoids  are  necessary  for  the 
normal  nutrition. 

According  to  Stepp  l  a  food  which  is  adequate  but  not  quite  genuine 
for  mice  can  be  made  genuine  by  the  addition  thereto  of  certain  substances 
soluble  in  alcohol-ether  from  milk,  egg-yolk,  brain,  etc.  These  substances, 
which  are  neither  fat  nor  cholesterin,  and  which  he  calls  lipoids,  are  partly 
heat-labile  and  correspondingly  lose  their  action  by  continuously  boiling 
with  alcohol  or  by  a  lengthy  boiling  of  the  natural  food-stuffs  with  alcohol 
or  water.  A  proper  food  for  mice  can  be  so  changed  by  continuous 
boiling  with  alcohol  so  that  all  animals  fed  with  it  die,  while  the  changes 
in  the  food  brought  about  in  this  way  can  be  counteracted  by  the  lipoids 
obtained  under  conditions  where  the  lengthy  action  of  heat  is  prevented. 
Mice,  which  die  with  an  otherwise  sufficient  food  but  free  from  lipoids 
may  be  kept  alive  by  the  addition  of  the  undestroyed  lipoids  to  the 
same  food. 

Recently  it  has  been  suggested  that  beside  the  foodstuffs  in  the  ordinary 
sense,  other  constituents  of  our  food  exist  which  are  of  the  very  greatest 
importance  for  life.  The  investigations  of  Funk  as  well  as  those  of 
Suzuki,  Shimamura  and  Odake  on  the  constituents  of  rice-bran  give  a 
specially  striking  proof  of  this.  According  to  C.  Funk  2  rice-bran  contains 
a  substance  called  vitamine,  C17H20X2O7,  which  belongs  to  the  pyrimidine 
group  and  which  also  occurs  in  yeast,  milk  residue  and  beef-brains.  This 
substance,  which  is  absent  in  polished  rice,  causes  the  disease  Beri-Beri 
in  man  and  polyneuritis  in  birds.  Suzuki,  Shimamura  and  Odake  have 
also  isolated  from  rice-bran  a  substance  which  they  call  oryzanine,  which 
is  soluble  in  alcohol  and  necessary  for  animal  life.  With  mixtures  of 
protein,  carbohydrates,  fat  and  salts  without  oryzanine  these  investiga- 
tors could  not  keep  hens,  pigeons  and  mice  alive  and  dogs  could  not  be 

1  Bioch.  Zeitschr.,  22,  and  Zeitschr.  f.  Biol.,  57  and  59. 

2  C.  Funk,  Journ.  of  Physiol.,  43  and  45;  Suzuki,  Shimamura  and  Odake,  Biocb. 
Zeitschr.,  43. 


906  METABOLISM. 

kept  alive  with  boiled  meat  and  polished  rice.     They  emaciate  quickly 
and  rapidly  recover  again  if  they  receive  oryzanine. 

It  follows  from  the  above  that  there  exists  a  certain  unexplainable 
contradiction  between  the  important  observations  of  Stepp  and  those 
of  the  other  investigators  on  the  one  side  and  the  very  interesting,  prolonged 
experiments  of  Osborne  and  Mendel  with  pure  foodstuffs  on  the 
other  side. 

ffl.     METABOLISM   WITH  VARIOUS  FOODS. 

For  carnivora,  as  above  stated,  meat  as  poor  as  possible  in  fat  may 
be  a  complete  and  sufficient  food.  As  the  proteins  moreover  take  a  special 
place  among  the  organic  nutritive  bodies  by  the  quantity  of  nitrogen  they 
contain,  it  is  proper  that  we  first  describe  the  metabolism  with  an  exclu- 
sively meat  diet. 

Metabolism  with  food  rich  in  proteins,  i.e.,  feeding  only  with  meat  as 
poor  in  fat  as  possible. 

By  an  increased  supply  of  proteins  the  catabolism  and  the  elimination 
of  nitrogen  is  increased,  and  this  in  proportion  to  the  supply  of  proteins. 

If  a  certain  quantity  of  meat  has  daily  been  given  to  carnivora  as 
food  and  the  quantity  is  suddenly  increased,  an  augmented  catabolism 
of  proteins,  or  an  increase  in  the  quantity  of  nitrogen  eliminated,  is  the 
result.  If  the  animal  is  daily  fed  for  a  certain  time  with  larger  quantities, 
of  the  same  meat,  a  part  of  the  proteins  accumulates  in  the  body,  but 
this  part  decreases  from  day  to  day,  while  there  is  a  corresponding  daily 
increase  in  the  elimination  of  nitrogen.  In  this  way  a  nitrogenous 
equilibrium  is  established;  that  is,  the  total  quantity  of  nitrogen  eliminated 
is  equal  to  the  quantity  of  nitrogen  in  the  absorbed  proteins  or  meat. 
If,  on  the  'contrary,  an  animal  in  nitrogenous  equilibrium,  having  been 
fed  on  large  quantities  of  meat,  is  suddenly  given  a  small  quantity 
of  meat  per  day,  it  uses  up  its  own  body  proteins,  the  amount  de- 
creasing from  day  to  day.  The  elimination  of  nitrogen  and  the  catab- 
olism of  proteins  decrease  constantly,  and  the  animal  may  in  this  case, 
also  pass  into  nitrogenous  equilibrium,  or  almost  into  this  condition 
These  relations  are  illustrated  by  the  following  table  (Voit):1 

Grams  of  Meat  in  the  Food  per  Day. 


1 

Before  the  Teat. 

500 

During  the  Test. 

1500 

1500 

1000 

Grams  of  Flesh  Metabolized  in  Body  per  Day. 

1 

1222 
1163 

2                   3                  4                   5 
1310             1390             1410              1440 
lOSC.              1088             1080             1027 

G                   7 
1450             1500 

1  Hermann's  Handbueh,  6,  Part  I,  110. 


METABOLISM  WITH   FOOD  RICH  IN  PROTEINS.  907 

In  the  first  case  (1)  the  metabolism  of  meat  before  the  beginning  of 
the  actual  experiment  on  feeding  with  500  grams  of  meat  was  447  grams, 
and  it  increased  considerably  on  the  first  day  of  the  experiment,  after 
feeding  with  1500  grams  of  meat.  In  the  second  case  (2),  in  which  the 
animal  was  previously  in  nitrogenous  equilibrium  with  1500  grams  of 
meat,  the  metabolism  of  flesh  on  the  first  day  of  the  experiment,  with 
only  1000  grams  meat,  decreased  considerably,  and  on  the  fifth  day  an 
almost  nitrogenous  equilibrium  was  obtained.  During  this  time  the 
animal  gave  up  daily  some  of  its  own  proteins.  Between  that  point  below 
which  the  animal  loses  from  its  own  weight  and  the  maximum,  which 
seems  to  be  dependent  upon  the  digestive  and  assimilative  capacity  of 
the  intestinal  canal,  a  carnivore  may  be  kept  in  nitrogenous  equilibrium 
with  varying  quantities  of  proteins  in  the  food. 

The  supply  of  proteins,  as  well  as  the  protein  condition  of  the  body, 
affects  the  extent  of  the  protein  metabolism.  A  body  which  has  become 
rich  in  proteins  by  a  previous  abundant  meat  diet  must,  to  prevent  a  loss 
of  proteins,  take  up  more  protein  with  the  food  than  a  body  poor  in  pro- 
teins. 

In  regard  to  the  rapidity  with  which  the  protein  catabolism  takes 
place  Falta  x  found  in  man  but  not,  or  at  least  not  to  the  same  extent, 
in  dogs,  that  quite  great  differences  exist  between  the  different  proteins. 
Thus  on  feeding  pure  proteins  the  chief  amount  of  the  nitrogen  is  more 
quickly  eliminated  after  feeding  casein  than  after  genuine  ovalbumin. 
This  latter  is  more  easily  demolished  after  a  previous  modification  by 
coagulation  than  in  the  native  state,  which  indicates  that  an  unequal 
resistance  of  the  different  proteins  toward  the  digestive  juices  plays  a 
part.  Hamalainen  and  Helme  2  have  also  obtained  similar  results. 
Even  on  feeding  with  easily  decomposable  proteins  it  always  takes  several 
days  before  the  total  nitrogen  corresponding  thereto  is  eliminated,  which 
depends,  according  to  Falta,  upon  a  progressive  demolition  of  the  pro- 
tein. From  the  unequal  rate  at  which  the  different  proteins  are  decom- 
posed it  follows  that  in  the  passage  from  a  diet  poor  in  protein  to  one 
rich  in  protein  the  time  within  which  nitrogenous  equilibrium  occurs 
depends  chiefly  upon  the  kind  of  protein  contained  in  the  food. 

Pettenkofer  and  Voit  have  made  investigations  on  the  ?netabolism 
of  fat  with  an  exclusively  protein  diet.  These  investigations  have  shown 
that  by  increasing  the  quantity  of  proteins  in  the  food  the  daily  metab- 
olism of  fat  decreases,  and  they  have  drawn  the  conclusion  from  these 
experiments,  that  there  may  even  take  place  a  formation  of  fat  under 
these   circumstances.     The  objections  presented  by   Pfluger   to   these 


1  Deutsch.  Arch.  f.  klin.  Med.,  86. 
1  Skand.  Arch.  f.  Physiol.,  19. 


908  METABOLISM. 

experiments,  as  well  as  the  proofs  of  the  formation  of  fat  from  proteins, 
are  also  given  in  Chapter  IX. 

According  to  Pfluger's  doctrine,  the  protein  can  influence  the  formation  of 
fat  only  in  an  indirect  way,  namely,  in  that  it  is  consumed  instead  of  the  non- 
nitrogenous  bodies  and  hence  the  fat  and  fat-forming  carbohydrates  are 
spared.  If  sufficient  protein  is  introduced  with  the  food  to  satisfy  the  total  nu- 
tritive requirements,  then  the  decomposition  of  fat  stops;  and  if  non-nitrogenous 
food  is  taken  at  the  same  time,  this  is  not  consumed,  but  is  stored  up  in  the  animal 
body,  the  fats  as  such,  and  the  carbohydrates  at  least  in  great  part  as  fat. 

Pfluger  defines  the  "  nutritive  requirement  "  as  the  smallest  quantity  of 
lean  meat  which  produces  nitrogenous  equilibrium  without  causing  any  decom- 
position of  fat  or  carbohydrates.  At  rest  and  at  an  average  temperature  it  is 
found  in  dogs  to  be  2.073  to  2.099  grams  of  nitrogen  1  (in  meat  fed)  per  kilo  of 
flesh  weight  (not  body  weight,  as  the  fat,  which  often  forms  a  considerable  fraction 
of  the  weight  of  the  body,  cannot  as  it  were  be  used  as  dead  measure).  Even 
when  the  supply  of  protein  is  in  excess  of  the  nutritive  requirements,  Pfluger 
found  that  the  protein  metabolism  increases  with  an  increased  supply  until 
the  limit  of  digestive  power  is  reached,  which  limit  is  about  2600  grams  of  meat 
with  a  dog  weighing  30  kilos.  In  these  experiments  of  Pfluger's  not  all  of  the 
excess  of  protein  introduced  was  completely  decomposed,  but  a  part  was  retained 
by  the  body.  Pfluger  therefore  defends  the  proposition  "  that  a  supply  of 
proteins  only,  without  fat  or  carbohydrate  does  not  exclude  a  protein  fattening." 

From  what  has  been  said  on  protein  metabolism  in  starvation  and 
with  exclusive  protein  food,  it  follows  that  the  protein  catabolism  in  the 
animal  body  never  stops,  that  the  extent  is  dependent  in  the  first  place 
upon  the  extent  of  protein  supply,  and  that  the  animal  body  has  the  prop- 
erty, within  wide  limits,  of  accommodating  the  protein  catabolism  to  the 
protein  supply. 

These  and  certain  other  peculiarities  of  protein  catabolism  have  led 
Voit  to  the  view  that  not  all  proteins  in  the  body  are  decomposed  with 
the  same  ease.  Voit  differentiates  between  the  proteins  fixed  in  the 
tissue-elements,  so-called  organized  proteins,  tissue-proteins,  from  those 
proteins  which  circulate  with  the  fluids  in  the  body  and  its  tissues  and 
which  are  taken  up  by  the  living  cells  of  the  tissues,  from  the  interstitial 
fluids  washing  them,  and  destroyed.  These  circulating  proteins  or  supply 
proteins  are,  he  claims,  more  easily  and  quickly  destroyed  than  the  tissue- 
proteins.  When,  therefore,  in  a  fasting  animal  which  has  been  previously 
fed  with  meat,  an  abundant  and  quickly  decreasing  decomposition  of 
proteins  takes  place,  while  in  the  further  course  of  starvation  this  protein 
catabolism  becomes  less  in  quantity  and  more  uniform,  this  depends  upon 
the  fact  that  the  supply  of  circulating  proteins  is  destroyed  chiefly  in  the 
first  days  of  starvation  and  the  tissue-proteins  in  the  last  days. 

The  tissue-elements  constitute  an  apparatus  of  a  relatively  stable 
nature,  which  has  the  power  of  taking  proteins  from  the  fluids  washing 
the  tissues  and  appropriating  them,  while  their  own  proteins,  the  tissue- 

1  See  Schondorff,  Pfluger's  Arch.,  71. 


METABOLISM  WITH  FOOD  RICH  IX  PROTEINS.  909 

proteins,  are  ordinarily  catabolized  to  only  a  small  extent,  about  1  per 
cent  daily  (Voit).  By  an  increased  supply  of  proteins  the  activity  of 
the  cells  and  their  ability  to  decompose  nutritive  proteins  is  also  increased 
to  a  certain  degree.  When  nitrogenous  equilibrium  is  obtained  after 
an  increased  supply  of  proteins,  it  indicates  that  the  decomposing  power 
of  the  cells  for  proteins  has  increased  so  that  the  same  quantity  of  proteins 
is  metabolized  as  is  supplied  to  the  body.  If  the  protein  metabolism  is 
decreased  by  the  simultaneous  administration  of  other  non-nitrogenous 
foods  (see  below),  a  part  of  the  circulating  proteins  may  have  time  to 
become  fixed  and  organized  by  the  tissues,  and  in  this  way  the  mass  of 
the  flesh  of  the  body  increases.  During  starvation  or  with  a  lack  of  pro- 
teins in  the  food  the  reverse  takes  place,  for  a  part  of  the  tissue  protein- 
is  converted  into  circulating  proteins  which  are  metabolized,  and  in  this 
case  the  flesh  of  the  body  decreases. 

Voit's  theory  has  been  criticised  by  several  investigators  and  espe- 
cially by  Pfluger.  Pfluger's  belief,  based  on  an  investigation  made 
by  one  of  his  pupils,  Schondorff,1  is  that  the  extent  of  protein  destruc- 
tion is  not  dependent  upon  the  quantity  of  circulating  proteins,  but 
upon  the  nutritive  condition  of  the  cells  for  the  time  being — a  view 
which  does  not  widely  differ  from  Voit  if  the  author  does  not  misunder- 
stand Pfluger.  Voit  2  has,  as  is  known,  stated  that  the  conditions  f < >r 
the  destruction  of  substances  in  the  body  exist  in  the  cells,  and  also  that 
the  circulating  protein  is  first  catabolized  after  having  beon  taken  up 
by  the  cells  from  the  fluids  washing  them.  Besides  this,  certain  inves- 
tigations conclusively  show  that  the  extent  of  protein  catabolism  is  depend- 
ent upon  the  concentration  of  the  decomposable  proteins  at  the  place 
where  the  decomposition  is  taking  place.  Thus  in  confirmation  with 
the  earlier  investigations  of  v.  Gebhardt  and  Krummacher,  Thomas, 
v.  Hoesslin  and  Lesser3  have  recently  shown  that  on  feeding  with 
a  certain  quantity  of  protein,  less  protein  was  catabolized  when  the  pro- 
tein was  supplied  piecemeal,  i.e.,  in  several  small  portions  during  the  day 
instead  of  at  one  time.  That  the  peculiarity  of  the  nitrogen  elimination 
in  starvation  and  after  sufficient  protein  supply  depends  essentially 
upon  the  concentration  of  the  decomposable  proteins  (or  more  correctly  the 
decomposable  nitrogenous  substances)  is  no  doubt  also  generally  admitted.4 


1  Pfluger,  Pfluger's  Arch.,  54;  Schondorff,  ibid.,  54. 

2  Zeitschr.  f.  Biologie,  11. 

3  K.  Thomas.  Arch.  f.  (Anat.  u.)  Physiol.,  1909;  H.  v.  Hoesslin  and  E.  J.  Lesser, 
Zci  schr.  f.  physiol.  Chem.,  73,  when  also  the  works  of  v.  Gebhardt  and  Kummacher  are 
cited. 

4  See  also  E.  Voit  and  A.  Korkunoff,  Zeitschr.  f.  Biol.,  32,  and  O.  Frank  and  R. 
Trommsdorff,  ibid.,  43. 


910  METABOLISM. 

Recent  investigations,  especially  those  of  Folin,1  which  show  that  the 
amount  of  certain  nitrogenous  urinary  constituents,  such  as  creatinine, 
uric  acid  and  the  combinations  containing  neutral  sulphur,  are  almost 
independent  of  the  quantity  of  protein  taken  as  food,  while  the  quantity 
of  urea  is  determined  by  the  protein  partaken  of,  tend  to  substantiate 
Voit's  view  that  we  must  differentiate  between  the  real  cell  protein 
and  the  food  protein.  This  has  also  led  Folin  to  differentiate  between 
endogenous  and  exogenous  protein  metabolism.  The  chief  point  in 
Voit's  theory  that  all  the  proteins  in  the  body  do  not  behave  alike 
and  that  the  organized  proteins  which  have  been  fixed  in  the  cells  and 
have  been  introduced  into  the  cell  structure  are  less  readily  catabolized 
than  the  proteins  occurring  in  the  nutritive  fluids  or  temporarily  taken 
up  from  these,  must  also  be  considered  as  not  disputed.  Rtjbner  2 
differentiates  also  between  the  deposited  protein  (growth  protein,  and 
deposited  by  the  activity  of  the  cells  melioration  protein)  in  the  body  on 
the  one  hand  and  the  protein  temporarily  incorporated  with  the  body 
(supply  protein  and  catabolized  in  passing  to  a  protein-poor  diet,  transitory 
protein)  on  the  other  hand. 

This  question  is  intimately  connected  with  another,  namely,  whether  the 
food  proteins  taken  up  by  the  cells  are  metabolized  as  such  or  whether  they  are 
first  organized,  i.e.,  are  converted  into  specific  cell  protein.  The  observations 
of  Panum,  Falck,  Asher  and  Haas  and  others  3  on  dogs  have  shown  that  the 
nitrogen  elimination  increases  almost  immediately  after  a  meal  and  in  the  fifth  or 
sixth  hour  according  to  these  experimenters,  when  according  to  Schmidt-Mulheim  * 
about  59  per  cent  of  the  eaten  protein  is  absorbed,  do  not  indicate  that  a  trans- 
formation of  the  food  protein  into  organized  protein  occurs  before  it  is  catab- 
olized. The  recent  investigations  upon  the  deep  cleavage  of  proteins  in  digestion 
and  the  generally  accepted  protein  syntheses  from  ammo-acids  have  made  this 
question  lose  its  special  interest. 

On  account  of  the  above-stated  action  of  the  concentration  of  the 
catabolizable  nitrogenous  material  upon  the  protein  decomposition  or 
nitrogen  elimination,  it  is  not  possible  to  replace  the  quantity  of  protein 
catabolized  in  starvation  by  the  exclusive  feeding  of  protein  administered 
at  one  time  and  in  quantities  corresponding  to  the  food  proteins.  This 
always  requires  large  amounts  of  protein.  Even  on  the  fractional  intro- 
duction of  natural  protein  v.  Hoesslin  and  Lesser  were  unable  to  pro- 
duce a  nitrogen  equilibrium  with  quantities  of  protein  equal  to  the  starva- 

1  Amer.  Journ.  of  Physiol.,  13. 

;  Anh.  f.  (Ariat.  u.)  Physiol.,  1911. 

*Panum,  Nord.  Med.  Arkiv.,  6;  Falck,  see  Hermann's  Handbuch,  6,  Part  I,  107; 
Asher  and  Hums,  Bioch.  Zeitsr-hr.,  12.  For  further  information  in  regard  to  the  curve 
of  nitrogen  elimination  in  man,  see  Tschenloff,  Korrespond.  Blatt  Schweiz.  Aerzte, 
1896;  Rosemann,  Pflliger's  Arch.,  65,  and  Veraguth,  Journ.  of  Physiol.,  21;  Schlosse, 
Maly's  Jahresber.,  31. 

4  Arch.  f.  (Anat.  u.)  Physiol.,  1879. 


NUTRITIVE  VALUE  OF  GELATIN.  911 

tion  protein;  the  elimination  of  nitrogen  was  always  somewhat  increased. 

On  the  fractional  introduction  <>f  protein,  Thomas1  was  nevertheless  able 
in  dogs  to  produce  nitrogenous  equilibrium  without  essentially  raising 
the  protein  metabolism  (in  comparison  with  the  starvation  value).  In 
experiments  upon  himself  he  was  not  able  to  produce  this. 

It  has  been  stated  above  that  other  foods  may  decrease  the  catab- 
olism  of  proteins.  Gelatin  is  such  a  food.  Gelatin  and  the  gelatin-formers 
do  not  seem  to  be  converted  into  protein  in  the  body,  and  this  last  cannot 
be  entirely  replaced  by  gelatin  in  the  food.  For  example,  if  a  dog  is  fed 
on  gelatin  and  fat,  its  body  sustains  a  loss  of  proteins  even  when  the 
quantity  of  gelatin  is  great  enough  so  that  the  animal  with  an  amount  of 
fat  and  meat  containing  just  the  same  quantity  of  nitrogen  as  the  gelatin 
in  question,  remains  in  nitrogenous  equilibrium.  On  the  other  hand, 
gelatin,  as  Voit,  Panum  and  Oerum2  have  shown,  has  great  value  as 
a  means  of  sparing  the  proteins,  and  it  may  decrease  the  catabolism  of 
proteins  to  a  still  greater  extent  than  fats  and  carbohydrates.  This  is 
apparent  from  the  following  summary  of  Voit's  experiments  upon  a  dog: 

Food  per  Day.  Flesh. 


Meat.  Gelatin.  Fat.  Sugar.  Catabolized.  On  the  Body. 

400  0  200  0                 450                   -50 

400  0  0  250              439                   -39 

400  200  0  0                 356                   +44 

I.  Munk  3  has  later  arrived  at  similar  results  by  means  of  more  deci- 
sive experiments,  and  the  recent  investigations  of  Krummacher  and 
Kirchmann  4  show  the  extent  of  the  sparing  action  of  gelatin  upon  pro- 
teins. The  extent  of  protein  destruction  during  gelatin  feeding  was  com- 
pared with  the  extent  of  protein  catabolism  in  starvation,  and  it  was 
found  that  35-37.5  per  cent  of  the  quantity  of  protein  decomposed  in 
starvation  could  be  spared  by  gelatin.  The  physiological  availability 
of  gelatin  was  found  by  Krummacher  to  be  equal  to  3.88  calories  for  1 
gram,  which  corresponds  to  about  72.4  per  cent  of  the  energy-content  of 
the  gelatin. 

The  value  of  gelatin  has  been  found  by  Murlin  5  to  be  dependent  to 
a  high  degree  upon  the  protein  condition  of  the  body,  on  the  calorific 
value  of  the  food  and  the  quantity  of  carbohydrates  in  the  latter.  If 
in  a  man  weighing  70  kilos,  51  calories  per  kilo  were  partaken,  the  quan- 
tity of  nitrogen  eliminated  wTas  10  per  cent  more  than  the  starvation 


1  v.  Hoesslin  and  Lesser,  1.  c;  Thomas,  Arch.  f.  (Anat.  u.)  Physiol.,  Suppl.  Bd.,1910. 
-  Voit,  1.  c,  123;  Panum  and  Oerum,  Nord.  Med.  Arkiv.,  11. 

3  Pfluger's  Arch.,  58. 

4  Krummacher,  Zeitschr.  f.  Biologie,  42;  Kirchmann,  ibid.,  40. 
*  Amer.  Journ.  of  Physiol.,  19. 


912  METABOLISM. 

value,  and  when  two-thirds  of  the  total  calories  partaken  of  were  sup- 
plied by  carbohydrates,  63  per  cent  of  the  total  nitrogen  could  be  replaced 
by  gelatin  nitrogen. 

The  reason  why  gelatin  cannot  entirely  replace  protein  has  been  sought 
for  in  the  fact  that  gelatin  does  not  contain  all  the  amino-acids  of  the 
proteins  (such  as  tyrosine  and  tryptophane),  or  does  not  contain  a  suf- 
ncient  amount  of  the  various  amino-acids.  The  correctness  of  this 
explanation  was  first  shown  by  Kaufmann  by  an  experiment  on  himself, 
where  he  showed  that  gelatin  after  addition  of  tyrosine,  tryptophane 
and  cystine  could  be  made  equivalent  to  protein.  The  conclusive  proof 
was  given  later  by  Abderhalden  1  when  he  showed  that  completely 
decomposed  gelatin  on  the  addition  of  a  mixture  of  amino-acids,  among 
them  also  tyrosine  and  tryptophane,  could  be  made  equivalent  to  proteins. 

As  it  has  been  possible  to  replace  the  proteins  in  the  food  by  their 
cleavage  products  or  mixtures  of  amino-acids,2  it  is  easily  understandable 
that  also  proteoses  or  peptones  can  completely  or  partly  replace  the 
protein.  Their  ability  in  this  regard  is  essentially  dependent  upon  their 
constitution,  i.e.,  their  content  of  the  different  amino-acids.  As  the 
proteoses  and  peptones  are  produced  by  cleavage  and  as  therefore  in 
one  proteose  we  hae  certain  atomic  comp  lcxes  and  in  others  again 
these  may  be  absent  or  only  exist  to  a  slight  extent,  it  is  conceivable  that 
different  investigators3  have  obtained  contradictory  results  because  of 
the  use  of  different  proteoses  and  peptones. 

We  have  a  number  of  investigations  on  the  action  of  amides  upon 
metabolism,  which  are  mostly  connected  by  the  use  of  asparagin.  These 
investigations  have  in  part  led  to  conflicting  results;  but  they  indicate 
that  carnivora  and  herbivora  act  differently,  that  the  results  are  depen- 
dent upon  the  rapidity  with  which  the  asparagin  is  absorbed  and  also 
upon  the  bacterial  action  in  the  intestine,  and  that  in  herbivora  a  protein- 
sparing  action  can  be  brought  about  by  asparagin.4     If,  as  is  generally 


1  Martin  Kaufmann,  Pfiuger's  Arch.,  109;  Abderhalden,  Zeitschr.  f.  physiol.  Chem., 
77. 

2  See  Abderhalden  and  collaborators,  Chapter  VIII;  also  Abderhalden,  Zeitschr. 
f.  physiol.  Chem.,  77,  and  especially  83. 

3  In  regard  to  the  literature  on  the  nutritive  value  of  the  proteoses  and  peptones 
see  Maly,  Pfliiger's  Arch.,  9;  P16sz  and  Gyergyay,  ibid.,  10;  Adamkiewicz,  'Die  Natur 
und  der  Niihrwerth  des  Peptones"  (Berlin,  1877);  Pollitzer,  Pfiuger's  Arch.,  37,  301; 
Zuntz,  ilrid.,  37,  313;  Munk,  Centralbl.  f.  d.  med.  Wissensch.,  1889,  20,  and  Deutsch. 
med.  Wochenschr.,  1889;  Ellinger,  Zeitschr.  f.  Biologie,  33  (literature).  Blum,  Zeitschr. 
f.  physiol.  Chem.,  30;  Henriques  and  Hansen,  Zeitschr.  f.  physiol.  Chem.,  48. 

4  Weiske,  Zeitschr.  f.  Biologie,  15  and  17,  and  Centralbl.  f.  d.  med.  Wissensch.,  1890, 
945;  Munk,  Virchow's  Arch.,  94  and  98;  Politis,  Zeitschr.  f.  Biologie,  28.  See  also 
Mauthner,  ibid.,  28;  Gabriel,  ibid.,  29;  and  Voit,  ibid.,  29,  125;  Kellner,  Maly's  Jahres- 


METABOLISM  WITH  A  MIXED   DIET.  913 

admitted,  the  amino-acids  can  serve  in  the  building  up  of  the  proteins, 
then  there  is  no  use  denying  that  their  amides  can  also  be  used  by  the 
animal  body. 

Recently  Grafe,  Abderhalden  l  and  their  collaborators  have  carried 
on  investigations  on  the  value  of  ammonia  and  of  urea  as  protein  sparers 
and  protein  formers.  These  investigations  have  shown  that  ammonia 
or  urea  under  special  conditions  of  experimentation  may  cause  a  nitrogen 
retention,  but  we  are  not  justified  in  believing  that  a  synthesis  of  protein 
from  ammonia  takes  place. 

Metabolism  on  a  Diet  Consisting  of  Protein,  with  Fat  or  Carbohydrates. 
As  the  various  foodstuffs  can  replace  each  other  as  sources  of  energy  in 
the  food  it  follows  that  the  non-nitrogenous  foodstuffs  can  be  used 
instead  of  the  proteins  and  can  reduce  the  catabolism  of  these.  Thus 
the  fat  cannot  completely  arrest  or  prevent  the  catabolism  of  proteins, 
but  it  can  decrease  it  and  so  spare  the  proteins.  This  is  apparent  from 
the  following  table  by  Voit.2  A  is  the  average  for  three  days,  and  B 
for  six  days. 

Food.  Flesh. 


A 

B 

Meat. 

1500 

1500 

Fat. 

0 
150 

Metabolized. 

1512 

1474 

On  the  Body. 

-12 
+26 

According  to  Voit  the  adipose  tissue  of  the  body  acts  like  the  food- 
fat,  and  the  protein-sparing  effect  of  the  former  may  be  added  to  that  of 
the  latter,  so  that  a  body  rich  in  fat  may  not  only  remain  in  nitrogenous 
equilibrium,  but  may  even  add  to  the  store  of  body  proteins,  while  in  a 
lean  body  with  the  same  food  containing  the  same  amount  of  proteins 
and  fat  there  would  be  a  loss  of  proteins.  In  a  body  rich  in  fat  a  greater 
quantity  of  proteins  is  protected  from  metabolism  by  a  certain  quantity 
of  fat  than  in  a  lean  body. 

Like  the  fats  the  carbohydrates  have  a  sparing  action  on  the  proteins. 
By  the  addition  of  carbohydrates  to  the  food,  carnivora  not  only  remain 
in  nitrogenous  equilibrium,  but  the  same  quantity  of  meat  which  in 
itself  is  insufficient  and  which  without  carbohydrates  would  cause  a  loss 


ber.,  27,  and  Zeitschr.  f.  Biologie,  39;  Pfliiger's  Arch.,  113;  Kellner  and  Kohler,  Chem. 
Centralbl.,  1,  1906;  Voltz,  Pfliiger's  Arch.,  107,  117,  with  Yakuwa,  ibid.,  121;  v. 
Strusiewicz,  Zeitschr.  f.  Biol.,  47;  Rosenfeld  and  Lehmann,  Pfliiger's  Arch.,  112; 
Lehmann,  ibid.,  115;  M.  Miiller,  ibid.,  117;  Henriques  and  Hansen,  Zeitschr.  f.  physiol. 
Chem.,  54. 

1  Grafe,  Zeitschr.  f.  physiol.  Chem.,  78,  82,  84,  with  Schlapfer,  ibid.,  77,  with 
Turban,  ibid.,  83;  Voltz,  ibid.,  74;  Abderhalden  with  Hirsch  or  Laupe,  ibid.,  80, 
82-84;  Peschek,  Bioch.  Zeitschr.  45. 

2  Voit,  in  Hermann's  Handb.,  6,  130. 


914  METABOLISM. 

of  weight  in  the  body  may  with  the  addition  of  carbohydrates  produce 
a  deposit  of  proteins.     This  is  apparent  from  the  following  table:1 

Food.  Flesh. 


Meat. 

Fat. 

Sugar. 

Starch. 

Metabolized. 

On  the  Body 

500 

250 

558 

-    58 

500 

300 

466 

+   34 

500 

200 

505 

-     5 

800 

250 

745 

+  55 

800 

200 

773 

+  27 

2000 

200^300 

1792 

+208 

2000 

250 

1883 

+  117 

The  sparing  of  protein  by  carbohydrates  is  greater,  as  shown  by  the 
table,  than  by  fats.  According  to  Voit  the  first  is  on  an  average  9  per 
cent  and  the  other  7  per  cent  of  the  administration  protein  without  a 
previous  addition  of  non-nitrogenous  bodies.  Increasing  quantities  of 
carbohydrates  in  the  food  decrease  the  protein  metabolism  more  regularly 
and  constantly  than  increasing  quantities  of  fat.  Atwater  and  Bene- 
dict 2  also  found  that  the  carbohydrates  had  a  somewhat  greater  sparing 
action  upon  proteins  than  fats. 

Because  of  this  great  protein-sparing  action  of  carbohydrates  the  her- 
bivora,  which  as  a  rule  partake  of  considerable  quantities  of  carbohydrates, 
assimilate  proteins  readily  (Voit). 

The  greater  protein-sparing  action  of  carbohydrates  as  compared  with 
that  of  the  fats  occurs,  as  shown  by  Landergren,3  to  a  still  higher  degree 
with  food  poor  in  nitrogen  or  in  nitrogen  starvation,  in  which  cases  the 
carbohydrates  have  double  the  protein-sparing  action  as  compared  with 
an  isodynamic  quantity  of  fat.  This  different  behavior  of  the  fats 
and  the  carbohydrates  is  also  shown  in  the  experiments  of  Rubner  and 
Thomas  4  that  on  the  exclusive  feeding  of  sugar  the  nitrogen  elimination 
is  reduced  to  the  wear  and  tear  quota  while  on  the  exclusive  feeding  of 
fats  the  nitrogen  requirement  was  about  two  to  three  times  as  great  as 
the  wear  and  tear  quota. 

The  protein-sparing  action  of  the  carbohydrates  and  fats  has  generally 
been  studied  through  the  one-sided  feeding  with  one  or  the  other  of  these 
two  groups  of  foodstuffs.  The  question  may  be  raised  whether  the  differ- 
ence observed  between  the  fats  and  carbohydrates  could  not  also  be 
brought  about  by  the  simultaneous  supply  of  carbohydrates  and  fat  in 
varying  proportions.     Tallquist  5  made  a  series  of  experiments  on  this 

1  Voit,  in  Hermann's  Handb.,  6,  page  143. 

2  See  Ergebnisse  der  Physiologie,  3. 

3 1.  c,  Inaug.-Diss.,  and  Skand.  Arch.  f.  Physiol.,  14.  Wimmer,  Zeitschr.  f.  Biol., 
57,  has  given  further  proofs  of  the  strong  protein-sparing  action  of  carbohydrates  in 
nitrogen  starvation. 

*  See  Thomas,  Arch.  f.  (Anat.  u.)  Physiol.  Suppl.  Bd.,  1910. 

6  Finska  Lakaresallskapets  Handl.,  1901.     See  also  Arch.  f.  Hygiene,  41. 


LIMIT  OF  PROTEIN   REQUIREMENT.  915 

subject.  In  one  of  the  periods  16.27  grams  X,  14  grama  fat,  and  166 
grains  carbohydrate  were  given;  in  a  second,  Hi. (is  grams  X,  1 10  grama 
fat,  and  250  grams  carbohydrate,  containing  almost  the  .same  number 
of  calorics,  namely,  2807  and  2873.  In  both  cases  an  almoal  complete 
nitrogenous  equilibrium  was  reached  and  the  carbohydrate  did  not 
spare  more  protein  than  the  fat.  It  is  therefore  possible  that  the  fat  has 
about  the  same  protein-sparing  action  as  an  isodynamic  amount  of  car- 
bohydrate when  the  quantity  of  carbohydrates  does  not  sink  below  a 
certain  minimum,  which  is  not  known  for  the  present. 

This  condition  as  well  as  the  extent  of  protein-sparing  action  of  the 
carbohydrates  stands,  according  to  Landergren,  in  close  relation  to 
the  formation  of  sugar  in  the  body.  The  animal  bod}-  always  needs 
sugar,  and  a  lack  of  carbohydrates  in  the  food  leads  to  a  part  of  the  pro- 
teins being  used  in  the  sugar  formation.  This  part  can  be  spared  by 
carbohydrates  but  not  by  fats,  from  which,  according  to  Landergren, 
the  carbohydrates  cannot  be  formed.  In  this  also  lies  the  probable 
reason  why  the  fats,  on  being  fed  exclusively  but  not  with  a  sufficient 
supply  of  carbohydrates,  have  a  much  lower  protein-sparing  action  than 
the  carbohydrates.  The  fats  cannot  prevent  the  protein  catabolism 
necessary  for  the  formation  of  sugar  on  a  diet  lacking  in  carbohydrates. 
The  law  as  to  the  increased  protein  catabolism  with  increased  pro- 
tein supply  also  applies  to  food  consisting  of  protein  with  fat  and  car- 
bohydrates. In  these  cases  the  body  tries  to  adapt  its  protein  catabolism 
to  the  supply;  and  when  the  daily  calorie-supply  is  completely  covered 
by  the  food,  the  organism  can,  within  wide  limits,  be  in  nitrogenous 
equilibrium  with  different  quantities  of  protein. 

The  upper  limit  to  the  possible  protein  catabolism  per  kilo  and  per 
day  has  been  determined  only  for  herbivora.  For  human  beings  it  is 
not  known,  and  its  determination  is  from  a  practical  standpoint  of  second- 
ary importance.  What  is  more  important  is  to  ascertain  the  lower  limit, 
and  on  this  subject  we  have  several  older  experiments  upon  man  as  well 
as  upon  dogs  by  Hirschfeld,  Kumagawa,  Klemperer,  Munk,  Rosen- 
heim,1 and  others.  It  follows  from  these  experiments  that  the  lower 
limit  of  protein  requirement  for  human  beings,  for  a  week  or  less,  is  about 
30-40  grams  or  0.4-0.6  gram  per  kilo  with  a  body  of  average  weight, 
v.  Noorden2  considers  0.6  gram  protein  (absorbed  protein)  per  kilo 
and  per  day  as  the  lower  limit  (threshold  of  protein  requirement).  The 
above-mentioned  figures  are  valid  only  for  short  series  of  experiments; 
still  there  exists  the  observation  of    E.  Voit  and  Constantinidi  3  on 

1  See  footnote  3,  page  903;  also  Munk,  Arch.  f.  (Anat.  u.)  Physiol.,  1S91  and  1896 
Rosenheim,  ibid.,  1891;  Pfluger's  Arch.,  54. 

2  Grundriss  einer  Methodik  der  Stoffwechseluntersuchungen.     Berlin,  1892. 
*  Zeitschr.  f.  Biologie,  2a. 


916  METABOLISM. 

the  diet  of  a  vegetarian  when  the  protein  condition  was  kept  almost 
but  not  completely  normal  for  a  long  time  with  about  0.6  gram  of  pro- 
tein per  kilo.  Caspari  x  has  also  made  observations  upon  a  vegetarian 
for  a  period  of  14  days  with  an  average  of  0.1  gram  nitrogen  (recalculated 
as  equal  to  0.62  gram  protein)  per  kilo,  where  a  nearly  complete  nitrog- 
enous equilibrium  was  observed  as  the  average  result. 

According  to  Voit's  normal  figures,  which  will  be  spoken  of  below, 
for  the  nutritive  need  of  man,  an  average  workingman  of  about  70  kilos 
weight,  requires  on  a  mixed  diet  about  40  calories  per  kilo  (true  calories 
or  net  calories).  In  the  above  experiments  with  food  very  poor  in  pro- 
tein the  demand  for  calories  was  considerably  greater;  as,  for  instance, 
in  certain  cases  it  was  51  (Kumagawa)  or  even  78.5  calories  (Klemperer). 
It  therefore  seems  as  if  the  above  very  low  supply  of  protein  was  pos- 
sible only  with  great  waste  of  non-nitrogenous  food;  but  in  opposition 
to  this  it  must  be  recalled  that  in  Voit  and  Constantinidi's  experiments 
upon  the  vegetarian,  who  for  years  was  accustomed  to  a  food  poor  in 
protein  and  rich  in  carbohydrate,  the  calories  amounted  to  only  43.7 
per  kilo.  In  the  case  studied  by  Caspari  a  supply  of  41  calories  per 
kilo  was  entirely  sufficient. 

Siven  has  shown  by  experiments  upon  himself  that  the  adult  human 
organism,  at  least  for  a  short  time,  can  be  maintained  in  nitrogenous 
equilibrium  with  a  specially  low  supply  of  nitrogen  without  increasing 
the  calories  in  the  food  above  the  normal.  With  a  supply  of  41-43 
calories  per  kilo  he  remained  in  nitrogenous  equilibrium  for  four  days 
with  a  supply  of  nitrogen  of  0.08  gram  per  kilo  of  body  weight.  Of  the 
nitrogen  taken,  a  part  was  of  a  non-protein  nature  and  the  quantity  of 
true  protein  nitrogen  was  only  0.045  gram,  corresponding  to  about  0.3 
gram  of  protein  per  kilo  of  body  weight.  That  this  low  limit,  which 
by  the  way  holds  only  for  a  short  time,  has  no  general  validity  follows 
from  other  observations.  Thus  Caspari  2  also,  in  an  experiment  on  him- 
self, could  not  attain  complete  nitrogenous  equilibrium  on  a  much  greater 
nitrogen  supply.  The  protein  minimum  also  seems  to  vary  in  different 
individuals. 

The  protein  minimum  can  also  be  different  for  other  reasons.  It 
varies,  as  mentioned  by  Rubner,  not  only  with  the  kind  of  foodstuffs, 
but  also  with  the  nutritive  condition  of  the  body.  The  needs  of  the 
cells  for  protein  varies  with  the  nutritive  condition  of  the  body.  Where 
the  protein  is  eagerly  demanded,  less  supply  of  protein  suffices,  and  where 
the  demand  is  low  more  protein  must  be  offered  (Rubner).     The  more  the 


1  Physiologische  Studien  iiber  Vegetarismus,  Bonn,  1905. 

2  Sivf'n,  Skand.  Arch.  f.  Physiol.,    10  and  11;    Caspari,  Arch.  f.  (Anat.  u.)  Physiol., 
1901. 


WEAR  AND  TEAR  QUOTA.  917 

body  has  become  reduced  the  lower  is  the  protein  minimum,  according  to 
RUBNBB.1 

As  mentioned  in  the  early  part  of  this  chapter,  the  body  always  suffers 
a  certain  loss  of  nitrogen  through  the  falling  out  of  the  hair  and  other 
epidermis  formations,  by  the  secretions,  etc.;  but  to  this  also  belongs  the 
constant  loss  of  nitrogenous  substance  which  every  cell  sustains  because 
of  its  activity.  This  unpreventable  loss  of  nitrogen  has  been  included 
by  Rubner  under  the  name  "  wear  and  tear  "  quota,  and  this  quota, 
which  corresponds  to  the  nitrogen  elimination  with  a  perfectly  nitrogen- 
free  diet,  and  hence  is  a  protein  minimum,  may  rise  to  4  to  6  per  cent  of 
the  total  calorific  needs.  The  energy  supply  of  the  food  is  under  these 
conditions  entirely  assumed  by  the  non-nitrogenous  foodstuffs,  and  when 
this  quota  is  replaced  by  protein  the  body  is  in  a  condition  of  lowest 
nitrogenous  equilibrium. 

All  proteins  do  not  have  the  same  value  in  replacing  the  protein 
minimum.  Michaud  2  determined  the  protein  minimum  in  dogs  by 
feeding  entirely  with  nitrogen-free  food,  and  he  found  that  this  min- 
imum can  be  covered  by  the  corresponding  quantity  of  protein  specific 
of  the  animal,  but  not  by  the  same  quantity  of  an  alien  protein,  like 
gliadin  and  edestin.  v.  Hoesslin  and  Lesser  have  found  on  the  con- 
trary in  experiments  with  dogs  that  proteins  specific  to  the  animal  were 
only  unessentially  superior  to  the  proteins  of  horse  flesh,  and  E.  Voit 
and  Listerer  found  for  the  three  kinds  of  protein,  beef-muscle,  aleuronat 
and  casein,  that  the  relation  was  100  :  106  :  121.  Thomas3  has  carried 
out  experiments  on  man  with  different  foods  and  has  found  that  the 
nitrogen  of  various  kinds  of  proteins  has  an  unequal  value  in  replacing 
the  wear  and  tear  quota.  By  the  expression  "  biological  equivalence  " 
of  the  nitrogenous  foodstuffs  he  denotes  the  number  of  parts  of  body 
nitrogen  which  can  be  replaced  by  100  parts  of  the  food-nitrogen  and 
he  found  the  following  equivalence:  for  beef  =104.7,  milk  =  99.7,  casein  = 
70.14,  wheat  flour  =  39.6,  potatoes  =  78.9,  peas  =  55.7,  and  corn  =  29.5. 
Also  in  consideration  of  the  different  content  of  nitrogenous  extractives 
in  the  food  these  figures  therefore  show  that  different  proteins  have  essen- 
tially different  values  for  the  replacement  of  the  nitrogen  minimum. 

The  purposes  of  the  protein  as  foodstuff  are,  according  to  Rubner,  as 
follows:  (1)  To  compensate  for  the  wear  and  tear  quota;  (2)  betterment 
of  the  condition  of  the  cells;  and  (3)  dynamogenic  purpose.  In  the 
accomplishment  of  this  third  purpose  the  protein  splits  into  a  nitrogenous 

1  Rubner,  Theorie  d.  Ernahrung  nach  Vollendung  des  Wachstums,  Arch.  f.  Hyg., 
66,    1-80,  and  Ernahrungsorgange  behn  Wachstum  des    Ivindes,   ibid.,   66,  81-126. 

2  Zeitschr.  f.  physiol.  Chem.,  59. 

1  v.  Hoesslin  and  Lesser,  1.  c,  E.  Voit  and  Listerer,  Zeitschr.  f.  Biol.,  53;  Thomas, 
Arch.  f.  (Anat.  u.)  Physiol.,  1909. 


918 


METABOLISM. 


and  a  non-nitrogenous  part.  The  potential  energy  set  free  immediately 
as  heat  in  the  combustion  of  the  nitrogenous  part,  which  is  quantitatively 
used  within  the  region  of  the  chemical  heat  regulation  but  is  otherwise 
lost,  has  been  called  the  specific  dynamic  action  by  Rubner.1  The 
remainder  of  the  energy  which  is  represented  by  the  non-nitrogenous 
part  of  the  proteins,  serves,  like  all  other  foodstuffs,  in  satisfying  the 
energy  requirement  of  the  cells.  According  to  Rubner  only  non-nitrog- 
enous groups  (of  the  proteins,  fats  and  carbohydrates)  come  almost 
entirely,  if  not  completely,  in  consideration  for  purposes  of  energy. 

In  close  relation  to  the  second  purpose,  the  betterment  of  the  condi- 
tion of  the  cells,  stands  the  question  as  to  the  conditions  favoring  the  deposi- 
tion of  flesh  in  the  body,  which  is  closely  associated  with  the  question  as 
to  the  conditions  of  fattening  the  body.  In  this  connection  it  must  be 
remembered  in  the  first  place  that  all  fattening  presupposes  an  overfeed- 
ing, i.e.,  a  supply  of  foodstuffs  which  is  greater  than  that  catabolized  in 
the  same  time. 

In  carnivora  a  flesh  deposition  may  take  place  on  the  exclusive  feeding 
with  meat.  This  is  not  generally  large  in  proportion  to  the  quantity  of 
protein  catabolized.  In  man  and  herbivora,  who  cannot  cover  their 
calorific  needs  by  protein  alone,  this  is  not  possible,  and  the  question  as 
to  the  conditions  of  fattening  with  a  mixed  diet  is  of  importance. 

These  conditions  have  also  been  studied  in  carnivora,  and  here,  as 
Voit  has  shown,  the  relation  between  protein  and  fat  (and  carbo- 
hydrates) is  of  great  importance.  If  much  fat  is  given  in  proportion 
to  the  protein  of  the  food,  as  with  average  quantities  of  meat  with  con- 
siderable addition  of  fat,  then  nitrogenous  equilibrium  is  but  slowly 
attained  and  the  daily  deposit  of  flesh,  though  not  large,  is  quite  constant, 
and  may  become  greater  in  the  course  of  time.  If,  on  the  contrary, 
much  meat  besides  proportionately  little  fat  is  given,  then  the  deposit 
of  protein  with  increased  catabolism  is  smaller  day  by  day,  and  nitrog- 
enous equilibrium  is  attained  in  a  few  days.  In  spite  of  the  somewhat 
larger  deposit  per  diem,  the  total  flesh  deposit  is  not  considerable  in 
these  cases.     The  following  experiment  of  Voit  may  serve  as  example: 


Number  of 
Days  of  Ex- 
perimentation. 

Food. 

Total 

Deposit  of 

Fleah. 

Daily 

Deposit  of 

Flesh. 

Meat,  Grams. 

Fat,  Grams. 

Equilibrium. 

32 

7 

.500 

1800 

250 
250 

1792 

8.54 

56 
122 

Not  attained 
Attained 

The  greatest  absolute  deposition  of  flesh  in  the  body  was  obtained  in 
these  cases  with  only  500  grams  of  meat  and  250  grams  of  fat,  and  even 

1  Rubner,  1.  c,  and  Gesetze  des  Energieverbrauch.es,  70. 


PROTEIN  FATTENING.  919 

after  32  days  nitrogenous  equilibrium  had  not  occurred.  On  feeding 
with  1800  grams  of  meat  and  250  grams  of  fat  nitrogen  us  equilibrium 
was  established  ;ifter  seven  days;  and  though  the  deposition  of  flesh 
per  day  was  greater,  still  the  absolute  deposit  was  not  one-half  as  great 
as  in  the  former  case. 

The  possibility  of  a  protein  fattening  in  man  and  animals  (dogs,  sheep) 
is  shown  by  the  series  of  experiments  of  Krug,  Bornstein,  Schreuer, 
Henneberg,  Pfeiffer  and  Kalb  and  others  1  and  there  is  no  doubt 
that  such  a  fattening  is  possible.  That  we  are  here  not  dealing  with  an 
increase  in  the  number  of  cells,  but  rather  an  enlargement  of  the  volume 
of  the  same  is  the  generally  accepted  view.  Theories  as  to  the  value  and 
nature  of  this  protein-fattening  are  still  divergent,  as  we  must  differentiate 
between  flesh  accumulation  or  actual  organ  formation  and  protein 
accumulation  or  deposition  of  dead  protein,  and  opinions  vary  in 
regard  to  the  question  how  far  the  one  or  the  other  of  these  occur. 
By  determining  the  relation  between  P2O5  and  N  in  muscles,  kidneys 
and  liver  in  dogs  and  hens  in  starvation  and  in  fattening,  Grund  2  has 
tested  this  possibility  experimentally.  If  we  are  dealing  with  the  deposi- 
tion of  dead  protein  then  the  relationship  of  the  P2O5  to  the  N  would 
change  in  favor  of  the  nitrogen;  Grund  found  only  a  very  slight  change 
of  this  kind,  which  was  not  conclusive,  and  according  to  him  the  various 
organs  have  correspondingly  a  certain  tendency  of  maintaining  the  rela- 
tion between  phosphorus  and  nitrogen  unchanged  in  starvation  as  well 
as  in  fattening. 

It  is  difficult  to  produce  a  permanent  flesh  deposit  in  adult  man  by 
overfeeding  alone.  It  is  to  a  much  greater  degree  a  function  of  the  specific 
growth  energy  of  the  cells  and  the  cell-work  than  the  excess  of  food. 
Therefore  there  is  observed,  according  to  v.  Noorden,  abundant  flesh 
deposition  (1)  in  each  growing  body;  (2)  in  those  no  longer  growing,  but 
whose  body  is  accustomed  to  increased  work;  (3)  whenever,  by  previous 
insufficient  food  or  by  disease,  the  flesh  condition  of  the  body  has  been 
diminished  and  therefore  requires  abundant  food  to  replace  it.  The 
deposition  of  flesh  is  in  this  case  an  expression  of  the  regenerative  energy 
of  the  cells.3  >  *<       T-/.I>  ' 

The  experiences  of  graziers  show  that  in  food-animals  a  flesh  deposit 
does  not  occur,  or  at  least  is  only  inconsiderable,  on  overfeeding.     The 


1Krug,  Cited  by  v.  Noorden,  Lehrb.  der  Path,  des  Stoffwechsel,  1.  Aufl.,  p.  120; 
Bornstein,  Berl.  klin.  Woehenschr.,  1898,  and  Pfluger's  Arch.,  83  and  106;  Bornstein 
and  Schreuer,  Pfliiger's  Arch.,  110;  Henneberg  and  Pfeiffer,  see  Maly's  Jahresb., 
20;  Pfeiffer  and  Kalb,  ibid.,  22. 

2  G.  Grund,  Zeitschr.  f.  Biol.,  54. 

J  See  also  Svenson,  Zeitschr.  f.  klin.  Med.,  43. 


920  METABOLISM. 

individuality  and  the  race  of  the  animal  are  of  importance  for  flesh  deposi- 
tion. 

The  conditions  in  young,  growing  individuals  differ  from  those  in 
adults.  In  the  first  the  protein  is  necessary  for  the  building  up  of  the 
growing  tissue  and  in  them  an  abundant  true  flesh  deposition  takes  place. 
For  this  protein  fattening  the  amount  of  supply  does  not  take  first  place, 
but  rather  the  energy  of  development. 

As  above  stated  (Chapter  IX) ,  in  regard  to  the  formation  of  fat  in  the 
animal  body,  the  most  essential  condition  for  a  fat  deposition  is  an  over- 
feeding with  non-nitrogenous  foods.  The  extent  of  fat  deposition  is 
determined  by  the  excess  of  calories  administered  over  those  actually 
needed.  But  as  protein  and  fat  are  expensive  nutritive  bodies  as  com- 
pared with  carbohydrates,  the  supply  of  greater  quantities  of  carbo- 
hydrates is  important  for  fat  deposition.  The  body  decomposes  less 
substances  at  rest  than  during  activity.  Bodily  rest,  besides  a  proper 
combination  of  the  three  chief  groups  of  organic  foods,  is  therefore 
also  an  essential  requisite  for  an  abundant  fat  deposit. 

E.  Grafe  and  D.  Graham  1  report  an  experiment  on  a  dog  in  which  they  were 
able  to  keep  the  body  weight  nearly  constant  for  about  two  months  by  excessive 
food  with  about  210  per  cent  of  the  minimum  need  of  calories  and  with  a  diet 
very  rich  in  non-nitrogenous  food-stuffs.  No  fattening  occurred  in  this  case; 
the  calories  produced  were  considerably  increased  and  the  author  considers  this 
case  as  an  accommodation  to  the  food  and  a  luxus-consumption  of  non-nitrogenous 
food-stuffs. 

Action  of  Certain  Other  Bodies  on  Metabolism.  Water.  If  a  quan- 
tity in  excess  of  that  which  is  necessary,  is  introduced  into  the  organism, 
the  excess  is  quickly  and  principally  eliminated  with  the  urine.  This 
increased  elimination  of  urine  causes  in  fasting  animals  (Voit,  Forster), 
but  not  to  any  appreciable  degree  in  animals  taking  food  (Seegen,  Sal- 
kowski  and  Munk,  Mayer,  Dubelir2),  an  increased  elimination  of 
nitrogen.  The  reason  for  this  increased  nitrogen  excretion  is  to  be  found 
in  the  fact  that  the  drinking  of  much  water  causes  a  complete  washing 
out  of  the  urea  from  the  tissues.  Another  view,  which  is  defended  by 
Voit,  is  that  because  of  the  more  active  current  of  fluids,  after  taking 
large  quantities  of  water,  an  increased  metabolism  of  proteins  takes  place. 
Voit  considers  this  explanation  the  correct  one,  although  he  does  not 
deny  that  by  the  liberal  administration  of  water  a  more  complete  washing 
out  of  the  urea  from  the  tissues  takes  place.  Opinions  on  this  subject 
are  not  yet  in  accord,   and  recently  Heilner    has  advocated    Voit's 

1  Zeitschr.  f.  physiol.  Chem.,  73. 

2  Voit,  Untersuch.  uber  den  Einfluss  des  Kochsalzes,  etc.  (Munchen,  1860);  Forster, 
cited  from  Voit  in  Plennann's  Handbuch,  6,  153;  Seegen,  Wien.  Sitzungsber.,  63; 
Salkowski  and  Munk,  Virchow's  Arch.,  71;  Mayer,  Zeitschr.  f.  klin.  Med.,  2;  Dubelir, 
Zeitschr.  f.  Biologie,  28. 


ACTION  OF  SALTS  AND  ALCOHOL  UPON   METABOLISM.      921 

view.  The  recent  investigations  of  Abderhalden  1  show  a  washing 
out  of  the  retained  nitrogen  by  the  partaking  of  water. 

We  have  the  thorough  investigations  of  Hawk2  and  his  co-workers 
on  the  action  of  drinking  of  water  upon  the  digestion  and  absorption  of 
foods  as  well  as  upon  the  putrefaction  processes  in  the  intestine  and  the 
elimination  of  allantoin  and  purine  bodies  in  the  urine. 

When  the  body  has  lost  a  certain  amount  of  water,  then  the  abstinence 
from  water  (in  animals)  is  accompanied  by  a  rise  in  the  protein  metabo- 
lism (Landauer,  Straib3).  In  regard  to  the  action  of  water  on  the 
formation  of  fat  and  its  metabolism,  the  theory  that  the  free  drinking 
of  water  is  favorable  for  the  deposition  of  fat  seems  to  be  generally 
admitted,  while  the  drinking  of  only  very  little  water  acts  against  its 
formation.  For  the  present  we  have  no  conclusive  proofs  of  the  correct- 
ness of  this  view. 

Salts.  In  regard  to  the  action  of  salts — for  example  sodium  chloride 
and  the  neutral  salts — which  partly  depends  upon  the  use  of  large  and 
varying  amounts  of  salt  in  the  experiments,  the  authors  disagree.  Inves- 
tigations of  Stratjb  and  Rost4  show  that  the  action  of  salts  stands  in 
close  relation  to  their  power  of  abstracting  water.  Small  amounts  of 
salt  which  do  not  produce  diuresis  have  no  action  on  metabolism.  On 
the  contrary,  larger  amounts,  which  bring  about  a  diuresis,  which  is 
not  compensated  by  the  ingestion  of  water,  produce  a  rise  in  the  pro- 
tein metabolism.  If  the  diuresis  is  compensated  by  drinking  water, 
then  the  protein  metabolism  is  not  increased  by  salts,  but  is  diminished 
to  a  slight  degree.  An  increased  nitrogen  excretion  caused  by  taking 
salts  can  be  increased  by  the  ingestion  of  water,  thus  increasing  the 
diuresis,  and  the  action  of  salts  seems  to  bear  a  close  relation  to  the 
demand  and  supply  of  water. 

Alcohol.  The  question  as  to  how  far  the  alcohol  absorbed  in  the 
intestinal  canal  is  burnt  in  the  bod}-,  or  whether  it  leaves  the  body 
unchanged  by  various  channels,  has  been  the  subject  of  much  discussion. 
To  all  appearances  the  greatest  part  of  the  alcohol  introduced  (95  per 
cent  or  more)  is  burnt  in  the  body  (Stubbotin,  Thudichum,  Bodlander, 
Benedicenti  5).     As  the  alcohol  has  a  high  calorific  value  (1  gram  =  7.1 


1  See  R.  Neumann,  Arch.  f.  Hygiene,  36;  Heilner,  Zeitschr.  f.  Biologie,  47  and  49; 
Hawk,  University  of  Pennsylvania  Med.  Bull.,  xviii;  Abderhalden,  Zeitschr.  f.  physiol. 
Chem.,  59. 

2  See  Journ.  of  biol.  Chem.,  10  and  11,  Arch,  of  internat.  Med.,  1911,Journ.  of 
Amer.  Chem.  Soc.,  33  and  34. 

3  Landauer,  Maly's  Jahresber.,  24;  Straub,  Zeitschr.  f.  Biologie,  37. 

4  W.  Straub,  Zeitschr.  f.  Biologie,  37  and  38;  Rost,  Arbeiten  aus  d.  Kaiserliche 
Gesundheitsamte,  18  (literature).     See  also  Griiber,  Maly's  Jahresber.,  30,  612. 

s  Arch.  f.  (Anat.  u.)  Physiol.,  1896.  which  contains  the  literature. 


922  METABOLISM. 

calories),  then  the  question  arises  whether  it  acts  sparingly  on  other 
bodies,  and  whether  it  is  to  be  considered  as  a  nutritive  substance.  The 
earlier  investigations  made  to  decide  these  questions  have  led  to  no 
decisive  result.  The  thorough  investigations  of  Atwater  and  Benedict, 
Zuntz  and  Geppert,  Bjerre,  Clopatt,  Neumann,  Offer,  Rosemann,1 
and  others,  seem  to  show  positively  that,  in  man,  alcohol  can  diminish  the 
consumption  not  only  of  fat  and  carbohydrates,  but  also  the  proteins, 
although  at  first,  due  to  its  poisonous  properties,  it  may  increase  the  pro- 
tein metabolism  for  a  short  time.  The  nutritive  value  of  alcohol  can 
be  of  special  importance  in  certain  cases  only,  as  large  amounts  of  alcohol 
taken  at  one  time,  or  the  continued  use  of  smaller  quantities,  has  an  injur- 
ious action  on  the  organism.  Alcohol  may  therefore  be  regarded  as  a 
foodstuff  only  in  exceptional  cases,  and  in  other  respects  must  be  con- 
sidered as  an  article  of  luxury. 

Coffee  and  tea  have  no  action  on  the  exchange  of  material  which  can 
be  positively  proven,  and  their  importance  lies  chiefly  in  their  action 
upon  the  nervous  system.  It  is  impossible  to  enter  into  the  effect  of 
various  therapeutic  agents  upon  metabolism. 

IV.     THE  DEPENDENCE   OF   METABOLISM   ON   OTHER   CONDITIONS. 

The  so-called  basal  requirement  which  was  previously  mentioned, 
i.e.,  the  extent  of  metabolism  with  absolute  rest  of  body  and  inactivity 
of  the  intestinal  tract,  serves  best  as  a  starting-point  for  the  study  of 
metabolism  under  various  external  circumstances.  The  metabolism 
going  on  under  these  conditions  leads  in  the  first  place  to  the  production 
of  heat,  and  it  is  only  to  a  subordinate  degree  dependent  upon  the  work 
of  the  circulatory  and  respiratory  apparatus  and  the  activity  of  the  glands. 
According  to  a  calculation  by  Zuntz,2  only  10-20  per  cent  of  the  total 
calories  of  the  basal  requirement  belongs  to  the  circulation  and  respira- 
tion work. 

The  magnitude  of  the  basal  requirement  depends  in  the  first  place 
upon  the  heat  production  necessary  to  cover  the  loss  of  heat,  and  this 
heat  production  is  in  turn  dependent  upon  the  relation  between  the  weight 
and  the  surface  of  the  body. 

Weight  of  Body  and  Age.  The  greater  the  mass  of  the  body  the  greater 
the   absolute   consumption  of  material;    while,   on  the   contrary,   other 

1  In  regard  to  the  literature  on  this  subject,  see  the  works  of  O.  Neumann,  Arch, 
f.  Hygiene,  36  and  41,  and  Rosemann,  Pfliiger's  Arch.,  86  and  94.  A  summary  of 
the  entire  literature  upon  alcohol  can  be  found  in  Abderhalden,  "  Bibliographie  der 
gesamten  wissenschaftlichen  Literatur  iiber  den  Alcohol  und  den  Alcoholismus," 
Berlin  and  Wien,  1904.  See  also  Rosemann  in  Oppenheimer's  Handb.  d.  Bioch.,  Bd. 
4,1. 

2  Cited  from  v.  Xoorden's  Handbuch,  1.  Aufl.,  page  97. 


DEPENDENCE  OF  WEIGHT  OF  BODY  AND  AGE.  923 

things  being  equal,  a  small  individual  of  the  same  species  of  animal  metab- 
olizes absolutely  less,  but  relatively  more  as  compared  with  the  unit  of 
the  weight  of  the  body.  With  increasing  bodily  weight  the  total  metab- 
olism per  kilo  of  animal  diminishes,  which  is  true  first  for  individuals  of 
the  same  species  of  animals,  but  also  seems  to  have  a  certain  correctness 
on  the  comparison  of  different  species  of  animals.  It  must  be  remarked 
that  the  relation  between  flesh  and  fat  in  the  body  exerts  an  important 
influence.  The  extent  of  the  metabolism  is  dependent  upon  the  quantity 
of  active  cells,  and  a  very  fat  individual  therefore  decomposes  less  sub- 
stance per  kilo  than  a  lean  person  of  the  same  weight.  According  to 
Rubner  l  the  importance  of  the  size  of  the  flesh  or  cell-mass  in  the  body 
is  overestimated.  In  his  investigations  on  two  boys,  one  of  whom  was 
corpulent  and  the  other  normally  developed,  and  on  comparing  the  food- 
need  with  that  found  by  Camerer  for  boys  of  the  same  weight,  Rubner 
came  to  theVesult  that  the  exchange  of  force  in  the  corpulent  boy  almost 
completely  corresponded  with  that  in  the  non-corpulent  boy  of  the  same 
weight.  By  approximately  estimating  the  quantity  of  fat  in  the  body 
Rubner  was  also  able,  from  the  protein  condition,  to  compare  the  cal- 
culated exchange  of  energy  with  that  actually  found.  The  exchange 
per  kilo  amounted  to  52  calories  in  the  lean  and  43.6  calories  in  the  fat 
boy,  while,  if  the  protein  condition  was  a  measure,  one  would  expect 
an  exchange  of  calories  of  only  35  calories  for  the  fat  person.  We  can- 
not therefore  admit  of  a  diminished  activity  of  the  cell-mass  in  the  fat  boy, 
but  rather  an  increased  activity.  According  to  Rubner  it  is  not  the 
flesh  -  mass  (protein  mass)  alone,  but  its  variable  functional  changes, 
which  determine  the  extent  of  decomposition.  In  women,  who  generally 
have  less  body  weight  and  a  greater  quantity  of  fat  than  men,  the  metab- 
olism in  general  is  smaller,  and  the  latter  is  ordinarily  about  four-fifths 
that  of  men. 

The  essential  reason  why  small  animals  catabolize  relatively  more 
substance  than  large  ones,  when  calculated  per  kilo  body  weight,  is  that 
the  bodies  of  smaller  animals  have  greater  surface  in  proportion  to  their 
mass.  On  this  account  the  loss  of  heat  is  greater,  which  causes  increased 
heat  production,  i.e.,  a  more  active  metabolism.  This  is  also  the  reason 
why  young  individuals  of  the  same  kind  show  a  relatively  greater  metab- 
olism than  older  ones.  If  the  heat  production  and  carbon-dioxide  elim- 
ination is  calculated  on  the  unit  of  surface  of  the  body,  we  find,  on  the 
contrary,  as  the  experiments  of  Rubner,  Richet,2  and  others  show, 
that  they  vary  only  slightly  from  a  certain  average  in  individuals  of 
different  weights. 


1  Beitrage  zur  Ernahrung  im  Knabenalter,  etc.     Berlin,  1902. 

*  Rubner,  Zeitschr.  f.  Biologie,  19  and  21;  Richet,  Arch,  de  Physiol.,  5  (2). 


924  METABOLISM. 

According  to  Rubner's  rule  as  to  the  influence  of  the  surface,  which 
has  been  recently  formulated  by  E.  Voit,  the  need  of  energy  in  homceo- 
thermic  animals  is  influenced  by  the  development  of  their  surface  when 
their  body  is  given  rest,  medium  surrounding  temperature,  and  relatively 
equal  protein  condition.  This  rule  applies  not  only  to  adult  human  beings, 
but  also  to  children  and  growing  individuals  (Rubner,  Oppenheimer, 
Schlossmann  and  Murschhauser)  .  The  surface  is  the  essential  factor 
in  determining  the  extent  of  exchange  of  energy.  In  order  to  show 
this  we  will  give  here,  from  a  work  of  Rubner,1  the  figures  representing 
the  quantity  of  heat  in  calories  for  1  square  meter  of  surface  for  twenty- 
four  hours: 

Adult,  medium  diet,  rest 1189  calories. 

Adult,  medium  diex,  work 1399      ' ' 

Suckling 1221      " 

Child  with  medium  diet 1447      ' ' 

Aged  men  and  women 1099      ' ' 

Women 1004      ' ' 

The  variation  in  the  calorific  values2  found  by  many  investigators, 
which  is  sometimes  not  very  small,  suggests  the  fact  that  the  surface 
rule  is  not  alone  decisive  for  the  exchange  of  material  in  resting  animals. 
Still  it  is  generally  considered  that  it  is  of  the  greatest  importance  in 
metabolism. 

The  more  active  metabolism  in  young  individuals  is  apparent  when 
we  measure  the  gaseous  exchange  as  well  as  the  excretion  of  nitrogen. 
As  example  of  the  elimination  of  urea  in  children  the  following  results  of 
Camerer  3  are  of  value : 


Age. 

Weight  of  Body 

Urea  in  Grams. 

in  Kilos. 

Per  Day. 

Per  Kilo. 

1 \  years 

10.80 

12.10 

1.35 

3       "     

13.30 

11.10 

0.90 

5       "     

16.20 

12.37 

0.76 

7       "     

18.80 

14.05 

17.27 

0.75 

9       "     

25.10 

0.69 

12J     "     

32.60 

17.79 

0.54 

15       "     

35.70 

17.78 

0.50 

In  adults  weighing  about  70  kilos,  from  30  to  35  grams  of  urea  per  day 
are  eliminated,  or  0.5  gram  per  kilo.  At  about  fifteen  years  of  age  the 
destruction  of  proteins  per  kilo  is  about  the  same  as  in  adults.  The 
relatively  greater  metabolism  of  proteins  in  young  individuals  is  explained 
partly  by  the  fact  that  the  metabolism  of  material  in  general  is  more 
active  in  young  animals,  and  partly  by  the  fact  that  young  animals  are, 
as  a  rule,  poorer  in  fat  than  those  full  grown. 

1  Rubner,  Ernahrung  im  Knabenalter,  page  45;    E.  Voit,  Zeitschr.  f.  Biologie,  41; 
Oppenheimer,  ibid.,  42;  Schlossmann  and  Murschhauser,  Bioch.  Zeitschr.,  18  and  26. 
1  See  Magnus-Levy,  Pfluger's  Arch.,  55;  Slowtzoff  (u.  Zuntz),  ibid.,  95. 
»  Zeitschr.  f .  Biologie,  16  and  20. 


SEX.     REST  AND  WORK.  925 

That  young  individuals  show  a  more  active  metabolism  than  adults, 
follows,  as  above  stated,  principally  from  the  relatively  greater  body 
surface  in  the  first  as  compared  to  the  latter.  According  to  Tigerstedt 
and  Sonden,  the  greater  metabolism  in  young  animals  depends  neverthe- 
less, also  in  part,  on  the  fact  that  in  these  individuals  the  decomposition 
in  itself  is  more  active  than  in  older  ones.  The  period  of  growth  has  a 
considerable  influence  on  the  extent  of  metabolism  (in  man),  and  indeed 
the  metabolism,  even  when  calculated  on  the  unit  of  surface  of  body,  is 
greater  in  youth  than  in  old  age.  This  view  is  strongly  disputed  by 
Rubner.  He  does  not  deny  that  differences  exist  between  young  and 
adult  individuals  which  may  be  considered  as  a  deviation  from  the  above 
rule;  still  these  differences  may,  he  claims,  be  dependent  upon  the  work 
performed,  the  food,  and  the  nutritive  condition.  Magnus-Levy  and 
Falk  l  have  reported  observations  which  support  the  conclusions  of 
Sonden  and  Tigerstedt. 

Nurslings  have  a  behavior  different  from  older  children,  as  with  them 
during  the  first  months  of  life,  and  especially  the  first  three  days,  the 
metabolism,  calculated  on  the  unit  of  surface,  is  strikingly  low,  and 
lower  than  with  adults.  After  about  two  weeks  it  attains  about  the  same 
height  as  adults  (Scherer,  Forster2). 

In  old  age  the  metabolism  is  very  much  reduced;  and  even  when  calcu- 
lated upon  the  square  meter  of  surface  of  body  it  is  lower  than  in  an 
individual  of  medium  age. 

The  question  as  to  what  extent  sex  specially  influences  metabolism 
remains  to  be  investigated.  Tigerstedt  and  Sonden  found  that  in  the 
young  the  carbon-dioxide  elimination,  per  kilo  of  body  weight,  as  well  as 
per  square  meter  of  body  surface,  was  considerably  greater  in  males 
than  in  females  of  the  same  age  and  the  same  weight  of  body.  This 
difference  between  the  sexes  seems  to  disappear  gradually,  and  at  old 
age  it  is  entirely  absent.  The  investigations  of  Magnus-Levy  and 
Falk  oppose  these  observations.  They  investigated  by  means  of  the 
Zuntz-Geppert  method,  not  only  children,  but  also  adults  and  old 
persons  of  both  sexes,  but  could  not  observe  any  positive  influence  of 
the  sex  upon  metabolism.3 


1  Tigerstedt  and  Sonden,  Skand.  Arch.  f.  Physiol.  6;  Rubner,  1.  c;  and  Arch.  f. 
Hygiene,  66;    Magnus-Levy,  Arch.  f.  (Anat.  u.)  Physiol.,  1899,  Suppl. 

1  Cited  by  A.  Loewy  in  Oppenheimer's  Handb.,  Bd.  4,  189. 

*  Tigerstedt  and  Sonden,  Skand.  Arch.  f.  Physiol.,  6;  Magnus-Levy  and  Falk, 
Arch.  f.  (Anat.  u.)  Physiol.,  1899,  Suppl.  In  regard  to  metabolism  and  its  relation 
to  the  phases  of  sexual  life  and  especially  under  the  influence  of  menstruation  and 
pregnancy,  see  the  investigations  of  A.  Ver  Eecke  (Bull.  acad.  roy.  de  m£d.  de  Bel- 
gique,  1897  and  1901,  and  Maly's  Jahresber.,  30  and  31).  See  also  Magnus-Levy 
in- v.  Noorden's  Handb.  d.  Pathol,  d.  Stoffwechsels. 


926  METABOLISM. 

As  the  metabolism  may  be  kept  at  its  lowest  point  by  absolute  rest 
of  body  and  inactivity  of  the  intestinal  tract,  it  is  manifest  that  work 
and  the  ingestion  of  focd  have  an  important  bearing  on  the  extent  of 
metabolism. 

Rest  and  Work.  During  work  a  greater  quantity  of  chemical  energy 
is  converted  into  kinetic  energy,  i.e.,  the  metabolism  is  increased  more  or 
less  on  account  of  work. 

As  explained  in  a  previous  chapter  (X),  work,  according  to  the  gen- 
erally accepted  view,  has  no  material  influence  on  the  excretion  of  nitro- 
gen. It  is  nevertheless  true  that  several  investigators  have  observed,, 
in  certain  cases,  an  increased  elimination  of  nitrogen;  this  increase  does 
not  seem  to  be  directly  related  to  the  work,  but  to  be  caused  by  secondary 
circumstances.  These  observations  have  been  explained  in  other  ways. 
For  instance,  work  may,  when  it  is  connected  with  violent  movements 
of  the  body,  easily  cause  dyspnoea,  and  this  last,  as  Frankel  j  has  shown, 
may  occasion  an  increase  in  the  elimination  of  nitrogen,  since  diminution 
of  the  oxygen  supply  increases  the  protein  metabolism.  In  other  series 
of  experiments  the  quantity  of  carbohydrates  and  fats  in  the  food  was 
not  sufficient;  the  supply  of  fat  in  the  body  was  decreased  thereby,  and 
the  destruction  of  proteins  was  correspondingly  increased.  Other  condi- 
tions, such  as  the  external  temperature  and  the  weather,2  thirst,  and 
drinking  of  water,  can  also  influence  the  excretion  of  nitrogen.  The 
prevailing  sentiment  is  that  muscular  activity  has  hardly  any  influence 
on  the  metabolism  of  proteins. 

On  the  contrary,  work  has  a  very  considerable  influence  on  the  elimi- 
nation of  carbon  dioxide  and  the  consumption  of  oxygen.  This  action, 
which  was  first  observed  by  Lavoisier,  has  later  been  confirmed  by 
many  investigators.  Pettenkofer  and  Voit3  have  made  investigations 
tions  on  a  full-grown  man  as  to  the  metabolism  of  the  nitrogenous  as  well 
as  of  the  non-nitrogenous  bodies  during  rest  and  work,  partly  while 
fasting  and  partly  on  a  mixed  diet.  The  experiments  were  made  on  a 
full-grown  man  weighing  70  kilos.  The  results  are  contained  in  the 
following  table: 


Fasting  .  . 
Mixed  diet . 


1  Virchow's  Arch.,  67  and  71.     In  regard  to  disputed  views  see  C.  Voit,  Zeitschr. 
f.  Biol.,  49,  and  Frankel,  ibid.,  50. 

1  See  Zuntz  and  Hchumburg,  Aroh.  f.  (Anat.  u.)  Physiol.,  1895. 
1  Zeitschr.  f .  Biologie,  2. 


- 

Ci 

jnsumpt 

ion  of 

Proteins. 

Fat. 

Carbohydrates.  CO2  Eliminated. 

O  Consumed. 

Rest .  . 

.       79 

209 

716 

761 

Work . 

...75 

380 

1187 

1071 

Rest.  . 

...137 

72 

352                    912 

831 

Work . 

. . . 137 

173 

352                   1209 

980 

WORK  AND  GAS  EXCHANGE.  927 

In  these  cases  work  did  not  seem  to  have  any  influence  on  the  destruc- 
tion of  proteins,  while  the  gas  exchange  was  considerably  increased. 

Zuntz  and  his  pupils  l  have  made  important  investigations  on  the 
extent  of  the  exchange  of  gas  as  a  measure  of  metabolism  during  work 
and  caused  by  work.  These  investigations  not  only  show  the  impor- 
tant influence  of  muscular  work  on  the  catabolism  of  material,  but  they 
also  indicate,  in  a  very  instructive  way,  the  relation  between  the  extent 
of  metabolism  of  material  and  its  utilization  for  work  of  various  kinds. 
We  can  refer  only  to  those  which  are  of  special  physiological  interest. 

The  action  of  muscular  work  on  the  gas  exchange  does  not  alone  appear 
with  hard  work.  From  the  researches  of  Speck  and  others  we  learn  that 
even  very  small,  apparently  quite  unessential  movements  may  increase 
the  production  of  carbon  dioxide  to  such  an  extent  that  by  not  observing 
these,  as  in  numerous  older  experiments,  very  considerable  errors  may 
creep  in.  Johansson  2  has  also  made  experiments  upon  himself,  and 
finds  that  on  the  production  of  as  complete  a  muscular  inactivity  as 
possible  the  ordinary  amount  of  carbon  dioxide  (31.2  grams  per  hour 
at  rest  in  the  ordinary  sense)  may  be  reduced  nearly  one-third,  or  to  an 
average  of  22  grams  per  hour. 

The  quantity  of  carbon  dioxide  eliminated  during  a  working  period 
is  uniformly  greater  than  the  quantity  of  oxygen  taken  up  at  the  same 
time,  and  hence  a  raising  of  the  respiratory  quotient  was  usually  con- 
sidered as  caused  by  work.  This  rise  does  not  seem  to  be  based  upon  the 
cl.aracter  of  the  chemical  processes  going  on  during  work,  as  we  have  a 
series  of  experiments  made  by  Zuntz  and  his  collaborators,  Lehmann,  Kat- 
zenstein  and  Hagemann,3  in  which  the  respiratory  quotient  remained 
almost  wholly  unchanged  in  spite  of  work.  According  to  Loewy  4  the 
combustion  processes  in  the  animal  body  go  on  in  the  same  way  in  work 
as  in  rest,  and  a  raising  cf  the  respiratory  quotient  (irrespective  of  the 
transient  change  in  the  respiratory  mechanism)  takes  place  only  with 
insufficient  supply  of  oxygen  to  the  muscles,  as  in  continuous  fatiguing 
work  or  excessive  muscular  activity  for  a  brief  period,  also  with  local 
lack  of  oxygen  caused  by  excessive  work  of  certain  groups  of  muscles. 
This  varying  condition  of  the  respiratory  quotient  has  been  explained  by 


1  See  the  works  of  Zuntz  and  Lehmann,  Maly'a  Jahresber.,  19;  Katzenstein,  Pfliiger's 
Arch.,  49;  Loewy,  ibid.;  Zuntz,  ibid.,  68;  Zuntz  and  Slowtzoff,  ibid.,  95;  and  especially 
the  large  work  "  Untersuch.  iiber  den  Stoffwechsel  des  Pferdes  bei  Ruhe  und  Arbeit," 
Zuntz  and  Hagemann,  Berlin,  1898;  Hohenklima  und  Bergwanderungen  by  Zuntz, 
Loewy,  Miiller  and  Caspari,  which  also  contains  a  bibliography. 

^ord.  Med.  Arkiv.  Festband,  1897;  also  Maly's  Jahresber.,  27;  Speck,  "Physiol, 
des  menschl.  Atmens,"  Leipzig,  1892. 

3  See  footnote  1 . 

4  Pfliiger's  Arch.,  49. 


928  METABOLISM. 

Katzenstein  by  the  statement  that  during  work  two  kinds  of  chemical 
processes  act  side  by  side.  The  one  depends  upon  the  work  which  is 
connected  with  the  production  of  carbon  dioxide,  also  in  the  absence 
of  free  oxygen,  while  the  other  brings  about  the  regeneration  which  takes 
place  by  the  taking  up  of  oxygen.  When  these  two  chief  kinds  of  chemical 
processes  make  the  same  progress  the  respiratory  quotient  remains 
unchanged  during  work;  if  by  hard  work  the  decomposition  is  increased 
as  compared  with  the  regeneration,  then  a  raising  of  the  respiratory 
quotient  takes  place.  If,  on  the  contrary,  moderate  work  is  continued 
and  performed  in  a  way  so  that  irregularities  and  occasional  changes 
in  the  circulation  and  respiration  are  excluded  or  are  without  importance, 
then  the  respiratory  quotient  may  correspondingly  remain  the  same 
during  work  as  in  rest.  Its  extent  is  thus  determined  in  the  first  place 
by  the  nutritive  material  at  its  disposal  (Zuntz  and  his  pupils). 

The  theory  of  Loewy  and  Zuntz,  that  the  raising  of  the  respiratory  quotient 
during  work  is  to  be  explained  by  an  insufficient  supply  of  oxygen,  is  opposed 
by  Laulanie.1  He  has  observed  the  reverse,  namely,  a  diminution  in  the 
respiratory  quotient  during  continuous  excessive  work,  and  this  is  not  reconcilable 
with  the  above  statements.  He  considers  that  sugar  is  the  source  of  muscular 
energy,  and  that  the  rise  in  the  respiratory  quotient  is  due  to  an  increased  combus- 
tion of  sugar.  Its  diminution,  he  explains,  is  caused  by  a  re-formation  of  sugar 
from  fat  which  takes  place  at  the  same  time  and  is  accompanied  by  an  increased 
consumption  of  oxygen. 

In  sleep  metabolism  decreases  as  compared  with  that  during  waking 
hours,  and  the  most  essential  reason  for  this  is  the  muscular  inactivity 
during  sleep.  The  investigations  of  Rubner  upon  a  dog,  and  of  Johans- 
son2 upon  human  beings,  teach  us  that  if  the  muscular  work  is  elim- 
inated the  metabolism  during  waking  hours  is  not  greater  than  in  sleep. 

The  action  of  light  also  stands  in  close  connection  with  the  question 
of  the  action  of  muscular  work.  It  seems  positively  proven  that  metabo- 
lism is  increased  under  the  influence  of  light.  Most  investigators,  such  as 
Speck,  Loeb,  and  Ewald,3  consider  that  this  increase  is  due  to  the  move- 
ments caused  by  the  light  or  an  increased  muscle  tonus,  and  in  man  an 
increase  in  metabolism  under  the  influence  of  light  with  complete  rest 
has  not  been  observed.  Divergent  results  have  been  obtained  in  animals, 
and  our  knowledge  of  the  truth  is  not  yet  complete.4 

Menial  activity  docs  not  seem  to  have  any  influence  on  metabolism 
according  to  the  means  at  hand  for  studying  this  influence. 


1  Arch,  de  Physiol.  (5),  8,  572. 

1  Rubner,   Ludwig-Festechr.,    1887;    Loewy,   Berl.   klin.   Wochenschr.,    1891,   434;, 
Johansson,  Skand.  Arch.  f.  Physiol.,  8. 

1  Speck,  1.  c;  Loeb,  Pfluger's  Arch.,  42;  Ewald,  Journ.  of  Physiol.,  13. 
4  See  larger  handbooks  for  the  literature  on  this  question. 


HEAT  REGULATION'   IN   ANIMALS.  929 

The  Action  of  the  External  Temperature  also  stands  in  close  relation 
to  muscular  work,  namely  to  the  question  as  to  whether  the  chemical 
heat  regulation  is  independent  of  the  muscular  activity.  The  heat 
regulation,  as  is  well  known,  is  of  two  kinds,  namely  the  chemical  heat 
regulation,  which  consists  in  a  change  in  the  metabolism  and  which  man- 
ifests itself  as  an  increased  heat  production  due  to  the  increased  metabo- 
lism at  low  temperatures,  and  the  physical  heat  regulation,  which  occurs 
generally  at  higher  temperatures  and  is  caused  by  changes  in  the  con- 
ditions in  the  heat  elimination  of  the  thermal  equilibrium. 

In  regard  to  the  chemical  heat  regulation,  which  will  only  be  discussed 
here,  we  must  differentiate  between  cold-blooded  and  warm-blooded 
animals.  In  the  first  the  metabolism  rises  with  an  increase  in  the  surround- 
ing temperature,  while  in  the  second  group  the  conditions  are  different. 
The  experiments  of  Speck,  Loewy  and  Johansson  j  on  human  beings 
have  shown  that  the  lowering  of  the  external  temperature  is  without 
influence  upon  the  extent  of  metabolism  (measured  by  the  gas  exchange) 
only  as  long  as  all  natural  and  non-voluntary  movements  of  the  muscles 
are  excluded;  otherwise  the  metabolism  is  raised.  A  chemical  heat  regu- 
lation, i.e.,  a  rise  in  metabolism  without  noticeable  movements  of  the 
muscles,  is  not  accepted  in  man,  or  at  least  it  has  not  been  proven.  The 
heat  regulation,  in  man,  at  lower  temperatures  seems  to  be  brought  about 
by  the  natural  or  reflex  production  of  muscle  action,  nor  has  a  chemical 
heat  regulation  in  the  reverse  sense,  namely,  a  fall  in  the  catabolism  by 
raising  the  external  temperature,  been  shown  in  man.  The  investigations 
of  Eykman  2  upon  inhabitants  of  the  tropics  also  show  the  same  result, 
namely,  that  in  human  beings  no  appreciable  chemical  heat  regulation 
occurs. 

In  animals  the  conditions  are  different  so  far  as  that  a  chemical  heat 
regulation  in  the  true  sense  has  been  positively  shown.  The  investiga- 
tions of  Rubner3  on  various  animals  have  shown  that  the  reduction 
of  the  external  temperature  with  these,  causes  a  considerable  chemical 
heat  regulation  by  increasing  the  metabolism  without  any  chill  or  shiver 
movements.  On  sufficient  cooling  the  temperature  of  the  body  may  fall 
irrespective  of  the  increased  metabolism,  and  at  a  certain  limit  of  body 
temperature  the  exchange  of  material  becomes  still  lower  with  decreasing 
temperature.  According  to  Rubner  many  animals  can  bear  a  temperature 
of  0°  C.  for  days  in  absolute  rest.  If  the  natural  muscular  activity  is 
eliminated  by  poisoning  with  curare  or  by  section  of  the  spinal  cord,  then, 
as  shown  by  Pfluger  4  and  his  pupils,  the  warm-blooded  animal  behaves 

1  Speck,  1.  c;  Loewy,  Pfliiger's  Arch.,  46;  Johansson,  Skand.  Arch.  f.  Physiol.,  7. 

2  Virchow's  Arch.,  133,  and  Pfluger's  Arch.,  64. 

'  Arch.  f.  Hyg.,  37,  and  Handbuch  d.  Hyg.,  Bd.  1,  Leipzig,  1911. 
4  See  footnote  2,  page  591. 


930  METABOLISM. 

like  a  cold-blooded  animal,  and  the  metabolism  decreases  parallel  with 
the  body  temperature.  In  normal  animals,  on  the  contrary,  the  body 
temperature  can  be  kept  constant,  on  lowering  the  external  temperature, 
by  an  increased  metabolism;  but  also  in  such  animals  because  of  a  rise 
in  the  external  temperature  a  rise  in  the  metabolism  above  a  certain 
limit  can  also  take  place. 

A  very  interesting  and  important  question  is  the  action  of  high  altitude 
upon  the  oxidation  processes,  the  economy  of  temperature,  the  protein 
exchange  and  the  general  metabolism.  The  results  of  the  laborious 
and  important  investigations  on  this  subject  may,  be  found  in  the  large 
work  of  N.  Zuntz,  A.  Loewy,  F.  Muller  and  W.  Caspari.1 

That  the  ingestion  of  food  raises  the  metabolism  has  been  known  for 
a  rather  long  time,  and  this  has  been  studied  by  Zuntz,  v.  Mering,  Mag- 
nus-Levy, Voit,  Rubner,  Johansson  and  collaborators,  also  by  Heilner 
and  by  Gigon.2  It  follows  from  these  investigations  that  this  rise  in 
metabolism,  which  in  man,  on  sufficient  supply  of  food,  amounts  to  a  rise 
of  10-15  per  cent  of  the  basal  requirement  and  with  abundant  supply  of 
food  may  be  still  larger  (35  per  cent  in  the  researches  of  Johansson, 
Tigerstedt  and  collaborators),  has  a  double  cause,  namely,  partly  a 
digestion  work  (Zuntz)  and  partly  a  chemical  decomposition  (specific 
dynamic  action  of  Rubner)  which  takes  place  at  the  same  time. 

The  sum  of  all  the  work  which  is  necessary  for  the  chemical  trans- 
formation of  the  foods,  as  well  as  for  the  mechanical  division  and  trans- 
portation of  the  food  in  the  intestinal  canal,  is  called  the  digestion  work  by 
Zuntz.  That  such  work  exists  has  been  shown  by  Zuntz  and  v.  Mering 
by  comparative  tests  of  the  different  action  upon  metabolism  by 
foods  introduced  per  os  and  intravenously,  and  recently  Cohnheim3 
has  shown  that  in  sham  feeding  an  increased  catabolism  of  non- 
nitrogenous  body  constituents  took  place.  The  influence  of  digestion 
work  in  Zuntz's  sense  is  especially  apparent  in  herbivora,  in  which  this 
work,  according  to  Zuntz  and  collaborators,  may  amount  to  the  consump- 
tion of  more  than  50  per  cent  of  the  total  energy  content  of  the  raw 
fodder. 


1  Hohenklima  und  Bergwanderungen  in  ihrer  Wirkung  auf  den  Menschen,  Berlin, 
1906. 

2  Zuntz  and  v.  Mering,  Pfluger's  Arch.,  15;  Zuntz,  Naturw.  Rundschau,  21  (1906), 
with  Hagemann,  1.  c,  with  Magnus-Levy,  Pfluger's  Arch.,  49;  Magnus-Levy,  ibid., 
55,  and  v.  Noorden's  Handbuch;  Voit,  Hermann's  Handbuch,  6;  Rubner,  Zietschr. 
f.  Biol.,  19  and  21;  and  Arch.  f.  Hyg.,  66;  Johansson,  Skand.  Arch.  f.  Physiol.,  21, 
with  Koraen,  ibid.,  13;  Heilner,  Zeitschr.  f.  Biol.,  48  and  50;  Gigon,  Pfluger's  Arch., 
140. 

'Arch.  f.  Hyg.,  57. 


SPECIFIC  DYNAMIC  ACTION.  931 

On  partaking  of  largo  amounts  of  food,  especially  proteins,  by  car- 
nivora,  the  digestion  work  in  the  above  sense  is  not  sufficient  to  account 
for  the  increase  in  metabolism,  and  in  these  cases,  besides  this,  we  must 
accept  an  increase  in  the  chemical  transformation  process  in  the  animal 
body  brought  on  by  the  foodstuffs  in  an  unknown  manner  (specific 
dynamic  action  of  foodstuffs,  according  to  Rubner).  The  only  real 
difference  in  opinion  between  the  various  experimenters  consists,  so  far 
as  Hammarsten  can  see,  in  that  according  to  the  Zuntz  school,  normally 
on  supplying  sufficient  food  it  is  the  digestion  work  in  the  above  sense 
which  chiefly  causes  the  rise  in  metabolism  after  taking  food,  while 
according  to  the  views  of  Voit-Rubner,  with  which  Heilner  agrees, 
it  is  on  the  contrary  the  specific  dynamic  action. 

That  the  proteins  or  their  cleavage  products,  without  regard  to  the 
digestion  work,  cause  a  rise  in  the  metabolism  seems  to  be  generally 
accepted.  This  rise,  according  to  Gigon,  is  not  proportional  to  the 
protein  supply,  as  on  supplying  quantities  of  protein  represented  by 
1:2:4:3  the  oxygen  absorption  was  in  the  proportion  1:3:6:9  and  the 
carbon  dioxide  elimination  was  in  the  proportion  1:4:8:12.  On  the 
introduction  of  glucose  Gigon  found,  as  first  shown  by  Johansson,1 
that  the  introduction  of  carbohydrate  caused  a  proportional  rise  in  the 
carbon  dioxide  elimination  to  a  maximal  limit  of  150  grams.  The 
conditions  on  supplying  fat  are  harder  to  judge,  but  Gigon  found  no  rise 
in  metabolism  on  introducing  oil. 

The  rise  in  the  gas  exchange  occurring  after  feeding  protein  and  sugar 
is  added,  according  to  Gigon,  entirely  to  the  basal  metabolism.  A 
substitution  in  the  basal  metabolism  of  the  catabolized  body  constituents 
by  the  food  taken  does  not  take  place  according  to  Gigon  and,  as  example, 
the  protein  is  not  replaced  from  catabolism  by  the  sugar  introduced. 
The  isodynamic  law  does  not  apply  to  the  metabolism  occurring  the 
first  few  hours  after  supplying  food,  as  shown  by  Johansson  and  Hell- 
gren,  and  Gigon  2  believes  that  the  foodstuffs  first  pass  into  the  various 
depots  of  the  body  to  be  later  used  for  purposes  of  energy.  Proteins 
serve  only  to  a  slight  degree  to  replace  the  catabolized  body  protein; 
the  remainder  is  stored  up  in  part  as  glycogen  and  in  part  as  fat.  The 
fat  is  deposited  as  such  and  the  carbohydrates  are  deposited  as  glycogen 
and  fat. 

As  the  three  foodstuffs  influence  the  metabolism  in  very  different  ways  we 
can,  according  to  Gigon,  speak  of  a  specific  action  of  the  foodstuffs.  This 
action,  according  to  him,  is  more  of  a  material  than  of  a  dynamic  kind,  and  the 
expression,  specific  dynamic  action,  may  lead  to  an  erroneous  conception. 


lSkand.  Arch.  f.  Physiol.,  21. 

2  Johansson  with  Hellgren,  Hammarsten's  Festschrift,  190C;  Gigon,  Skand.  Arch.  f. 
Physiol.,  21. 


932  METABOLISM. 


V.     THE  NECESSITY  OF  FOOD  BY  MAN  UNDER  VARIOUS  CONDITIONS. 

Various  attempts  have  been  made  to  determine  the  daily  quantity  of 
organic  food  needed  by  man.  Certain  investigators  have  calculated 
from  the  total  consumption  of  food  by  a  large  number  of  similarly  fed 
individuals — soldiers,  sailors,  laborers,  etc. — the  average  quantity  of 
foodstuffs  required  per  head.  Others  have  calculated  the  daily  demand 
for  food  from  the  quantity  of  carbon  and  nitrogen  in  the  excreta,  or  cal- 
culated it  from  the  exchange  of  force  of  the  persons  experimented  upon. 
Others,  again,  have  calculated  the  quantity  of  nutritive  material  in  a 
diet  by  which  an  equilibrium  was  maintained  in  the  individual  for  one  or 
several  days  between  the  consumption  and  the  elimination  of  carbon 
and  nitrogen.  Lastly,  still  others  have  quantitatively  determined,  dur- 
ing a  period  of  several  days,  the  organic  foodstuffs  daily  consumed  by 
persons  of  various  occupations  who  chose  their  own  food,  by  which  they 
were  well  nourished  and  rendered  fully  capable  of  work. 

Among  these  methods  a  few  are  not  quite  free  from  objection,  and 
others  have  not  as  yet  been  tried  on  a  sufficiently  large  scale.  Neverthe- 
less the  experiments  collected  thus  far  serve,  partly  because  of  their 
number  and  partly  because  the  methods  correct  and  control  one  another, 
as  a  good  starting-point  in  determining  the  diet  of  various  classes  and 
similar  questions. 

If  the  quantity  of  foodstuffs  taken  daily  be  converted  into  calories 
produced  during  physiological  combustion,  we  then  obtain  some  idea  of 
the  sum  of  the  chemical  energy  which  under  varying  conditions  is  intro- 
duced into  the  body.  It  must  not  be  forgotten  that  the  food  is  never 
completely  absorbed,  and  that  undigested  or  unabsorbed  residues  are 
always  expelled  from  the  body  with  the  feces.  The  gross  results  of  calo- 
ries calculated  from  the  food  taken  must  therefore,  according  to  Rubner, 
be  diminished  by  at  least  8  per  cent.  This  figure  is  true  at  least  when 
the  human  being  partakes  of  a  mixed  diet  of  about  60  per  cent  of  the 
proteins  as  animal,  and  about  40  per  cent  of  the  proteins  as  vegetable 
foodstuffs.  With  more  one-sided  vegetable  food,  especially  when  this 
is  rich  in  undigestible  cellulose,  a  much  larger  quantity  must  be  sub- 
tracted. 

The  following  summary  contains  a  few  examples  of  the  quantity  of 
food  which  is  consumed  by  individuals  of  various  classes  of  people  under 
different  conditions.  In  the  last  column  we  also  find  the  quantity  of 
living  force  which  corresponds  to  the  quantity  of  food  in  question,  calcu- 
lated as  calories,  with  the  above-stated  correction.  The  calories  are 
therefore  net  results,  while  the  figures  for  the  nutritive  bodies  are  gross 
results. 


FOOD  REQUIREMENT  IN  MAN.  933 

Proteins.    Fat.      ^J^tea"    Calories.     Authority. 

Soldier  during  peace 119  40  529  2784  Playfair.'- 

Soldier  light  service 117  35  447  2424  Hildesheim. 

Soldier  in  field 146  40  504  2852  Hildesheim. 

Laborer 130  40  550  2903  Moleschott. 

Laborer  at  rest 137  72  352  2458  Pettenkofer  and  Voit. 

Cabinetmaker  (40  years).  131  68  494  2835  Forster.-" 

Young  physician 127  89  302  2002  Forster. 

Young  phvsi.ian 134  102  292  2476  Forster. 

Laborer  (36  years) 133  95  422  2902  Forster. 

English  smith 176  71  666  3780  Playfair. 

English  pugilist 288  88  93  2189  Playfair. 

Bavarian  wood-chopper. .  135  208  876  5589  Liebig. 

Laborer  in  Silesia 80  16  552  2518  Meinert.3 

Seamstress  in  London ...  54  29  292  1688  Playfair. 

Swedish  laborer 134  79  485  3019  Hultgren  and  Landergren.4 

Japanese  student 83  14  622  2779  Eijkma.v5 

Japanese  shopman 55  6  394  1744  Tawara.5 

We  have  a  very  large  number  of  complete  investigations  upon  the 
diet  of  people  of  different  vocations  in  America,  but  they  are  too  exten- 
sive to  enter  into,  hence  we  must  refer  to  the  original  publications  of 
Atwater.6 

It  is  evident  that  persons  of  essentially  different  weight  of  body 
who  live  under  unequal  external  conditions  must  need  essentially  dif- 
ferent food.  It  is  also  to  be  expected  (and  this  is  confirmed  by  the  table) 
that  not  only  the  absolute  quantity  of  food  consumed  by  various  persons, 
but  also  the  relative  proportion  of  the  various  organic  nutritive  substances, 
shows  considerable  variation.  Results  for  the  daily  need  of  human 
beings  in  general  cannot  be  given.  For  certain  classes,  such  as  soldiers, 
laborers,  etc.,  results  may  be  given  which  are  valuable  for  the  calculation 
of  the  daily  rations. 

Based  on  extensive  investigations  and  a  very  wide  experience,  Voit 
has  proposed  the  following  average  quantities  for  the  daily  diet  of  adults: 

Proteins.  Fat.  Carbohydrates.  Calories. 

For  men 118  grams  56  grams  500  grams  2810 

But  it  should  be  remarked  that  these  data  relate  to  a  man  weighing 
70  to  75  kilos  and  who  was  engaged  daily  for  ten  hours  in  not  too  fatiguing 
labor. 

The  quantity  of  food  required  by  a  woman  engaged  in  moderate  work 

1  In  regard  to  the  earlier  researches  cited  in  this  table,  we  refer  the  reader  to  Voit, 
in  Hermann's  Handbuch,  6,  519. 

2  Ibid.,  and  Zeitschr.  f.  Biologie,  9. 

3  Armee-  und  Volksernahrung,  Berlin,  1880. 

4  Untersuching  uber  die  Ernahrung  schwedischer  Arbeiter  bei  frei  gewahlter  Kost, 
Stockholm,  1891.     Maly's  Jahresber.,  21. 

5  Cited  from  Kellner  and  Mori  in  Zeitschr.  f .  Biologie,  25. 

6  Report  of  the  Storrs  Agric.  Expt.  Station,  Conn.,  1891-1895,  and  1896,  and  U.  S. 
Depart,  of  Agriculture,  Bull.  53,  1898. 


934  METABOLISM. 

is  about  four-fifths  that  of  a  laboring  man,  and  we  may  consider  the 
following  as  a  daily  diet  with  moderate  work : 

Proteins.  Fat.  Carbohydrates.         Calories. 

For  women 94  grams  45  grams  400  grams  2240 

The  proportion  of  fat  to  carbohydrates  is  here  as  1:8-9.  Such  a 
proportion  often  occurs  in  the  food  of  the  poorer  classes  who  chiefly  live 
upon  the  cheap  and  voluminous  vegetable  food,  while  this  ratio  in  the 
food  of  wealthier  persons  is  1 :3-4.  It  would  be  desirable  if  in  the  above 
rations  the  fat  were  increased  at  the  expense  of  the  carbohydrates,  but 
unfortunately  on  account  of  the  high  price  of  fat  such  a  modification 
cannot  always  be  made. 

In  examining  the  above  figures  for  the  daily  rations  it  must  not 
be  forgotten  that  those  for  the  various  foodstuffs  are  gross  results. 
They  consequently  represent  the  quantity  of  those  which  must  be  taken 
in,  and  not  those  which  are  really  absorbed.  The  figures  for  the  calories 
are,  on  the  contrary,  net  results. 

The  various  foods  are,  as  is  well  known,  not  equally  digested  and 
absorbed,  and  in  general  the  vegetable  foods  are  less  completely  consumed 
than  animal  foods.  This  is  especially  true  of  the  proteins.  When, 
therefore,  Voit,  as  above  stated,  calculates  the  daily  quantity  of  pro- 
teins needed  by  a  laborer  as  118  grams,  he  starts  with  the  supposition 
that  the  diet  is  a  mixed  animal  and  vegetable  one,  and  also  that  of  the 
above  118  grams  about  105  grams  are  absorbed.  The  results  obtained 
by  Pfluger  and  his  pupils  Bohland  and  Bleibtreu  x  on  the  extent  of 
the  metabolism  of  proteins  in  man  with  an  optional  and  sufficient  diet 
correspond  well  with  the  above  figures,  when  the  unequal  weight  of  body 
of  the  various  persons  experimented  upon  is  sufficiently  considered. 

As  a  rule,  the  more  exclusively  a  vegetable  food  is  employed,  the 
smaller  is  the  quantity  of  proteins  in  it.  The  strictly  vegetable  diet 
of  certain  people,  as  that  of  the  Japanese  and  of  the  so-called  vegeta- 
rians, is  therefore  a  proof  that,  if  the  quantity  of  food  be  sufficient,  a 
person  may  exist  on  considerably  smaller  quantities  of  proteins  than 
Voit  suggests.  It  follows  from  the  investigations  of  Hirschfeld,  Kuma- 
gawa  and  Klemperer,  Siven,  and  others  (see  pages  903,  915)  that  an 
almost  complete  or  indeed  a  complete  nitrogenous  equilibrium  may  be 
attained  by  the  sufficient  administration  of  non-nitrogeneous  nutritive 
bodies  with  relatively  very  small  quantities  of  proteins. 

If  we  bear  in  mind  that  the  food  of  people  of  different  countries 
varies  greatly,  and  that  the  individual  also  takes  essentially  different 
nourishment  according  to  the  external  conditions  of  living  and  the  influence 
of  climate,  it  is  not  remarkable  that  a  person  accustomed  to  a  mixed 

1  Bohland,  Pfluger's  Arch.,  36;  Bleibtreu,  ibid.,  38. 


PROTEIN   REQUIREMENT.  935 

diet  can  exist  for  some  time  on  a  strictly  vegetable  diet  deficient  in  pro- 
teins. No  one  doubts  the  ability  of  man  to  adapt  himself  to  a  heteroge- 
neously  composed  diet  when  this  is  not  too  difficult  of  digestion  and  is 
sufficient  in  quantity;  nor  can  we  deny  that  it  is  possible  for  a  man 
to  exist  for  a  long  time  with  smaller  amounts  of  protein  than  Voir 
suggests,  namely  118  grams.  Thus  0.  Neumann  '  experimented  on  him- 
self during  746  days  in  three  series  of  experiments,  and  his  diet  consisted 
of  74.2  grams  protein,  117  grams  fat,  and  213  grams  carbohydrates  =  23G7 
gross  calories,  with  a  weight  of  70  kilos  and  with  ordinary  laboratory 
work.  These  figures  cannot  be  compared  with  those  obtained  by  Voit's 
worker,  weighing  70  kilos,  whose  work  was  harder  than  a  tailor's  and 
easier  than  a  blacksmith's;  for  example,  the  work  of  a  mason,  carpenter, 
or  cabinet-maker.  The  very  extensive  investigations  recently  performed 
by  Chittenden  2  on  the  determination  of  the  extent  of  protein  necessary 
are  of  great  interest.  These  investigations,  upon  a  total  of  twenty- 
six  persons,  extended  over  a  period  of  five  to  twenty  months,  and  con- 
sisted of  careful  observations  upon  the  manner  of  living,  food  taken, 
nitrogen  elimination,  and  the  ability  of  performing  work.  The  individuals 
were  divided  into  three  groups.  The  first  consisted  of  five  professional 
men  (four  assistants  and  one  professor).  The  second  group  was  composed 
of  thirteen  soldiers  (of  the  sanitary  corps  of  the  United  States  army)  who 
besides  their  daily  work  were  given  gymnastic  exercises  for  six  months. 
The  third  group  consisted  of  eight  athletic  students  who  were  trained  in 
different  kinds  of  sport. 

In  all  the  persons  experimented  upon  the  original  nitrogen  content 
of  the  food,  which  corresponded  to  Voit's  value  or  were  somewhat  higher, 
was  gradually  reduced  more  or  less.  The  total  calories  supplied  were 
not  increased  above  the  original  value,  but  rather  diminished  to  a  reason- 
able extent.  The  bodily  as  well  as  the  mental  ability  was  repeatedly 
tested.  As  it  is  not  possible  to  enter  into  the  details  of  the  investiga- 
tion the  following  will  be  sufficient  to  show  the  results.  With  a  diet 
corresponding  to  Voit's  values  the  amount  of  urine  nitrogen  per  day  is 
16  grams,  corresponding  to  a  total  protein  catabolism  in  the  body  of  100 
grams,  or  1.43  grams  per  kilo.  The  corresponding  results  for  the  above 
three  groups  may  be  found  in  the  following  table,  where  for  comparison 
Hammarsten  also  includes  the  figures  for  Voit's  diet: 

Urine  Nitrogen.  Catabolized  Protein.  Protein  per  Kilo. 

Mm.  Max.  Min.  Max.  Min.  Max. 

Group  1          5.69  8.99  35.6           56.19  0.61           0.86 

Group  2         7.03  8.39  43.9           52.44  0.74           0.87 

Group  3         7.47  11.06  46.7           69.10  0.75           0.92 

Voit's  figures  16                              100                                 1.43 

1  Arch.  f.  Hygiene,  45. 

*  R.  H.  Chittenden,  Physiological  Economy  in  Nutrition,  New  York,  1904. 


936  METABOLISM. 

The  chief  results  from  these  investigations  are  that  on  partaking  of 
amounts  of  protein  much  smaller  than  Voit's  figures,  without  changing 
the  original  supply  of  calories  and  indeed  diminishing  the  same,  the 
persons  experimented  upon  remained  not  only  in  nitrogenous  equilibrium, 
but  in  perfect  health,  and  were  not  only  able  to  perform  ordinary  work, 
but  were  indeed  regularly  able  to  perform  much  greater  work. 

From  these  investigations,  which  extended  over  a  long  period  and 
were  carried  on  with  special  care  in  exactitude,  it  cannot  be  denied  that 
man  can  for  a  long  time  exist  with  much  smaller  quantities  of  protein 
than  Voit's  figures  call  for,  which  is  also  derived  from  the  experience  of 
vegetarians,  and  from  people  living  almost  entirely  upon  vegetable  food. 
On  the  other  hand  it  must  not  be  forgotten  that  Voit's  figures  represent 
average  results  not  theoretically  necessary,  but  which  have  been  shown 
to  be  the  actual  diet  developed  from  habit,  custom,  conditions  of  life  and 
climate,  with  sufficient  nourishment  and  free  selection  for  centuries  in 
Middle  and  North  Europe.  A  rational  change  in  this  food  requirement 
based  upon  scientific  facts  is  just  as  difficult  to  determine  as  it  is  to  carry 
out  practically.  Certain  standard  figures  for  the  general  needs  of  nutri- 
tion cannot  be  established  because  the  conditions  in  various  countries 
are  different  and  must  necessarily  be  so.  The  numerous  compilations 
(of  Atwater  and  others  l)  on  the  diet  of  different  families  in  America 
have  given  the  figures  97-113  grams  protein  for  a  man,  and  the  very 
careful  investigations  of  Hultgren  and  Landergren  have  also  shown 
that  the  laborer  in  Sweden  with  moderate  work  and  an  average  body 
weight  of  70.3  kilos,  with  optional  diet,  partakes  134  grams  protein,  79 
grams  fat,  and  522  grams  carbohydrates.  The  quantity  of  protein 
is  here  greater  than  is  necessary,  according  to  Voit.  On  the  other  hand 
Lapicque  2  found  67  grams  protein  for  Abyssinians  and  81  grams  for 
Malaysians  (per  body  weight  of  70  kilos),  materially  lower  figures. 

If  we  compare  the  figures  on  page  933  with  the  average  figures  pro- 
posed by  Voit  for  the  daily  diet  of  a  laborer,  it  would  seem  at  the  first 
glance  as  if  the  food  consumed  in  certain  cases  was  considerably  in  excess 
of  the  need,  while  in  other  cases,  as,  for  instance,  that  of  a  seamstress 
in  London,  it  was  entirely  insufficient.  A  positive  conclusion  cannot, 
therefore,  be  drawn  if  we  do  not  know  the  weight  of  the  body,  as  well 
as  the  labor  performed  by  the  person,  and  also  the  conditions  of  living. 


1  Atwater,  Report  of  the  Storrs  Agric.  Expt.  Station,  Conn.,  1891-1895  and  1896; 
also  Nutrition  investigations  at  the  University  of  Tennessee,  1896  and  1897;  U.S 
Dept.  of  Agriculture,  Bull.  53,  1898.  See  also  Atwater  and  Bryant,  ibid.,  Bull.  75. 
Jaffa,  ibid.,  84;  Grindley,  Sammis,  and  others,  ibid.,  91. 

2  Hultgren  and  Landergren,  1.  c;  Lapicque,  Arch,  de  Physiol.  (5),  6. 


WORK   AND   FOOD  REQUIREMENT.  937 

It  is  certainly  true  that  the  amount  of  nutriment  required  by  the  body 
is  not  directly  proportional  to  the  body  weight,  for  a  small  body  consume! 
relatively  more  substance  than  a  larger  one,  and  varying  quantities  of 
fat  may  also  cause  a  difference;  but  a  large  body,  which  must  maintain 
a  greater  quantity,  consumes  an  absolutely  greater  amount  of  substance 
than  a  small  one,  and  in  estimating  the  nutritive  need  one  must  also 
always  consider  the  weight  of  the  body.  According  to  Voit,  the  diet 
for  a  laborer  with  70  kilos  body  weight  requires  40  calories  for  each  kilo. 
Ekholm  1  calculates,  basing  it  upon  his  experiments,  that  for  a  man 
weighing  70  kilos,  busied  with  reading  and  writing,  the  net  calories  are 
24 .",0  and  the  gross  calories  2700,  or  35  and  38.6  calories  per  kilo.  In  the 
ordinary  sense  for  a  resting  man,  the  general  food  requirement  is  calculated 
in  round  numbers  as  30  calories  for  every  kilo.  The  minimum  figure 
for  metabolism  during  sleep  and  in  as  complete  rest  as  possible  has  been 
found  by  Sonden,  Tigerstedt  and  Johansson2  to  be  24-25  calories. 

As  several  times  stated  above,  the  demands  of  the  body  for  nourish- 
ment vary  with  different  conditions  of  the  body.  Among  these  condi- 
tions two  are  especially  important,  namely,  work  and  rest. 

In  a  previous  chapter,  in  which  muscular  labor  was  spoken  of,  it  was 
seen  that  all  foodstuffs  have  almost  the  same  power  of  serving  as  a  source 
of  muscular  work,  and  that  the  muscles,  it  seems,  select  that  foodstuff 
which  is  supplied  to  them  in  the  greatest  quantity.  As  a  natural  sequence 
it  is  to  be  expected  that  muscular  activity  requires  indeed  an  increased 
supply  of  foodstuffs,  but  no  essential  change  in  their  relation  as  compared 
to  rest. 

Still  this  does  not  seem  to  hold  true  in  daily  experience.  It  is  a  well- 
known  fact  that  hard-working  individuals — men  and  animals — require 
a  greater  quantity  of  proteins  in  the  food  than  less  active  ones.  This 
contradiction,  is  however,  only  apparent,  and  it  depends,  as  Voit  has 
shown,  upon  the  fact  that  individuals  used  to  violent  work  are  more 
muscular.  For  this  reason  a  person  performing  severe  muscular  labor 
requires  food  containing  a  larger  proportion  of  proteins  than  an  individual 
whose  occupation  demands  less  violent  exertion.  Another  fact  is  that 
the  diet  rich  in  proteins  is  often  concentrated  and  less  bulky,  and  also 
that  in  many  cases  of  training,  a  diet  yielding  as  little  fat  as  possible  is 
selected. 

If  we  compare  the  results  for  the  needs  of  food  in  work  and  rest  which 
are  obtained  under  conditions  which  can  be  readily  controlled,  it  is  found 
that  the  above  statements  are  in  general  confirmed.     As  example  of  this 


1  Skand.  Arch,  f .  Physiol.,  11. 

*  Sonden  and  Tigerstedt,  Skand.  Arch.  f.  Physiol.,  6;  Johansson,  ibid.,  7;  Tigerstedt, 
Nord.  Med.  Arkiv.  Festband.,  1897. 


938  METABOLISM. 

the  following  tables  give  the  rations  of  soldiers  in  peace  and  in  the  field 
and  the  average  figures  from  the  detailed  data  of  various  countries : 1 

A.   Peace  Ration.  B.   War  Ration. 


Fat. 

Carbohydrates. 

Calories. 

40 

551 

2900 

59 

557 

3250 

Fat. 

Carbohydrates. 

Calories. 

80 

500 

3013 

100 

500 

3218 

Proteins.  Fat.     Carbohydrates.     Proteins.       Fat.     Carbohydrates. 

Minimum. .  .    108  22  504  126  38  484 

Maximum. .  .    165  97  731  197  95  688 

Mean 130  40  551  146  59  557 

The  following  figures  for  the  daily  ration  are  obtained  from  the  above 
averages : 

Proteins. 

In  peace 130 

In  war 146 

If  we  calculate  the  fat  in  its  equivalent  quantity  of  starch,  then  the 
relation  of  the  proteins  to  the  non-nitrogenous  foods  is : 

In  peace 1  :  4 .  97 

In  war 1  :  4 .  79 

The  relation  in  both  cases  is  nearly  the  same.  Similar  results  are 
obtained  when  we  start  with  Voit's  figures  for  a  soldier  in  manceuver  A 
(hard  work)  and  B  (strenuous  work)  in  war. 

Proteins. 

A 135 

B 145 

The  relation  here,  when  the  fat  is  recalculated  as  starch,  in  both  cases  is 
the  same,  or  equal  to  1 :  5. 

If  we  calculate  that  portion  of  the  total  calories  supplied  which  falls 
to  each  group  of  the  foodstuffs,  it  is  found  that  16-19  per  cent  comes 
from  the  protein  in  rest  as  well  as  with  medium  and  strenuous  work. 
For  the  fat  and  the  carbohydrates  the  variations  are  greater;  the  chief 
quantity  of  calories  comes  from  the  carbohydrates.  Of  the  total  calories 
16-30  per  cent  comes  from  the  fat  and  50-60  per  cent  from  the  carbo- 
hydrates. 

The  importance  of  the  food-demand  for  working  individuals  is  shown 
by  the  figures  given  on  page  933  for  a  wood-chopper  in  Bavaria.  A 
need  of  more  than  4000  calories  occurs  but  seldom,  and  with  very  hard 
work  the  demand  may  rise  even  to  7000  calories  (Atwater  and  Bryant, 
Jaffa2). 


1  Germany,  Austria,  Switzerland,  France,  Italy,  Russia,  and  the  United  States. 
It  is  not  known  by  the  author  whether  these  figures  have  been  changed  in  the  last 
few  years  in  the  various  countries,  and  hence  whether  they  must  be  modified  or  not. 

*  flee  footnote  1,  page  936. 


WORK  AND  FOOD  REQUIREMENT.  939 

As  more  work  requires  an  increase  in  the  absolute  quantity  of  food, 
so  the  quantity  of  food  must  he  diminished  when  little  work  is  performed. 
The  question  as  to  how  far  this  can  be  done  is  of  importance  in  regard 
to  the  diet  in  prisons  and  poorhouses.  We  give  below  the  following  as 
example  of  such  diets: 

Proteins.  Fat.  Carbohydrates.  Calories. 

Prisoner  (not  working)..   87  22  305  1667     Schuster.1 

Prisoner  (not  working)..   85  30  300  1709     Voit. 

Man  in  poorhouse 92  45  332  1985     Forster.2 

Woman  in  poorhouse.  .  .  80  49  266  1724     Forster. 

The  figures  given  by  Voit  are,  he  says,  the  lowest  reported  for  a  non- 
working  prionser.  He  considers  the  following  as  the  lowest  diet  for  old 
non-working  people: 

Proteins.  Fat.  Carbohydrates.  Calories. 

Men 90  40  350  2200 

Women 80  35  300  1723 

In  calculating  the  daily  diet  it  is  in  most  cases  sufficient  to  ascertain 
how  much  of  the  various  foodstuffs  must  be  administered  to  the  body 
in  order  to  keep  it  in  the  proper  condition  to  perform  the  work  required 
of  it.  In  other  cases  it  may  be  a  question  of  improving  the  nutritive 
condition  of  the  body  by  properly  selected  food;  and  there  are  also  cases 
in  which  it  is  desired  to  diminish  the  mass  or  weight  of  the  body  by  an 
insufficient  nutrition.  This  is  especially  the  case  in  obesity,  and  all  the 
dietaries  proposed  for  this  purpose  are  chiefly  starvation  cures,  which 
is  readily  apparent  if  we  study  such  dietaries. 


1  See  Voit,   Unters.  der  Kost,   Munchen,    1877,  page   142.     See  also  Hirschfeld, 
Maly's  Jahresber.,  30. 
*  Ibid.,  page  186. 


940 


FOOD  TABLES. 


TABLE  I.— FOODS.1 


1.  Animal  Foodstuffs. 


1000  Parts  contain 


a  o 

0)  > 

m  -^ 

IS 


O  V 


Relation  of 
1:2:3. 


a.  Meat  without  Bones. 

Fat  beef  2 

Beef  (average  fat  J) 

Beef2 

Corned  beef  (average  fat) 

Veal 

Horse,  salted  and  smoked 

Smoked  ham 

Pork,  salted  and  smoked  3 

Meat  from  hare 

"        "     chicken 

"        "     partridge 

"        "     wild  duck 

b.  Meat  with  Bones. 

Fat  beef  2 

Beef  (average  fat1) 

Beef,  slightly  corned 

Beef,  thoroughly  corned 

Mutton,  very  fat 

' '        average  fat 

Pork,  fresh,  fat 

' '      corned,  fat 

Smoked  ham 

c.  Fishes. 

River  eel,  fresh,  entire 

Salmon,  "      

Anchovy,  

Flounder,      "         "      

River  perch,  fresh,  entire 

Torsk  "         "     

Pike,  "         "     

Herring,  salted,  entire 

Anchovy,     "  "    

Salmon  (side),  salted 

Kabeljau  (salted  haddock) 

Codfish  (dried  ling) 

"   (dried  torsk) 

Fish-rneal  from  variety  of  Gadtjs 


183 

166 

196 

98 

190 

120 

218 

115 

190 

80 

318 

65 

255 

365 

100 

660 

233 

11 

195 

93 

253 

14 

246 

31 

156 

141 

167 

83 

175 

93 

190 

100 

135 

332 

160 

160 

100 

460 

120 

540 

200 

300 

89 

220 

121 

67 

128 

39 

145 

14 

100 

2 

86 

1 

82 

1 

140 

140 

116 

43 

200 

108 

246 

4 

532 

5 

665 

10 

736 

7 

11 

18 

18 

117 

13 

125 

100 

40 

12 

11 

14 

12 


9 

15 

85 

100 

8 
10 

5 
60 
70 


6 
10 
11 

11 


6 
100 
107 
132 
178 
106 
59 
87 


640 
688 
672 
550 
717 
492 
280 
130 
744 
701 
719 
711 


544 
585 
480 
430 
437 
520 
365 
200 
340 


352 
469 
489 
580 
440 
455 
461 
280 
334 
460 
472 
257 
116 
170 


150 

150 

167 

180 

88 

150 

70 

80 

90 


333 
333 
333 
250 
450 
450 
450 
340 
400 
100 
100 
100 
150 


100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 


100 
100 
100 
100 
100 
100 
100 
100 
100 


100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 


90 

50 

63 

53 

42 

20 

143 

660 

5 

48 

6 

13 


90 

49 

53 

53 

246 

100 

460 

450 

150 


246 

56 

31 

9 

2 

1 

1 

100 

37 

54 

1 

1 

1 

1 


'  The  re'sults  in  the  following  tables  are  chiefly  compiled  from  the  summary  of  Alm^n  and  of 
KoNi'i  We  here  designate  as  "  waste  "  that  part  of  the  foods  which  is  lost  in  the  preparation 
or  that  which  is  not  used  by  the  body;  for  instance,  bones,  skin,  egg-shells,  and  the  cellulose 
vegetable  foods.  . 

»  Meat  such   as  is  ordinarily  sold   in   the  markets  in   bweden. 

•  Pork,  chiefly  from  the  breast  and  belly,  such  as  occurs  in  the  rations  of  Swedish  soldiers. 


ANIMAL  FOODS. 
TABLE  I.— FOODS— (Continued). 


941 


1.  Animal  Foodstuffs. 


1000   Parts   contain 


Relation  of 
11:2:  3. 


1 

100 

89 

10!) 

2S 

100 

50 

100 

65 

100 

17 

100 

113 

1U 

1 

d.  Inner  Organs  (Fresh). 

Brain 

Beef-liver 

Beef-heart 

Heart  and  lungs  of  mutton 

Veal-kidney 

Ox  tongue  (fresh) 

Blood    from    various    animals 

(average  results) 


e.  Other  Animal  Foods. 
Variety  of  pork-sausage  (Mett- 

wurst) 

Same  for  frying 

Butter 

Lard 

Meat  extract 

Cow's  milk  (full) 

"        "      (skimmed) 

Buttermilk 

Cream 

Cheese  (fat) 

"       (poor) 

Whey  cheese  (poor) 

Hen's  egg,  entire 

"        "     without  shell 

Yolk  of  egg 

White  of  egg 


2.  Vegetable  Foodstuffs. 

Wheat  (grains) 

Wheat-flour  (fine) 

' '      (very  fine) , 

Wheat-bran 

Wheat-bread  (fresh) 

Macaroni 

Rye  (grains) , 

Rye-flour 

Rye-bread  (dry) 

(fresh,  coarse)..  .  . 

(fresh,  fine) 

Barley  (grains) 

Scotch  barley 

Oat  (grains) 

' '    (peeled) 

Corn 

Rice  (peeled  for  boiling) 

French  beans 

Peas  (yellow  or  green,  dry) .  . 
Flour  from  peas 


116 
196 
184 
163 
221 
150 

182 


190 

220 

7 

3 

304 

35 

35 

41 

37 

230 

334 

89 

106 

122 

160 

103 


123 

110 

92 

150 

88 

90 

115 

115 

114 

77 

80 

111 

110 

117 

140 

101 

70 

232 

220 

270 


103 
56 
92 

106 
38 

170 


150 
160 
850 
990 

35 

7 

9 

257 

270 

66 

70 

93 

107 

307 


17 
10 
11 

39 
10 

3 
17 
15 
20 
10 
14 
21 
10 
60 
60 
58 

7 
21 
15 
15 


11 


50 

50 

38 

35 

40 

50 

456 

4 

5 


676 
740 
768 
439 
550 
768 
688 
720 
725 
480 
514 
654 
720 
563 
660 
656 
770 
537 
530 
520 


50 
55 
15 

175 

7 

7 

7 

6 

60 

50 

56 

8 

10 

13 


18 

8 

3 

50 

17 

8 

18 

20 

15 

16 

11 

26 

7 

30 

20 

17 

2 

36 

25 

25 


770 
720 
714 
721 
728 
670 

807 


610 
565 
119 
7 
217 
873 
901 
905 
665 
400 
500 
329 
654 
756 
520 
875 


140 
120 
120 
130 
330 
131 
140 
110 
110 
400 
370 
140 
146 
130 
100 
140 
146 
137 
150 
125 


135 


26 
12 

6 
192 

5 

22 
20 
16 

17 
11 
48 

7 

100 

20 

28 

5 
37 
60 
45 


100 
100 

100 
100 

100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 


100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 
100 


79 

73 

12 1C0 

33000 

100 
20 
22 
695 
117 
19 
79 


192 

7 


0 

0 

100 

0 

143 

143 

93 

95 

17 

15 

512 

4 

4 

0 

7 


549 
654 
835 
292 
625 
853 
600 
626 
634 
623 
634 
589 
654 
481 
471 
662 
1100 
231 
240 
192 


942 


FOOD  TABLES. 
TABLE  I.— FOODS— (Continued). 


1000  Parts  contain 

Relation  of 
1:2:3. 

1 

2 

3 

4 

5 

6 

1 

■  2 

:3 

2.  Vegetable  Foodstuffs. 

■a  m 

CD  "o 

"O  cS 

°r1 

A~W 

o  H 

id 

■Si 

Potatoes 

20 
14 

2 
2 

200 
74 

10 

7 

760 
893 

8 
10 

100 
100 

10 
14 

1030 

Turnips 

529 

Carrot  (yellow) 

10 

2 

90 

10 

873 

15 

100 

20 

900 

Cauliflower 

25 
19 
27 
31 

4 
2 
1 
5 

50 
49 
66 
33 

8 
12 

6 
19 

904 
900 

888 
908 

9 
18 
12 

8 

100 
100 
100 
100 

16 

11 

4 

16 

200 

Cabbage 

258 

Beans 

244 

Spinach 

106 

Lettuce 

14 
10 

3 
1 

22 
23 

10 

4 

944 
956 

7 
6 

100 
100 

21 
10 

157 

Cucumbers 

230 

Radishes 

12 
32 

1 

4 

38 
60 

7 
9 

934 

877 

8 
18 

100 
100 

8 
12 

317 

Edible  mushrooms  (average)..  . 

188 

Same  dried  in  the  air  (average). 

219 

25 

412 

61 

160 

123 

100 

12 

188 

Apples  and  pears 

4 

130 

3 

832 

31 

100 

3250 

Various  berries  (average) 

5 

90 

6 

849 

50 

100 

1800 

Almonds 

242 

537 

72 

29 

54 

66 

100 

222 

30 

Cocoa 

140 

480 

180 

50 

55 

95 

100 

343 

129 

TABLE  II.— MALT  LIQUORS 


1000  Parts  by  Weight  contain 


Porter 

Beer  (Swedish) 

'  ■     (Swedish  export). . 

Draught-beer 

Lager-beer 

Bock-beer 

Weiss-beer 

Swedish  "  Svagdricka  " 


0> 

o?5 
2  * 
■£.2 

o 
A 

o 

o 

o 

S3 

u 

X 

W 

13 
O 

871 

2 

54 

76 

7 

887 

28 

— 

15 

885 

32 

— 

7 

911 

2 

35 

55 

8 

903 

2 

40 

58 

4 

881 

2 

47 

72 

6 

916 

3 

25 

59 

5 

945 

— 

22 

— 

7 

13 


65 
73 


10 

7 

13 


31 

47 


d 

a 

<u 

T3 

< 

O 

3.0 

— 

2.0 

2 

1.5 

2 

1.7 

— 

4.0 

— 

— 

— 

23  —       — 


WINES  AND  OTHER  ALCOHOLIC  LIQUORS. 


943 


TABLE  III.— WINES  AND  OTHER  ALCOHOLIC  LIQUORS 


1000  Parts  by  Weight  contain 


Bordeaux  wine 

White  wine  (Rheingau) 

Champagne 

Rhine  wine  (sparkling). 

Tokay 

Sherry 

Port  wine 

Madeira 

Marsala 

Swedish  punch 

Brandy 

French  cognac 

Liqueurs 


1 

It 

o 
JO 

o 
a 

ti 

85 

111 

a  a  £ 

c 
a 

3 

■  o  . 

g  CO 

03 

G 

M 

w 

Ml 

~  — — 
< 

>> 
O 

A 

■ 
< 

as  :  — 

o 

883 

94 

23 

6 

5.9 



2.0 

863 

115 

23 

4 

5.0 

— 

2.0 

776 

90 

134 

115 

6.0 

1.0 

1  0 

}  60-70 

801 

94 

105 

87 

6.0 

1.0 

2.0 

SOS 

120 

72 

51 

7.0 

9.0 

3.0 

795 

170 

35 

15 

5.0 

6.0 

5.0 

774 

164 

62 

40 

4.0 

2.0 

3.0 

791 

156 

53 

33 

5.0 

3.0 

3.0 

790 

164 

46 

35 

5.0 

4.0 

4.0 

479 

263 
460 

— 

332 

— 

— 

— 

— 

550 

442-590 

— 

260-475 

— 

— 

— 

S44  INDEX  TO  SPECTRUM  PLATE. 


SPECTRUM  PLATE 

1.  Absorption  spectrum  of  a  solution  of  oxyhemoglobin. 

2.  Absorption  spectrum  of  a  solution  of  haemoglobin,  obtained  by  the  action  of  an 

ammoniacal  ferro-tartrate  solution  on  an  oxyhemoglobin  solution. 
-3.  Absorption  spectrum  of  a  faintly  alkaline  solution  of  melhcemoglobin. 
A.  Absorption  spectrum  of  a  solution  of  hoemalin  in  ether  containing  oxalic  acid. 

5.  Absorption  spectrum  of  an  alkaline  solution  of  hcematin. 

6.  Absorption  spectrum  of  an  alkaline  solution  of  hcemochromoaen,  obtained  by  the 

action  of  an  ammoniacal  ferro-tartrate  solution  on  an  alkaline-haematin  solution 

7.  Absorption  spectrum  of  an  acid  solution  of  hamatoporphyrin. 

8.  Absorption  spectrum  of  an  ammoniacal  solution  of  urobilin  after  the  addition  of  a 

zinc-chloride  solution. 


INDEX   OF   AUTHORS 


Abderhalden,  E.,  enzymes,  50,  53,  54,  62, 
65,  525,  protein  hydrolysis,  84,  106, 
107,  109,  114,  121-125,  132,  133,  142, 
146,  154,  262,  288,  507,  605,  634,  662; 
polypeptides,  86-90,  132,  133,  509; 
protein  reaction,  101;  ichthylepidin, 
121;  proteoses.  127;  cystine  and  cys- 
tinuria,  148,  776;  tryptophane,  157, 
158;  histidine,  160,  161;  diamino- 
trioxydodecanoic  acid,  165;  glyco- 
proteid,  167;  proteoses  in  the  blood, 
263,  264;  blood  serum,  266,  269,  270; 
blood  corpuscles,  275,  277,  304;  blood 
analyses,  328,  334,  338;  iron  per- 
parations.  340;  blood  and  high  altitudes, 
341;  adrenalin,  376,  380;  melanin,  380; 
cholesterin,  445,  448;  gastric  contents, 
481;  digestion,  484,  513;  duodenal 
secretion,  489,  490;  absorption  and 
synthesis  of  proteins,  529,  530;  fat, 
559;  milk,  646,  659,  662,  667;  urea 
formation,  682;  nuclein  metabolism, 
702,  705;  amino-acid  storage,  721,  722; 
alcaptonuria,  735-737;  urinary  sul- 
phur, 752;  polypetides  in  the  urine, 
763;  amino  acids  in  the  urine,  756,  827: 
pyridine,  787;  Bence-Jones  proteid, 
792,  793;  tunicin,  839;  nutritive  value 
of  gelatin,  912;  ammonia  as  protein 
sparer,  912;  water  and  metabolism 
920;  alcohol,  921;  nin  -  hydrin,  101; 
caprine,  144;  tyrosine  reagent,  154 

Abel,  J.,  379,  621,  683 

Abeles,  M.,  266,  749 

Abelmann,  M.,  532,  539,  540 

Abelous,  J.,  757,  877 

Abelsdorf,  G.,  616 

Ach,  L.,  698 

Achalme,  307 

Achard,  Ch.,  265 

Acheles,  \V.,  698,  757 

Ackermann,  D.,  putrefaction  bases,  47; 
histidine,  82,  159,  160;  proline,  154; 
aporrhegmen,  166;  blood  corpuscles, 
274,  275;  meat  bases,  578;  lysine  in 
the  urine,  828 

Ackrovd,  H.,  717 

Adam,  H.,  311 

Adamkiewicz,  A.,  100,  912 

Adamson,  17 


v.  Anrep,  R.,  287,  723 

Adler,  O.,  proteoses,  209;    fructose,  217, 

814;  ochronosis,  550;  blood,  797 
Adler,  R.,  proteoses,  134;    pentoses,  209; 

fructose,  217;  blood,  797 
Adrian,  C,  730 
Adriance,  J.,  662,  665 
Adriance,  V.,  662,  665 
Aducco,  V.,  675,  757 
Agulhon,  50 
Albanese,  M.,  711 
Albert,  R.,  41 

Albertoni,  P.,  415,  502,  533 
Albrecht,  E.,  273 
Albro,  A.,  506 
Albu,  A.,  gastric  juice,  466;   urine  poison, 

758;     mineral    metabolism,    758,    769, 

899,  901,  902 
v.  Alder,  L.,  792 
Aldrich,  J.  B.,  846 
Alexander,  F.,  133,  652 
v.  Alfthan,  K.,  749 
Allard,  E.,  825 
Allaria,  G.  B.,  659 
Allen,  S.,  471 
Alleis,  R.  A.,  157,  380 
Allihn,  F.,  656,  811 
Allison,  F.  G.,  689 
Almagia,  M.f  410,  583,  706 
Almen,  A.,  xanthine,     188;      sugar     test, 

214;    meat,  599,  600;    food-stuffs,  940 
Aloy,  J.,  877 
Alsberg,  C.,  104,  184 
Altmann,  R.,  179 
Amberg,  S.,  71,  642,  643 
Ambronn,  H.,  839 
Amerman,  G.,  470 
Ameseder,  Fr.,  239,  627,  844 
Amiradzibi,  S.,  575 
Amthor,  K.,  237,  559 
Andersen,  A.  C.,  154,  804,  809,  810 
Anderson,  R   J.,  580 
Anderson,  N.,  332 
Andersson,  J.,  376 
Andryewskij,  P.,  874 
Anselm,  R.,  435 
Ansiaux,  G.,  97,  253 
Anthon,  213 
Appleyard,  J.  R.,  28 
Araki,  T.,  blood  pigments,  285;    nucleic 

945 


946 


INDEX  OF  AUTHORS 


acids,  508;    lactic  acid,  582-585,  748, 

749-  chitin,  839 
Ardin-Delteil,  P.,  848 
Argiris,  A.,  112,  114,  606 
Argutinsky,  P.,  600,  848,  883 
Arinkin,  M.,  43 
Armstrong,   E.   T.,   enzyme,   55,   56,   58, 

59,  65;  glucoside,  200;  osone,  203 
Armstrong,  H.  E.,  59,  65 
Arnheim,  J.,  408 
Arnold,  J.,  377 
Arnold,  V.,  protein  reaction,  101;  cystein, 

149;    urine  reaction,  696;    nephrosein, 

734;     hffimatoporphyrin,    797;     aceto- 

acetic  acid,  824,  825 
Arnold,  W.,  2S8 
Arnschink,  L.,  537 
Arnstein,  R.,  715 
Aron,  H.,  281,  552,  554,  555 
Arrhenius,     S.,     dissociation     theory,     4; 

catalysis,  33,  34;  enzymes,  53;  Schutz's 

rule,  57;    toxin-antitoxin  combination, 

67,  68 
Arronet,  H.,  328 
Ateaga,  T.  F.,  400 
Arthus,  M.,  blood  coagulation,  250,  251, 

313,    314,    316-318,    320;     fibrinolysis, 

255;   serum  lipase,  265,  glycolysis,  332; 

casein,  ,648,  650 
Artmann,  P.,  23 
Asahina,  Y.,  297 
Ascher,  E.,  145 
Ascherson,  646 
Ascoli,  A.,  antolysis,  43;    nucleic  acids, 

172,   185;    uracil  194;    glutinase,  505; 

protein  absorption,  526;   placenta,  642; 

urea   formation,    682;     uric   acid,    706 
Asher,  L.,  blood  sugar,  264;    lymph,  364, 

349-352;    spleen,    372;    thyroids,    374; 

liver,   381,   absorption,  .528;    nitrogen 

elimination,  910 
Aso,  K.,  97 
Astaschewsky,  593 
Athanasiu,  J.,  384,  561 
Atwater,    W.   O.,    metabolism,   595,   597, 

879,  891,   914;    respiration  apparatus, 

868,869;     urine    quotient    C:N,    884; 

alcohol,    921;     diets,    932,    936,    939 
Aubert,  H.,  849 
Auche,  A.,  267,  431 
Austin,  A.  E.,  392 
Austrian,  C.  R.,  702 
Autenrieth,  W.,  766 
Aynand,  M.,  308,  314,  315 
Ayres,  W.  C,  615 
Akerman,  J.,  477 

Baas,  H.,  501,  720 

Babcock,  653 

Babkin,  B.,  107,  499,  682 

Bach,    A.,     oxygenases,     peroxides    and 
peroxidases,  871,  873;    catalases,  873;  j 
oxidation    processes,    875;     philothion,   ' 
877;  reduction  processes,  877  I 


Bacmeister,  439 

Baer,  J.,  43;  cystin,  148;  thiolactic  acid, 
151;  lactic  acid,  583;  ammonia  elimina- 
tion, 675,  676;  acetone  bodies,  818,. 
819,  822 

v.  Baeyer,  A.,  37,  38,  212 

Baginsky,  A.,  438,  555 

Baglioni,  S.,  333 

Bailie,  A.,  387 

Bainbridge,  F.  A.,  53,  351 

Baisch,  C,  749,  808 

Baker,  J.  L.,  226 

Balch,  A.,  414 

Baldi,  D.,  385 

Baldoni,  A.,  509,  783 

Balean,  H.,  301 

Balke,  P.,  purine  bases,  188,  193,  714; 
phosphocarmic  acid,  578 

Bang,  B.,  671 

Bang,  1.,  histone,  108;  guanylic  acid, 
183,  185;  nucleohistone,  307;  blood 
sugar,  329;  blood,  329;  estimation  of 
sugar,  329,  808-811;  lymph  glands 
and  thymus,  365-368;  glycogen,  402; 
saliva,  457;  rennin  enzyme,  473;  chlor- 
ine estimation  in  the  urine,  759;  pro- 
teoses in  the  urine,  792;  sugar  tests,  803, 
804;  sugar  formation,  399 

Barbara,  A.  G.,  356,  349,  415 

Barbieri,  J.,  152,  448 

Barbieri,  N.  A.,  615,  630 

Barcroft,  J.,  277-279 

Bardach,  Fr.,  726 

Bardach,  K.,  684  i 

Bardier,  E.,  757 

Barendrecht,  H.  P.,  65 

Barker,  B.,  307  < 

Barker,  L.  F.,  827 

Barral,  332 

Barratt,  W.,  849 

Barszczewski,  C,  210 

Bartholomans,  E.,  297,  295,  429 

Bartoschewitsch,  S.  F.,  724 

Basch,  K.,  667-669 

Baserin,  O.,  443 

Bashford,  E.,  722 

Baskoff,  385,  386 j 

Bass,  727 

Basso w,  461 

Bastianelli,  G.,  492 

Batelli,  F.,  fermentation,  407;  uricolysis, 
706;  peroxidases,  873,  874;  oxidation 
processes,  874,  875 

Baudrimont,  838 

Bauer,  Fr.,  182 

Bauer,  H.,  646 

Bauer,  J.,  525,  561 

Bauer,  K.,  757 

Bauer,  M.,  160 

Bauer,  R.,  113,  815 

Baum,  Fr.,  159 

Baumann,  E.,  diamines,  47;  cystine,  and 
cystinuria,  149,  827,  828;  thiolactic 
acid,    149,    150;     carbohydrates,    215; 


INDEX  OF  AUTHORS 


947 


iodothyrin,  376;  deamidation,  410; 
intestinal  putrefaction,  514, 720: ethereal 
sulphuric  acids,  515,  724-729,  784; 
hippuric  acid,  720;  oxyacids,  734, 
homogentisic  acid,  735,  73ii,  73s,  7:;'."; 
carbohydrates  in  the  urine,  749,  SOS; 
urinary  sulphuric  acids,  764;  sarcosine, 
77t>;  behavior  of  aromatic  bodies,  77s, 
780,    783:  mercapturic   acids,    786 

Baumann.  L.,  157,  288,  484,  513 

Baumgarten,  A.,  360 

Baumgarten,  O.,  403,  758 

Baumstark,  v.,  brain  constituents,  605, 
606,  612,  613;    urinary  pigments,  799 

Baumstark,  Rob.,  512,  524 

Haver.  H.,  136 

Bayer,  R..  372 

Bavliss.  W.  M.,  enzvme,  54,  enterokinase, 
492,  496;  secretin.  492,  498;  intestinal 
enzymes,  492,  493,  503;  trypsinogen 
and  trypsin,  496,  497,  503,  506;  casein 
digestion,  652 

Beattv.  W.  A.,  89 

Beaumont,  W.,  461,  481 

Bebeschin,  K.,  673 

Beccari,  L.,  435 

Bechamp,  A.,  633,  655,  661 

Bechhold,  H.,  colloids,  17,  18,  30,  32,  51; 
uric  acid,  70S;  sugar  determination,  804 

Becht,  F.  C,  349 

Beck.  A.,  744 

Beck.  C,  595 

Beckmann.  E.,  4 

Beckmann,  Ernst.  846 

Beckmann.  \Y.,  768 

Becquerel,  A.,  33S,  665 

Beger,  C,  667 

Behrend,  R..  698,  699 

v.  Behring,  E.,  66 

Beier,  Karl,  441 

Beitler,  C,  152 

Bellamv.  H..  496 

Bellori,  E..  653 

van  Bemmelen,  J.  M.,  29,  31 

Bence,  J.,  311 

Bence  Jones,  H.,  792 

Bendix,  E.,  208,  209,  394 

Benedicenti,  A.,  126,  729,  921 

Benedict,  F.  G.,  metabolism  in  work, 
595,  597;  respiration,  868;  calorimetry, 
885;  sparing  of  proteins,  914;  alcohol, 
921 

Benedict,  H.,  595.  752,  897 

Benedict,  S.  R.,  689,  693,  752 

Benedikt,  R.,  236 

Benedikt.  St..  214 

Benrath,  A..  477 

Berard.  E..  97.  633 

Berdez.  J.,  841 

Berenstein,  M.,  521 

Bergell.  P.,  carbohydrates  in  proteins, 
84,  262;  lecithin,  245;  polvpeptides, 
508:  placenta.  642;  casein,  662; 
oxybutyric  acid,  826 


Berber,  W.  M.,  456 
Bergh,  E.,  116 

Ber^holz,  R.,  50!) 

Bergin,  T.  J.,  519 

v.  Bergmann,  G.,  266,  267,  442 

Bergmann,  P.,  376,  466,  542-543 

Bergmann,  VVolfg,  761 

Berlioz,  A.,  758 

Berlinerblau,  M.,  334 

Bernard,  Claude,  blood  sugar,  332;  gly- 
colysis, 332;  glycogen,  389;  390,  398, 
sugar  puncture,  402;  diabetes,  405; 
pancreas,  495,  501,  502,  fat  absorption, 
538;  muscle  glycogen,  592 

Bernert,  R.,  83,  233,  358 

Bernheim,  A,  357,  359 

Bernheim,  R.,  766 

Bernstein,  J.,  602 

Bernstein,  N.  O.,  495 

Bert,  P.,  mammary  glands,  643,  669; 
gases  of  blood,  851,  861 

Bertagnini,  C.,  783 

Bertarelli,  E.,  64 

Berthelot,  M.  P.  E.,  division  law,  27; 
fat  cleavage,  501;  tunicin,  839,  calor- 
imetry, 885 

Bertin-Sans  H.,  285,  287 

Bertrand,  G.,  arsenic,  72,  373,  838; 
xylonic  acid,  210;  sugar  determina- 
tion, 808,  811;  tyrosinase,  842;  reptile 
poison,  846;  oxidases,  873 

Bertz,  F.,  558 

Berzelius,  J.  J.,  catalytic  reactions,  33; 
saliva,  458  -i 

Besbokaia,  M.,  496 

Best,  Fr.,  481 

Bezzola,  C.,  706 

Bial,  M.,  pentoses.  208,  209,  816;  glucur- 
onic acids,  221;  diastase,  265,  346, 
398;  glvcogen,  396 

Bialobrzeski.  M.,  292 

Bialocour,  F.,  485 

v.  Bibra,  E.,  389 

Bickel,  A.,  464,  465 

Bidder,  F.,  buccal  mucus,  453,  554; 
saliva,  458;  gastric  juice,  465;  pan- 
creatic juice,  499;  bile,  518;  fat 
absorption,  538 

Biedert,  Ph.,  660 

Biehler,  A.,  124 

Biel,  J.,  658,  662 

Bielfeld,  P.,  387 

Bienenfeld,  B.,  660 

Bienstock,  B.,  519,  520 

Biernacki,  E.,  blood,  309,  326;  pepsin, 
467;  trypsin,  503;  intestinal  putrefac- 
tion, 517,  519,  724 

Bierry,  H.,  cataphoresis,  50;  filtration.  51 ; 
enzvmes,  53,  71,  231;  pancreatic  juice, 
500 

Biffi,  U.,  267,  652,  744 

Billard,  G.,  45 

Billitzer,  J.,  26,  27 

Biltz,  W.,  glvcogen,  19,  20;    colloids,  22, 


948 


INDEX  OF   AUTHORS 


23;     adsorption,    28,    29,   69;    dextrin 
B,  230 
Binet,  P.,  541 

Bing,  H.  J.,  245,  331,  385,  398 
Bingel,  A.,  266 
Bing,  C,  777 
Biondi,  C,  42 
Biot,  J.  B.,  867 
Birchard,  Fr.,  134,  144 
Bisearo,  G.,  653 
Bischoff,  Th.,  879 
Bizio,  G.,  849 
Bizio,  J.,  390 
Bizzozero,  J.,  307,  314 
Bjerre,  P.,  921 
Bjorn-Andersen,  H.,  769 
Blachstein,  A.,  582 
Blanck,  F.  C,  696 
Blankenhorn,  E.,  606 
Blanksma,    J.,    amino-sugar,    167;     219, 
oxymethylfurfurol,   211,  215,  217;   ace- 
tone, 825 
Bleibtreu,  L.,  326,  690,  934 
Bleibtreu,  M.,  326,  563,  636 
Bleile,  A.  M.,  332 
Blendermann,  H.,  410,  734,  780 
Bliss,  C.  L.,  126 
Blix,  M.  G.,  326 
Bloch,  Br.,  735,  736 
Blondlot,  N.,  518 
Blood,  A.,  52 

Blum,  F.,  haloginized  protein,  82;    Mil- 
Ions  reaction,  99;   adrenalin  glycosuria, 
380 
Blum,  L.,  autolysis,  45;  protein  nitrogen, 
77;    protogon,    126;    alcoptonuria,  735, 
cystin,  776;  tvrosin  cleavage,  779,  ace- 
tone bodies,  818,  819,  822;    food  value 
of  albumoses,  912 
Blumenthal,   F.,   gelatin,   83;    indol  and 
skatol,   159;    pentoses,  208,  815,  816; 
nucleoproteins,     383;      glycogen,     394; 
assimilation  limit,  534;    urine  indican, 
728;  acetone,  818 
Boas,  J.,  488 
Bocarius,  N.,  621 
Bocchi,  O.,  741 
Bock,  C.,  398,  400 
Bock,  J.,  285 
Bode,  A.,  658 
Boden,  E.,  707 
Bodliinder,  G.,  921 
Bodon,  K.,  356 
Bodong,  A.,  318,  323 
Boedeker,  G\,  727 
Bddtker,  E.,  680,  759 
Boehm,  P.,  381 
Boehm,  I!..  448,  581,  590,591 

Moaner,   H.,  154 

Boehtlingk,  R.,  895,  896 

Hofikfjlmann.  W.  A.,  826 
Bor-ri,  C,.,  71 11,  752 
Boettger,  214 
Bogdanow,  E.,  5X6,  596 


Bogdanow-Beresowski,  455 

Bogen,  H.,  464 

Bogomoloff,  Th.,  743 

Bohland,  K.,  urea  nitrogen,  680;  urea, 
690;  uric  acid,  701;  ammonia,  768; 
protein  need,  934 

Bohm,  V.,  402 

Bohmansson,  G.,  803,  804 

Bohr,  Chr.,  blood  pigments,  276,  278, 
279,  282,  287;  egg  hatching,  637;  gases 
of  blood,  850-853;  metabolism  in 
lungs,  858,  869;  oxygen  tension,  859, 
861-863,  867;  carbon  dioxide  tension, 
865,  866;  specific  action  of  lungs,  864, 
867;  air-bladder,  867;  oxygen  capacity, 
868 

Du  Bois-Reymond,  E.,  592,  602 

Du  Bois,  Reymond,  R.,  312 

Bokorny,  T.',  212 

Bolafno,  C,  630 

Boland,  G.  W.,  365 

Boldyreff,  W.,  gastric  digestion,  481,  511; 
intestinal  juice,  490-493 

Boljarski,  N.,  694 

Bolin,  J.,  873 

Boll,  F.,  615 

Bonamartini,  G.,  96 

Bonanni,  A.,  437,  438,  785 

Bondi,  J.,  642,  643 

Bondi,  S.,  lipoproteids,  87;  sericin,  122, 
123;  bile  acids,  419,  421,  423;  acetoace- 
tic  acid,  825 

Bondzinski,  St.,  oxyproteic  acid,  83; 
koprosterin,  448;  ovalbumin,  633; 
urine  purines,  711;  urochrom,  741; 
oxyproteic  acids  in  urine,  753,  754 

Bonnema,  A.,  646 

Bookman,  S.,  721,  722 

Boos,  P.,  454 

Borchardt,  L.,  elastin  albumose,  263, 
527;  sugar  formation,  398;  fructose 
uria,     814;      acetone,     818,    819,    825 

Bordet,  J.,  antienzymes,  64;  sensibiliza- 
tors,  69;  blood  coagulation,  314,  318, 
320 

Borissow,  P.,  462,  717 

Borkel,  C.,  136 

Bornstein,  K.,  595,  918 

Boruttau,  H.,  581 

Bosshard,  E.,  149 

Bostock,  G.,  676 

Bottazzi,  Ph.,  freezing  point,  lowering  of 
blood,  12;    blood  corpuscles,  304;    gly- 
cogen, 384,  390,  391;  heart  muscle,  569; 
smooth  muscle,  602;  placenta,  640 
Bouchard,  Ch.,  393,  757,. 758 
Bouchet,  A.,  680 
Bouchez,  767,  772 
Boudet,  264 

Boulud,  glucuronic  acids,  221,  331;    pen- 
toses, 264;    sugar  in  blood,  264,  329- 
332;  glycolysis,333;  maltose  in  urine,  814 
Bouma,  .1.,  indican,  730;    bile  pigments, 
801,  oxybutyric  acid,  826 


INDEX  OF  AUTHORS 


949 


Bourcet,  P.,  269,  336 

Bourquelot,  E.,  395 

Bouveault,  L.,  144 

Brach,  H.,  839 

Bradley,  H.  C,  500 

Brahm,  C,  559,  785 

Brahm,  B.,  179,  182,  183,  211 

Brand,  J.,  414,  437,  438 

Brandberg,  J.,  793 

Brandenburg,  K.,  310 

Brandl,  J.,  Ml 

Brasch,  VV.,  144,  534 

Brat,  H.,  209 

Brauer,  L.,  140 

Braun,  K.,  04 

Braunstein,  A.,  408 

Brautlecht,  C,  78 

Bredig,  G.,  colloidal  metals  14;  surface 
tension,  26;  catalysis,  33-37;  assym- 
etric  synthesis,  59 

Brienl,  F.,  103 

Brenzinger,  K.,  827 

Bretschneider,  A.,  329,  330 

Brewster,  J.  F.,  155 

Brieger,  L.,  putrefaction  products,  47; 
neurine,  246;  intestinal  putrefaction, 
514;  skatol,  521;  neuridine,  606,  612, 
628;  urine  indican,  727,  729;  skatoxyl- 
sulphuric  acid,  732  \  cystinuria,  827; 
perspiration,  848 

Briggs,  C.  E.,  402 

Brig],  P.,  179,  183 

Brion,  A.,  774 

Brodie.  T.  G.,'256,  400,  509 

Brodlev,  H.  C,  72 

Brook,  F.  \Y  ,  82 

Brooks,  CI.,  399 

Browinski,  J.,  266,  741,  754 

Brown.  A.  J.,  55,  56,  65 

Brown,  E.  W  .,  705,  717 

Brown,  H.  T.,  inverting  enzymes,  55; 
starch  hydrolysis,  226,  229,  456,  sacha- 
rase,  492 

Brown.  R.,  19 

Brown,  T.  Graham,  594 

Browne,  C.  A.  (jr.),  647 

Brubacher,  H.,  554,  557 

v.  Brucke.  E.,  blood  coagulation,  316; 
glycogen,  392;  pepsin  467-469,  fat 
emulsion,  511;  protein  absorption, 
525;    carbohydrates  in   urine,   749 

Brugsch,  Th.,  bile  pigments,  443;  pan- 
creas, 532;  uric  acid,  700,  701;  hip- 
puric  acid,  721;  amino-acids  in  urine, 
757;  urine  in  hunger,  897 

Bruhns,  G.,  193 

Brunner,  E.,  36 

Brunner,  Th.,  665 

Bruno,  G.,  501,  502,  506,  510,  758 

Brunton-Blaikie,  572 

de  Bruyn,  Lobrv,  19,  201,  220 

Bryant,  A.  P.,  936,  938 

Buchanan,  A.,  256 

Buchner,    E.,    alcohol    fermentation,    41, 


205;  lactic  ackl  fermentation,  207; 
sugar  test,  213 

Buchner,  H„  41,  856 

Buchtala,  H.,  113-115,  147,  426 

Budde,  V.,  Sl_> 

Billow,  K.,  <).-»,  227 

Biinz,  R.,  612,  613 

Burger,  L.,  545 

Burker,  K.,  314,  315 

Bufalini,  339 

Bugarszky,  St.,  271,  326 

Buglia,  G.,  510,  572,  602,  603 

Bull,  H.,  237 

v.  Bunge,  G.,  serum,  270;  blood  cor- 
puscles, 304;  blood  coagulation,  313; 
blood  analysis,  328;  iron  preparations, 
340;  blood  and  high  altitude,  341; 
iron  in  liver,  387;  gastric  juice,  485; 
cartilage,  550;  hamatogen,  629,  637; 
milk,  657,  664,666-668;  hippuric  acid, 
722,  723;  mineral  requirement,  900, 
901 

Bunsen,  R.,  690 

Buntzen,  J.,  339 

Buraczewski,  J.,  83 

Burchard,  H.,  447 

Burckhardt,  A.  E.,  270 

Burian,  R.,  purine  bases  and  their 
enzymes,  193,  195,  371,  373,  572,  594, 
702,  703,  713;  uric  acid  formation,  700, 
792,  703,  705;  uric  acid  cleavage,  706; 
histone  in  urine,  795 

Burow,  R.,  371,  663 

Busch,  P.  W.,  349,  350 

Butlerow,  A.,  211,  212 

Butterfield,  E.,  277,  303 

Bywaters,  H.  W.,  260,  263 

Cade,  A.,  464 

Cahn,  A.,  476,  615 

Camerer,  W.,  milk,  656,  657,  662,  664- 
666;  urine  nitrogen,  680;  uric  acid  elim- 
ination, 700;  purine  bases,  715;  meta- 
bolism, 922,  924 

Camerer,  W.,  jr.,  847 

Cameron,  A.  T.,  78,  109 

Cam  is,  M.,  279,  592 

Cammidge,  P.  J.,  815,  827 

Campani,  A.,  763 

Campbell,  G.,  628,  629,  633,  634 

Campbell,  J.  F„  432-434 

Camps,  R.,  739 

Camus,  L.,  pancreatic  juice,  496;  secretin, 
498;  vesiculase,  622 

Cannon,  W.  B.,  479-484,  524 

Cappelli,  J.,  304,  602 

Cappezzuoli,  C,  369,  557 

Capranica,  St..  190.  848 

Carbone,  D.,  613 

Carlier,  E.  \\\,  313,  347 

Carlinfanti,  E.,  235,  236 

Carlini,  C,  381 

Carlson,  A.  J.,  348,  349,  454 

Carlson,  C.  E.,  873 


950 


INDEX  OF  AUTHORS 


Carnot,  Ad.,  552,  558 

Carvallo,  J.,  485,  48(5 

Casali,  A.,  847 

Caspari,    W.,    high    altitude    341,    888; 

protein  metabolism,  595,  916,  milk  fat; 

669;  vegetable  diet,  915-916 
Castoro,  N.,  161 
Catheart,  E.  P.,  autolysis,  44;    glycogen, 

396;    stomach,  479;    gastric  digestion, 

4S1;    protein  absorption,  527;    creatine 

and   creatinine,  594,  693;    milk  sugar, 

670;  starvation  urine,  897 
Cavazzani,   E.,   cerebrospinal  fluid,   361; 

glycogen    cleavage,    399;     absorption, 

535,  phosphocarnic  acid,  578;  muscular 

work,  592;  semen,  620 
Cernj,  C,  838 
Cerny,  T.,  583,  792 
Chabbas,  J.,  617 
Chabrie,  C,  557 
Chandelon,  Th.,  592 
Chaniewski,  563 
Charnas,  D.,  746,  747 
Chassevant,  A.,  706 
Chauveau,     A.,     sugar    formation,     412; 

fat    formation,    562;     muscular    work; 

592,  597 
Cherry,  Th.,  68 
Chigin,  P.,  462 
Chistoni,  A.,  314,  315 
Chittenden,  R.  H.,  keratin,  119;    elastin, 

116,  117;   gelatin,  118,  120;   albumoses 

and    peptones,    128-131,    137;     saliva, 

455-458;  pepsin,  470,  471;  trypsin,  506; 

tendon    mucoid,     545,     551;      myosin, 

567,  568;    neurokeratin,  605,  613,  614; 

protein  requirement,  934 
Chodat,     R.,     oxidases,     peroxides     and 

peroxidases,  871;    oxidation  processes, 

875;  ,"  philothion,"  877 
Chossat,  Th.,  892,  896 
Christenn,  G.,  662 
Christensen,  A.,  793 
Ciamician,  G.,  159 
Cingolani,  M.,  699 
Citron,  H.,  766 
Clapp,  S.  H.,  89,  107 
Clar,  C,  701 
Clarke,  T.  W.,  287 
Claus,  R.,  408 

Clausmann,  P.,  693,  552,  558 
Clemens,  Paul,  785.  827 
Clemm,  C.  G.,  665 
Clerk,  B.,  265 
Cleve,  P.  T.,  422,  423 
Cloetta,  M.,  290,  292,  293,  753 
Cloez,  441 
Clopatt,  A.,  921 
Closson,  O.  E.,  400 
Cobliner.  332 
Cohn,  Felix,  485 
Cohn,  Max,  500 
Cohn,  Michael,  455 
Cohn,  R.,  leucinimid,  145;    carbohydrate 


formation,  410,  411;  fate  of  aromatic 
substances  in  the  animal  organism,  778, 
783,   784;    furfurol,   784,  pyridin,   787 

Cohn,  Th.,  621,  717 

Cohnheim,  J.,  456 

Cohnheim,  O.,  lipoid  action,  10;  proteins, 
76,  95;  blood  and  high  altitudes,  341, 
340;  glycolysis;  407,  408;  gastric 
juice,  465;  peptic  digestion,  481,  484; 
absorption,  484,  528,  542-543;  erepsin, 
491,  493;  pancreatic  juice,  497;  con- 
nective tissue,  512;  peristalsis,  524; 
digestion  work,  930;  accommodation 
of  digestive  enzymes,  53 

Cohnstein,  J.,  336 

Cohnstein,  W.,  309,  352 

Colasanti,  G,  591;  593,  698,  748,  749 

Cole,  S.  W.,  protein  reaction,  100;  trypto- 
phane, 155-158 

Collmann,  849 

Comaille,  A.,  653,  668 

Comesatti,  G.,  380 

Comiotti,  L.,  816 

Connstein,  W.,  265 

Conradi,  H.,  45,  324,  520 

Constantinidi,  A.,  535,  915,  916 

Constantino,  A.,  572 

Contejean,  Ch  ,  phlorhizin  diabetes, 
400;  gastric  juice,  465,  476;  pyloric 
secretion,  477 

Copemann,  M.,  797 

Coranda,  G.,  682 

Cordua,  H.,  301 

Corin,  G.,  protein  coagulation,  97;  fibrino- 
gen, 253;    proteins  of  egg-white,   633 

Coronedi,  G.,  560 

Corper,  H.  J.,  372,  449-450,  701,  706 

Corvisart,  L.,  502 

Costantino,  A.,  586,  602,  603,  748 

Le  Count,  E.  R.,  114 

Courant,   G.,   milk,   644,   649,   650,    659 

Cousin,  H.,  242,  248 

Couvreur,  E.,  393 

Cramer.  C.  D.,  253 

Crameri  E.,  122,  848 

Cramer,  Tr.,  531 

Cramer,  W.,  absorption,  525,  528,  530; 
protagon,  606-608;  placenta,  640; 
creatine,  692;  hippuric  acid,  722; 
blood  coagulation,  320 

Cremer,  M.,  glycogen,  58;  332,  390, 
393,  395;  pentoses,  208;  glycolysis, 
332;  phlorizin-diabetes,  400;  sugar 
formation,    412;     fat    formation,    562 

Cristea,  G.,  336 

Crittenden,  A.  L.,  454 

Croftan,  A.,  334 

Croft-Hill,  A.,  58,  225 

Croner,  W.,  488.  536 

Cronheim,  W..  488 

Croockewitt,  J.  H.,  122 

Csdkae,  J.,  648,  658 

v.  Csonka,  F.,  657 

Cummis,  G.  W.,  506,  567,  568 


INDEX   OF  AUTHORS 


951 


Cunningham,  K.  II  ,  ">10 
Curtius,  Th.,  85,  So,  422 
Cutler,  W.  D.,  168,  544,  545 
Cybulski,  N.,  379 
Czernecki,  W.,  266,  358 
Czerny,  Adalb,  307 
Czernv,  F.,  407, 
Czerny,  V.,  485,  525 
v.  Czyhlarz,  E.,  873 

Daddi,  L.,  339,  669 

Daenhardt,  C,  856 

Dakin,  H.  D.,  autolysis,  44;  mandelic 
acid  ester,  62;  arginase,  89,  161,  574, 
681,  682;  protamine,  109,  110;  valine, 
141;  serine,  145;  proline,  154;  color 
reactions,  157;  lysin,  163;  hexon  bases, 
164;  oxalic  acid,  716,  773;  alcap- 
tonuria,  737;  cleavage  of  fatty  acids 
774,  781;  uramino  acids,  786;  acetone 
formers,  822;  sugar  formation,  412; 
lactic   acid,   583;    a-amino  acids,   775 

Daland,  J.,  329 

van  Dam,  W.,  474,  650 

Danilewski,  A.,  plasteines,  58,  135;  pro- 
tein sulphur,  79;  retarding  substances, 
487,  492;  muscle  protein,  566-569; 
milk  globules,  646 

Danilewsky,  B.,  885 

Danilewsky,  W.,  241 

Dareste,  C,  620,  628 

Darmstadter,  E.,  826 

Darmstadter,  J.,  846 

Darmstadter,  L.,  448 

Dastre,  A.,  fibrinogen,  252;  fibrinolysis, 
255;  glycogen,  307,  346;  blood  coagula- 
tion, 314;  liver,  383,  384,  388;  gly- 
cogen, 395,  397,  399;  bile,  414,  434, 
435,  511;  enterokinase,  496,  497;  fat 
absorption,  538 

Dantzenberg,  P.  J.  W.,  783 

Danwe,  F.,  49 

Davidoff,  W.,  75 

Day,  H.,  480 

Dean,  A.  L.,  228,  529 

Decaisne,  E.,  667 

Deetjen,  H.,  308,  314,  315 

Dehn,  W.  M.,  760 

Dekhuysen,  C,  13 

Delezenne,  C  ,  enzyme  retardation,  65; 
papain,  65;  blood  coagulation,  250, 
313,  318,  320,  324,  325,  351;  intestinal 
juice,  490-492,  enterokinase,  and  pan- 
creatic juice,  493,  495-497,  509;  secre- 
tin, 498 

Delfino,  A.,  640 

Demant,  B.,  491 

Detnoor,  J.,  453 

Denig^s,  G.,  tyrosin,  154;  indol  and 
skatol  reactions,  158,  159;  inosite, 
580;  homogentisic  acid,  739 

Denis,  P.  S.,  257 

Denis,  W.,  tyrosin,  154;  urea  in  blood, 
333,  336;     hypophysis,  592;     creatine, 


692,    606:    uric  acid,  708,  711,  334; 
urine  sulphur,  752 

Denk,  \\\,  336 

Dennemark,  L.,  658 

Derrien,  E.,  glucose,  215;  blood  pig- 
ments, 281,  285,  oxymethylfurfurol, 
419;  Bence-Jones,  protein,  792 

Desgrez,  A.,  393 

Dencher.  P.,  532,  539,  540 

Devilard,  P.,  360 

Devoto,  L.,  792 

Dewitz,  J.,  843 

Diakanow,  C,  243-245 

Diamare,  V.,  494 

Dick,  AL,  702 

Diels,  O.,  445,  446,  448 

Diesselhorst,  G.,  848 

Dietrich,  M.,  104,  652 

Dietschv,  R.,  792 

Dietz,  VV.,  56,  60 

Dietze,  Alb.,  506 

Dillner,  H.,  633 

Dimitz,  L.,  248,  614 

Disque,  L.,  740,  742,  744 

Ditthorn,  Fr.,  167,  173,  219 

Dittrich,  P.,  285 

Ditz,  H.,  726 

Doblin,  A.,  329,  333 

Dorpinghaus,  Th.,  carbohydrates  in  pro- 
tein substances,  84;'  protein  hydrolysis, 
113,  141,  142,  252 

Dohm,  M.,  406 

Dombrowsky,  St.,  urinary  pigments, 
740,  741;  oxyproteic  acids  in  urine, 
753,  754;  urinary  bases,  757 

de  Domenicis,  A.,  287 

de  Dominicis,  N.,  404 

Donath,  J.,  360 

Donne,  A.,  799 

Donv-Henault,  O.,  873 

Donze,  G.,  680 

Doree,  Ch.,  448 

Dormann,  E.,  298 

Dorner,  G.,  574 

Douglas,  C.  G.,  286 

Douglas,  Gordon  C,  864 

Doyon,  M.,  fibrinogen,  252,  253,  335; 
serum  lipase,  265;  blood  coagulation, 
324,  325;  glycolysis,  332;  bile,  415,  416, 
434,  439 

Dragendorff,  D„  800 

Drechsel,  E.,  proteins,  76,  79,  94,  123; 
diamino-acetic  acid,  163;  lysin,  163, 
164;  purin  bases,  188;  thyroid  gland, 
375;  jecorin,  385;  urea  formation, 
681,  683;  carbamic  acid,  683;  silicic 
acid  ester,  838 

Dreser,  H.,  13,  71 

Dreyfus,  G.  L.,  465 

Droop-Richmond,  H.,  646 

Drosdoff,  W.f  335 

Dubelir,  D.,  920 

Ducceschi,  V.,  475,  569 

Duclaux,  E.,  97,  647 


952 


INDEX  OF  AUTHORS 


Ducleau,  E.,  55 
Dudley,  H.  W.,  583,  775 
During,  Fr.,  554 
Dufau,  E.,  80S 
Dufourt,  415,  416,  439,  592 
Duggan,  C.  W.,  97 
Dull,  G.,  229 
Dumas,  J.  A.,  684 
Dunham,  E.,  180,  239,  673 
Dunlop,  J.  C,  595,  715 

Ebbeke,  U.,  757 

Ebstein,  E.,  114,  208,  209 

Ebstein,  W.,  457,  727,  831 

Eckhard,  C,  452 

Edelstein,  E.,  43 

Edelstein,  F.,  657,  662 

Edic,  E.,  246,  402 

Edkins,  J.  S.,  463,  477,  509 

verEecke,  A.,  925 

Ehrenfeld,  R.,  proteins,  83;  leucine,  143; 
tyrosin,  153 

Ehenreich,  M.,  49,  505 

Ehrenthal,  W.,  521 

Ehrlich,  F.,  amino-acids,  143,  144,  153, 
206;  fermentation,  157;    fusel  oil,  206 

Ehrlich,  P.,  side  chain  theory,  67, 
68;  amboceptors,  69;  dimethylamino- 
benzaldehyde,  159,  219,  746,  827; 
bilirubin,  431;  urine  test,  826 

Ehrmann,  R.,  405 

Ehrstrom,  R.,  histone,  108;  phosphate, 
761,762,796;  albumoseuria,  791 

Eichholz,  A.,  260,  633,  743 

Eichhorst,  H.,  525 

Einhorn,  M.,  159 

Ekbom,  A.,  426 

Ekehorn,  G.,  760 

van  Ekenstein,  A.,  amino-sugar,  167, 
219;  carbohydrates,  201,  oxymethyl- 
furfurol,  211,  215,  217;    acetone,  825 

Ekholm,  K.,  936 

Elias,  H.,  609 

Ellenberger,  W.,  479,  480,  658,  659 

Ellinger,  A.,  isoserin,  145;  tryptophan, 
155,  156;  arginin,  163;  blood  coagula- 
tion, 324;  lymph  formation,  351,  352; 
pancreatic  secretion,  500;  urine  in- 
dican,  728,  730;  tri-indylmethane  pig- 
ments, 734;  kynurenic  acid,  739; 
oxyphenyllactic  acid,  780;  food  value 
of.albumoses,  912 

Ellmer,  A.,  237 

Ely,  J.,  455,  457 

Embden,  G.,  cystein  and  cystine,  79, 
147,  149;  serine,  145;  glycolysis,  333, 
408;  carbohydrate  formation.  410; 
liver  blood  perfusion,  529,  530,  775, 
786;  lactic  acid,  583,  584,  333;  gly- 
cocoll  in  urine,  756;  acetone  bodies,  819 
822,  825,  826 

Embden,  H.,  735 

Emerson,  R.  L.,  507 

Emich,  Fr.,  517 


Emmerling,  A.,  113 

Emmerling,  O.,  58,  59,  225 

Emsmann,  Otto,  463 

Engel,  662 

Engel,   H.,  Upases,   57,  476;    lactic    acid 

formation,   585;    acetone    bodies,   818, 

821 
Engel,  St.,  658,  663 
Engeland,  R.,  agmantin,  162;    aporrheg- 

men,  166;  carnitin,  577;  methvl-guani- 

din,  698;  urine  bases,  757,  826^  827 
Engelmann,  G.  J.,  595 
Engler,  C.,  871,  875 
Eppinger,  H.,  375,  406,  676,  799 
Eppinger,  P.,  290,  292,  293,  354 
Epstein,  A.,  721,  722 

Erben,  Fr.,  oxystearic  acid,  233;    leuco- 
cytes,  307,  342;    blood  analysis,   342; 

chyle     fat,  346;    urine  nitrogen,  681, 

urein,  691 
Erdelvi,  A.,  482 
Erikson,  A.,  50,  63 
Erlandsen,    A.,    phosphatides,    242,    243, 

245,  246,  586;   cuorin,  248,  249;  phlor- 

hizindiabetes,  400 
Erlanger,  J.,  541 
Erlenmeyer,  E.,  142,  152 
Erlenmeyer,  E.  (jr.)  145,  148,  152 
d'Errico,  G.,  391 
Esbach,  G.,  793 
Escher,  Heins,  H.,  623,  631 
Estor,  A.,  867 
Etard,  A.,  578 
Etti,  C.,  642 
Ettinger,  J.,  43 
Eulenburg,  398 
v.  Euler,  H.,  activators,  52;  enzymes,  53,. 

56,    70;     phosphoric   acid    ester,    205; 

erepsin,  493;    oxidation  processes,  873 
Eves,  F.,  457 
Ewald,  Aug.,  proteins,  112,  119;   haema- 

toidin,  301;      digestion,     508;      visual 

purple,  615;  corpora  lutea,  623 
Ewald,  C.  A.,  857,  928 
Ewins,  A.  I.,  380 
Eykman,  C.,  326,  929,  932 
Eymonnet,  757 

Fabian,  E.,  396 

Fahr,  G.,  587 

Fajans,  K.,  35 

Falck,  910 

Falk,  Edm,  642 

Falk,  Ernst,  924,  925 

Falk,  Fr.,  248,  613 

Falloise,    A.,    bile,    416;     gastric    lipase,. 

476;      intestinal    enzyme;    492,     493; 

chloralsecretin,  499;   blood  gas  tension, 

861,  866 
Falta,  W.,  thyroid  gland,  375;    diabetes 

405,  406,  409,  412;    alcaptonuria,  735, 

736;  protein  metabolism,  882,  883,  907 
Fano,  G.,  251 
Farkas,  K.,  638 


INDEX  OF  AUTHORS 


953 


Farmer,  Ch.,  688 

Farwik,  B.,  599 

Fasal,  H.,  106,  107,  158,  652 

Faust,  E.,  sepsine,  47;  gelatin,  118: 
poisons  in  the  secretion  of  the  skin  and 
salivary  glands,  847 

Favre,  P.  A.,  848 

Fawitzki,  A.,  685 

Fedeli,  C,  686 

Feder,  L.,  682 

Fehling,  H.,  214,  808 

Fehrsen,  A.,  338 

Fetgin,  P.,  721 

Feinshmidt.  J.,  408 

v.  Fenyvessy,  Bela,  751,  785 

Fermi,  CI.,  255,  486,  505 

Fernet,  E.,  855 

Ferrv,  E.,  904 

Kick,  A.,  596 

Fiessinger,  N.,  307,  364 

Fiiehne,  \V.,  440 

Filhol,  670 

De  Filippi,  F.,  397,  534,  757 

Fine,  M.  S.,  692 

Fingerling,  G.,  667 

Fink,  H.,  295 

Finkler,  D.,  41 

Fischer,  Ch.,  140 

Fischer,    Kuril,  enzymes,  58,  59;    specif- 
icity of  enzymes  action,  62,  65;  amino- 
acids,    85,      140-145,     148,       152-155 
polypeptides,    86-89,    124,    132,    133 
136;    protein  hydrolysis,  113,  119,  120 
132,     133,     136,    507;     ornithin,     162 
lysin,     163;      diaminotrioxydodecanoic 
acid,   165;    purin  bases,  186,   188-191 
pyrimidine    bases,     194,     195;     carbo- 
hydrates, 197-202,  211,  212,  215,  216 
glucosamine,  218;  glucuronic  acid,  220 
isomaltose,  225;    lactose  fermentation 
654;  uric  acid,  698;  urine  purines,  711 
conjugated  glucuronic  acids,  751 

Fischer,  Hans,  blood  pigment  derivatives 
295-297,  295;  bile  acids,  425,  427 
bilirubin,  hydrobilirubin,  urobilinogen 
and  urobilin,  429,  744,  746,  747,  429 
koprosterin,  448;  urobilinoids,  743 
bilirubic  acid,  429 

Fischer,  H.  W.,  587 

Fischer,  Martin,  400 

Fischler,  E.,  686 

Fischler,  M.,  434,  744 

Fisher,  H.  L.,  694 

Fiske,  P.  S.,  36,  38 

Fitzgerald,  476 

Flack,  M.,  374 

Flacher,  Fr.,  379 

Planum.!,  CI.,  152,  153,  734 

Flanders,  Fr.,  723 

Flatow,  L.,  738,  739,  779,  780 

Flatow,  R.,  711 

Fleckseder,  R.,  455 

Fleig,  C.  416,  498,  499 

Fleischer,  R.,  402 


Fleischl,  E.,  303,  441 

Fleischmann,  W.,  (>47 

Fleischer,  M.  S.,  320 

Fleitmann,  79 

Fletcher,  W.  M.,  459,  585,  593 

Flint,  A.,  448,  488 

Floresco,  N.f  3X4,  388,  434 

Fluckiger,  M.,  749,  750 

Foa  C,  455,  64.^,  659 

Folin,  ().,  cystine,  149;  tyrosine  test,  154; 
blood  alkalinity,  309;  urea,  333,  335; 
urine  acidity,  677;  nitrogen  determina- 
tion, 688;  urea  determination,  688- 
690;  urein,  691;  methylurea,  691; 
creatinine  and  creatine,  691-693,  696, 
697;  uric  acid,  700,  708,  711,  334; 
hippuric  acid,  723;  urinary  sulphur, 
752,  766;  ammonia,  768,  769;  acetone, 
825;  protein  metabolism,  910 

Fordos,  M.,  365 

La  For-e,  F.,  179,  180,  185 

Forrest,  J.  R..  553 

Forschbach,  J.,  396 

Forssner,  G.,  756,  819,  820 

Forster,  J.,  mineral  metabolism,  72,  899; 
transfusion,  344;  water  and  metabol- 
ism, 920;  metabolism  of  sucklings,  925; 
diets,  932,  939 

Fosse,  R.,  681 

Foster,  M.  L.,  159 

Foster,  N.  B.,  694 

Franckel,  P.,  465 

Frankel,  A.,  685,  860 

Frankel,  Sigm.,  proteins,  79,  102;  pep- 
tones, 131;  thiolactic  acid,  151;  his- 
tidin,  160;  albumin,  219;  cephalin, 
248;  thyroids,  375;  adrenalin,  380; 
glycogen,  396;  gastric  juice,  476; 
chondrosin,  548;  brain  phosphatides, 
605,  609;  brain  analyses,  613,  614; 
neottin,  630,  kidney  phosphatides, 
673;  homogentisic  acid,  738;  chitin, 
839;  protein  cleavage,  925 

Frankel,  W.,  34 

Framm,  F.,  120,  213,  332 

Franchimont,  A.  P.,  839 

Frank,  E.,  265,  329-332 

Frank,  Fr.,  702,  706 

Frank,  O.,  535-537,  591.  910 

Frankland,  E.,  885 

Franz,  Fr.,  251 

Frauenberger,  Fr.,  547 

Frazer,  J.  C.  W.,  3 

Fredericq,  L.,  proteincoagulation  97; 
serumglobulin,  260;  haemocyanin,  303, 
304;  blood  coagulation,  313"  blood 
gases,  852,  861,  862,  864,  866 

Frehn,  A.,  663 

Fremv,  E.,  603,  636 

Frenkel-Heiden,  360 

Frenzel,  J.,  glvcogen,  391,  393;  work  and 
fat  destruction,  596,  598;  meat,  600; 
calories  and  nitrogen,  892 

Frerichs,    F.   Th.,    synovia,    362,    human 


^54 


INDEX  OF  AUTHORS 


bile,  437;  saliva,  458;  uric  acid  de- 
composition, 705 

Freudberg,  A.,  309 

Freund,  E.,  serumglobulins,  259;  albu- 
moses  in  blood,  263;  haematinof:  en,  299; 
blood  coagulation,  313,  320;  glycogen, 
395;  .digestion  blood,  530;  chlorine 
determination,  759;  lungs,  870;  starva- 
tion metabolism,  987 

Freund,  O.,  897 

Freundlich,  H.,  22;  surface  tension,  28; 
adsorption,  29 

Prey,  W.,  756 

Frevtag,  Fr.,  cerebrosides,  364,  607,  610, 
protagon,  606,  607,  610 

Fridericia,  L.  S.,  597 

Friedenthal,  PL,  467,  525 

Fried jung,  J.  K.,  664 

Friedlander,  G.,  525 

Frienlander,  P.,  843 

Friedmann,  E.,  protein  sulphur,  79; 
thiolactic  acid,  113,  151;  albumoses, 
134;  isoleucine,  144;  cystine,  148; 
cysteinic  acid  and  taurine,  148,  151, 
442;  adrenalin,  379;  demolition  of 
fatty  acids,  774,  776,  781;  of  aromatic 
substances,  779-781,  736;  furfurol, 
784;  acetone  former,  820,  822 

Friend,  W.  M.,  274,  356 

Fries,  H.,  593 

Frohlich,  379 

Fromherz,  K.,  735,  736,  780 

Fromholdt,  G.,  743,  744 

Fromm,  E.,  785 

Fromme,  A.,  57,  476 

Frommer,  V.,  823 

Frouin,  A.,  thyroid  gland,  377;  gastric 
juice,  463,  464;  intestinal  juice,  490- 
492;  pancreatic  juice,  496 

Frugoni,  C.,  405 

Fubini,  S.,  849 

Fuchs,  D.,  114,  154 

Fiirbringer,  P.,  715,  791 

v.  Furth,  O.,  peroxyproteic  acids,  83; 
xanthoprotein,  83;  nucleic  acids,  184; 
cholin,  247;  iodothyrin,  376;  supra- 
renin,  379;  adrenalinglucosuria,  405; 
cholic  acid,  424;  secretin,  498;  bile 
and  fat  splitting,  502;  muscles,  566- 
572,  589,  590;  camosin,  575;  smooth 
muscles,  602;  chitosin,  839;  melanins, 
841-843;  tyrosinase,  843,  peroxydase, 
873 

Fuld,  E.,  rennin  action,  57,  474,  650; 
fibrin  formation,  256,  318,  319,  324; 
pepsin  determination,  470;  womans' 
milk,  660,  662 

Fuller,  J.  G.,  902 

Funk,  C.,  amino-aoid  combination,  87, 
88;  glutamic  acid,  146;  urinary  sul- 
phur, 752;  sugar  determination,  809; 
vitamin'',  905 

v.  Funke,  595 


Gabriel,  S.,  cystine,  148;  bones,  552; 
teeth,  558;  ovalbumin,  633;  food- 
value  of  aspargin,  912 

Gache,  J.,  496 

Gaglio,  G.,  334,  582,  773 

Galdi,  F.,  354 

Galeotti,  G.,  96,  706 

Galimard,  J.,  113 

Galli,  P.,  267 

Gallois,  580 

Gamgee,  A.,  nucleoproteins,  175;  nucleic 
acids,  182;  blood  pigments,  280; 
intestinal  juice,  490;  protagon,  606, 
607;  pseudocerebrin,  611 

Gammeltoft,  S.  A.,  689,  690,  767 

Ganassini,  D.,  455,  456,  709 

Gansser,  E.,  278 

Gardner,  J.  A.,  448 

Garrod,  A.  E.,  hsematoporphyrin,  295, 
299,  797;  alcaptonuria,  738;  homo- 
gen  tisic  acid,  738,  739;  urochrome,  740, 
741;  urobilin,  743,  745;  uroerythrin, 
748;  cystinuria,  827 

Gaskell,  J.  F.,  828 

Gassmann,  Th.,  552,  557,  558 

Gatin-Gruzewska,  Z.,  glycogen,  19,  20, 
390,  391;  seralbumin,  261 

Gaube,  T.,  848 

Gaule,  J.,  340 

Gaunt,  41 

Gautier,  A.,  ptomaines,  47;  arsenic,  72, 
269,  336,  368,  373,  838;  glycogen,  392; 
fat  formation,  562;  muscles,  578; 
protein  of  hen  egg,  633,  634;  xantho- 
creatininine,    698;     fluorine,    552,    558 

Gautier,  CI.,  252,  253,  654,  744 

Gawinski,  W.,  756 

v.  Gebhardt,  F.,  591,  909 

Geelmuvden,  H.  C.,  sugar  in  urine,  814, 
816;  acetone  bodies,  819,  820,  822,  826 

Geiger,  W.,  145 

Geissler,  791 

Generali,  F.,  374 

Gengou,  O.,  64,  314,  318,  320 

v.  Genser,  665 

Gentzen,  M.,  728 

Geoghegan,  E.  G.,  609,  610,  614 

Gephart,  F.,  245,  680,  689,  769 

Geppert,  J.,  blood  alkalinity,  309;  respira- 
tion, 860,  869,  890;  alcohol,  921 

Gerard,  E.,  446,  699 

Gerber,  N.,  662 

Gerhardt,  C.,  824 

Gerhardt,  D.,  744,  745 

Gerhartz,  H.,  870 

Gerngross,  O.,  195 

Gertner,  W.,  416 

Gessard,  C.,  843 

Gewin,  J.  W.  A.,  475 

Geyer,  J.,  806 

Geyger,  A.,  735 

Giacosa,  P.,  mucins,  167;  blood  pigments, 
302;  frog  egg,  636;  iron  in  urine,  770; 
aromatic  substances,  779 


INDEX   OF  AUTHORS 


955 


Giaja,  J.,  71,  231 

Gibson,  R.,  103 

Giertz,  H.,  104,  177 

Gies,  W.  J.,  elastin,  116-1  IS;  hexone 
bases,  164;  mucin  substances,  16,N, 
L89,  472,  644,  645,  551;  lymph,  349 
351;  pancreatic  juice,  ■"><>(>;  ligaments 
and  tendons,  545;  bones,  f>f)l;  pro- 
tagon,  606,  608;  phrenosin,  611; 
urein,  691 

Gigon,  A.,  polypeptides,  65;  diabetes, 
405,  409;  amino-acid  supply,  721, 
722;  amino-acide  in  urine,  756;  basal- 
requirement,  898,  931;  metabolism, 
929-931 

Gilbert,  531,  563 

Cull,  F.  W.,  689,  752 

Gilson,  E.,  242,  245,  839 

Ginsberg,  S.,  534 

Ginsberg,  VV.,  754,  756 

Githens,  Th.,  St.,  270 

Giunti,  L.,  773 

Gizelt,  A.,  498 

Gjaldbak,  I.  K.,  58,  135,  165 

Glaessner,  K.,  pseudopepsin,  466,  489, 
490;  erepsin,  493;  pancreatic  juice, 
500,  509 

Gleiss,  W.,  593 

Glendinning,  T.  A.,  55 

Gley,  E.,  iodine  in  blood,  269;  blood 
coagulation,  324;  lymphagogues,  351; 
thyroidea,  374;  pancreatic  juice,  496, 
498;  heart  muscle;  599,  vesiculase, 
622 

Glikin,  W.,  fat,  233;  lecithin,  241,  244, 
245,  663;  liver  nitrogen,  387;  choles- 
terin,  445;  milk,  663 

Glund,  W.,  87 

Gmelin,  L.,  432,  458 

Gmelin,  W.,  465 

Gogitidse,  S.,  669 

Goldmann,  E.,  ovstine  and  cystinuria, 
149,827,828;  iodothyrin,  376 

Goldmann,  F.,  813 

Goldschmidt,  C,  687 

tioldschmidt,  F.,  133 

Goldschmidt,  H.,  36,  456 

Golodetz,  L.,  844 

Gompel,  M.,  65 

Gonnermann,  M.,  140,  508 

Goodbodv,  W.,  515 

Goodwin,  R.,  130 

Gorodecki,  443 

Gortner,  R.  A.,  841,  842 

v.  Gorup-Besanez,  E.  F.,  356,  437,  838 

Gossmann,  H.,  495 

Gosio,  B.,  582 

Goto,  M.,  110,  111,  709 

Gottlieb,  R.,  urea,  333;  bile,  435;  creatine 
and  creatinine,  573,  692,  698;  urine 
purines,  711;  oxyproteic  acid,  754; 
iron  in  urine.  770 

Gouban,  F.,  366,  367 

Gourhay,  F.,  373 


•  IrCraaf,  C.  J.  H.,  792,  793 
Grabowski,  J.,  297 

Graebe,  C.,  524 

Graffenberger,  L.,  554 

Grafe,  E.,  54,  912,  919 

Graham,  I).,  919 

Graham,  Th.,  13,  14,  18,  31,  90 

Granstrdm,  E.,  718,  769 

Grawite,  E.,  340 

Grebe,  F.,  396 

Green,  E.  H.,  121 

Green,  J.  R.,  255,  316 

Greer,  James  Richard,  348,  349 

Gregersen,  J.  P.,  903 

Gregor,  A.,  698 

Gregor,  G.,  766 

G  reliant,  N.,  333,  335,  684,  773 

Griesbach,  W.,  333 

Griffiths,  A.  B.,  614,  758 

Grimaux,  E.,  85,  86,  323 

Grimbert,  L.,  677,  746,  747 

Grimm,  F.,  744 

Grimmer,  W.,  482,  490,  874 

Crindley,  H.  S.,  689,  752,  936 

Griswofd,  W.,  457 

Grober,  J.,  467 

Grober,  A.,  732 

Groho,  B.,  485 

Groll,  S.,  339 

Grosjean,  A.,  324 

Gross,  A.,  358,  629 

Gross,    O.,    pepsin    determination,    469; 
trypsin  determination,  506;    lens,  619; 
alkaline  earths  in  urine,  769 
Gross,  W.,  463,  780,  818 
Grossenbacher,  H.,  372 
Grosser,  P.,  733 
Grossmann,  H.,  808 
Grouven,  H.,  599 
Grube,  K.,  396,  400 
Gruber,  D.,  229 
Gruber,  M.,  8S1,  920 
Griibler,  G.,  94 
Griinbaum,  D.,  642 
Griindler,  J.,  777 
Griinhagen,  A.,  361 
<  initzner,  B.,  790 

Gri'itzner,  P.,  pepsin  determination,  469; 
sast'ic   contents,  479,   480;    Brunner'a 
glands,  489,  490;    pancreatic  diastase, 
501 
Grund,  G..  208,  209,  389,  919 
Grutterink,  A.,  792,  793 
Gryns,  G.,  7 
Gscheidlen,   R.,   rhodan,   455,   456,   751; 

lactic  acid,  582;  urea,  684 
Gubler,  A.,  348,  349 
Gudzent,  F.,  707,  708 
Gumbel,  Th.,  77 
Gunther,  G.,  622 

Gurber,    A.,    ion    permeability,    8,    310; 
serumalbumin,   261,   263;    serum,   267; 
bile,  440;  amniotic  fluid,  642 
Guerin,  G.,  758 


956 


INDEX  OF  AUTHORS 


Guest,  H.  H.,  106,  107 

Guggenheim,  M.,  50,  380 

Guigan,  H.,  Mc,  399 

Guillemonat,  A.,  371,  387 

Guinochet,  E.,  358 

Guldberg,  C.  M.,  32 

Gulewitsch,  W.,  arginine,  161,  162; 
thymin,  195;  choline,  247;  trypsin, 
508;    meat   extractive,  bases,  575-578 

Gullbring,  A.,  421 

Gumilewski,  491 

Gumlich,  E.,  680,  685,  761 

Gundermann,  K.,  82 

Gunning,  J.  W.,  823 

Gusserow,  A.,  716,  717 

Guth,  F.,  233 

Guyenot,  E.,  457 

Gyergyai,  A.,  912 

de  Haan,  J.,  306 

Haas,  E.,  528,  910 

Haberlandt,  L.,  265 

Habermann,  J.,  proteins,  83;  amino- 
acids,  143,  146,  153 

Hamalainen,  J.,  glucuronic  acid  conjuga- 
tion, 785;  sulphur  elimination,  882, 
883;  "protein  catabolism,  907 

Handel',  M.,  549 

Hansel,  E.,  487,  663 

Haser,  771 

Hausermann,  E.,  340 

Hafner,  A.,  233 

Hagemann,  O.,  skin  breathing,  849; 
blood  gases,  851;  metabolism,  926, 
927,  929 

Hagen,  W.,  193 

Hahn,  M.,  fermentation,  41;  casein 
digestion,  651,  urea  formation,  683, 
684 

Haig,  A.,  701 

Haiser,  F.,  inosinic  acid,  178-180;  182, 
183;  carnine,  577 

Haldane,  J.,  blood  pigments,  285,  286, 
302;  amount  of  blood,  343;  oxygen 
tension,  862,  863 

Hall,  W.,  purine  bases,  193,  520,  572; 
iron   absorption,   340;    amino-acids  in 

urine,  757 

Hallauer,  B.,  440 

Halle,  W.  L.,  380 

v.  Hallervorden,  E.,  682,  685,  767 

Halliburton,  W.  D.,  protein  coagula- 
tion, 97;  dextrins,  234,  choline;  246; 
fibrin  ferment,  256;  eerum  albumin, 
261;  blood  serum,  270;  stroma  of 
blood  corpuscles,  274;  tetronerythrin, 
304,  843;  leucocytes,  306;  blood 
coagulation,  323;  pericardial  fluid, 
356;  cerebro-spinal  fluid,  360;  liver, 
382;  glycogen,  391;  pancreatic  renin, 
509;  myxcedema,  545;  bone  marrow, 
553;  muscles,  566-570;  brain  proteins, 
604,  605;  diseases  of  the  nervous  sys- 
tem, 614;  kidneys,  673 


Halpern,  H,  372 

Halsey,  J.  T.,  152 

Ham,  C.  E.,  301 

Hamburger,  C.,  265,  456 

Hamburger,  E.  W.,  435 

Hamburger,  H.  J.,  blood  corpuscles,  6-8, 
273,  304,  305,  311;  blood  alkalinity, 
269;  blood  serum,  271;  phagocytoses, 
306;  blood  alkalinity,  309,  310;  lymph 
formation,  350,  351;  ascitic  fluid,  359; 
intestinal  juice,  490,  491;  enterokinase, 
496;  absorption,  542-543 

Hammarsten,  O.,  rennin  action,  54,  63, 
473,  474,  475,  650;  nucleoaJbumin, 
105;  mucin  substances,  168-169; 
helicoproteid,  173;  nucleoproteins, 
208,  383,  494,  495  ;  fibrinogen, 
fibrin  and  blood  coagulation,  253, 
254,  257,  263,  316;  fibrin  globulin, 
258;  globulins,  259-261,  263,  serum- 
albumin,  262;  blood  plasma  and  serum, 
267-270,  hsematoporphyrin,  299,  797, 
798;  gases  of  lymph,  346,  856;  transu- 
dates, 354,  356,  357,  359;  synovia,  362; 
human  bile,  414,  437,  438;  bile  of 
various  animals,  417,  427,  439;  bile  acids 
417,  420-427,  438;  bile  pigments,  267, 
432,  434,  801;  phosphatides,  435, 
439;  saliva,  457;  pepsin,  466,  467; 
trypsin,  503;  pseudomucin,  625,  626; 
perch  eggs,  630,  636;  casein,  649; 
lactoprotein,  653;  area  in  bile,  679; 
protein  in  urine,  793;  sugar  determina- 
tion in  urine,  806 

Hammerbacker,  F.,  458 

Hammerl,  H,  521 

Hammerschlag,  A.,  308,  309 

Hammerschlag,  G.,  637 

Handovsky,  H,  18 

Henriot,  M.,  lipases,  59,  265;  djiabetes, 
404;  respiration  quotient,  563;  respira- 
tion, 869 

Hansen,  C.,  lecithin,  242;  protein  syn- 
thesis, 529;  fatty  tissue,  559;  fat  of 
yolk,  630;  fat  of  milk,  669;  food  value 
of  albumoses,  912;  asparagin,  912 

Hansen,  W.,  233 

Hanssen,  O.,  amyloid,  171,  172,  173, 
547 

Harden,  A.,  co-enzyme,  52,  205;  phos- 
phoric acid  ester,  60,  205;  dioxyacetone, 
205;  glycolysis,  407,  583 

v.  Hardt-Stremayr,  77 

Hardy,  W.  B.,  colloids,  15,  20,  21,  25,  26, 
30;  ovalbumin,  634 

Hari,  P.,  54 

Harkink,  I.,  594,  693 

Harley,  V.,  sugar  of  blood,  398;  intestinal 
putrefaction,  515;  pancreas,  532,  539, 
540;  large  intestines,  541,  urinary 
pigments,  740,  744 

Harms,  H,  552 

Harnack,  E.,  ash-free  protein,  [95;  iodo- 
spongin,    122;     blood    pigments,    286, 


INDEX  OF  AUTHORS 


957 


287;  hydramnion,  642;  oxalic  acid 
poisoning,  728,  729;  sulphur  in  urine, 
752;  demolition  of  halogen  substi- 
tuted methane,  777;  galic  and  tannic 
acid,  785 

Harries,  C,  S3 

Harris,  J.  F.,  77,  179,  185 

Hart,  A.  8.,  116,  117 

Hart,  E.,  protein  nitrogen,  77;  albumoses, 
134;   histidine,  160;   hexone  bases,  164; 

Hart,  E.  B..  902 

Hartley,  P.,  384,  385 

Hartogh,  412 

Hartung,  C,  636 

Hartwell,  J.  A.,  130 

Hasebroek,  K.,  lecithin,  245,  515;  peri- 
cardial fluid,  356;  digestion  products, 
472 

Haskins,  H.  D.,  680,  683,  691 

Haslam,  H.  C,  salting  out  of  proteins, 
95,  250;  albumoses,  139 

Hasselbalch,  K.  A.,  reaction  of  blood,  75, 
310;  methsemoglobin,  283,  284;  egg- 
hatching,  637;  sugar  determination, 
814;  oxygen  intake,  859;  oxygen 
tension,  867 

Hatcher,  R.  A.,  397 

Hauff,  665 

Hausmann,  W.,  nitrogen  in  proteins,  77, 
119;  haematoporphyrin,  295;  kopros- 
terin,  448;  cholesterin,  449-450 

Hawk,  P.  B.,  keratin,  112;  bones,  551; 
Eck's  fistula,  683;  sugar  determina- 
tion, 804;  hair,  838;  metabolism,  892, 
897,  920 

Hay,  M.,  358 

Haycroft,  J.  B.,  protein  coagulation,  97; 
blood  coagulation,  251,  313;  diabetes, 
403;  biliverdin,  433 

Havem,  G.,  307,  342 

Heckel,  F.,  141 

Hedenius,  116,  434 

Hedin,  S.  G.,  blood  corpuscles,  7,  8; 
autolysis,  42-44;  adsorption,  49,  50; 
enzymes,  54-57;  retardation  of  enzyme 
action,  63-65;  rennin-antirennin  bind- 
ing, 68;  elastin,  116;  histidine,  159, 
160;  arginine,  161;  lysine,  163;  blood, 
326;  haematocrit,  326;  lienases,  371; 
rennin  zymogen.  473;  rennin  enzyme, 
474;  muscle  protease,  571 

Hedon,  E.,  pancreas  diabetes,  406; 
absorption  of  sugar,  533;  of  fat,  539, 
540 

Heffter,  A.,  liver,  385;  muscle,  565,  589; 
lactic  acid,  593;  hyposulphite  in  urine, 
753;  foreign  substances  in  urine,  773; 
hippuric  acid  synthesis,  783;  reduc- 
tions, 877 

Heger,  P.,  381 

Heidenhain,  M.,  101 

Heidenhain,  R.,  lvmphagogues  and  lymph, 
11,  345,  349-351;  transudates,  353; 
bile,  414,  415;  saliva,  453,  459;   stom- 


ach, 461,  476,  477;  pyloric  secretion, 
477;  pancreas  and  its  secretion,  494, 
495,  497,  503;  absorption,  528,  534; 
smooth  muscles,  602 

Heilner,  E.,  protein  assimilation,  526; 
metabolism,  920,  929;  specific  dynamic 
action,  930 

Heinemann,  H.  N.,  597 

Heinsheimer,  Fr.,  476 

Heintz,  W.,  235,  582 

Heiss,  E.,  555 

Heitzmann,  C.,  555 

Heckma,  E.,  intestinal  juice,  490-493, 
enterokinase,  496,  497 

Hele,  T.  Sh.,  738,  828 

Heller,  Fl.,  protein  test,  99,  789;  uroxan- 
thin,  727;  urinary  pigments,  740; 
blood  test,  796;  urinary  calculi,  834- 
835 

Hellgren,  W.,  931 

Hellwig,  602 

Helm,  461 

Helme,  W.,  882,  883,  907 

Hetper,  J.,  292 

Hemmeter,  J.  C.,  463,  464,  519 

Hempel,  E.,  894 

Henderson,  L.  J.,  309,  675,  677 

Henderson,  Y.,  77,  402,  529 

Henkel,  Th.,  647 

Henneberg,  W.,  510,  918 

Henninger,  A.,  137 

Henogque,  A.,  303 

Henri,  V.,  cataphoresis,  50,  51;  enzymes, 
56;  white  of  egg,  65;  saccharase,  65; 
diastase,  71 

Henriques,  R.,  239 

Henriques,  v.,  plastein,  58,  135;  formol 
titration,  165;  lecithin,  242;  lecithin 
sugar,  331,  332;  protein  synthesis, 
529;  fat  tissue,  559;  fat  of  yolk,  630; 
fat  of  milk,  669;  urea  determination, 
689,  690;  hippuric  acid,  723;  urine  nitro- 
gen, 756;  ammonia,  768:  gases  of  blood, 
851;  metabolism  in  lungs,  85S;  food 
value,  albumoses,  912;   asparagin,  912 

Hensel,  Marie,  726 

Hensen,  V.,  349,  856 

Henze,  M.,  gorgonin  and  iodogorgonic 
acid,  123,  146;  protein  of  octopoilcs, 
175;  hsemocyanin,  303,  304;  bloo  1 
corpuscles  in  ascitic  fluid,  304;  liver, 
388;   spongosterin,  448;   muscles,  603 

Heptner,  F.  K.,  438 

l'Heritier,  665 

Herlaut,  I...  185 

Herlitzka,  A.,  455 

Hermann,  E.,  72 

Hermann,  L.,  blood  in  starvation,  339; 
formation  of  feces,  521;  muscular 
work,  592;  allantoin,  716,  717 

Heron,  J.,  229,  456,  492 

Herrmann,  A.,  507,  700,  701 

Hermann,  Edm.,  336 

Herry,  A.,  325 


958 


INDEX  OF  AUTHORS 


Herter,  C.  A.,  indol  and  skatol,  159,  732; 

urorosein,  732,  733 
Herter,  E.,  saliva,  458;  ethereal  sulphuric 

acids,  724,  726,  784;    oxybenzoic  acids, 

783;  oxvgen  tension,  861 
Herth,  R.,*  137 
Hervierux,  Ch.,  indol  and  indican,  266, 

728-732;     skatol    red    and    urorosein, 

733;  uroerythrin,  748;  glucuronic  acids, 

817 
v.  Herwerden,  M.,  651 
Herzen,   A.,    spleen   and   digestion,    372; 

gastric  juice  secretion,  462;   pancreatic 

juice,  496,  498 
Herzfeld,  A.,  203,  224 
Herzfeld,  E.,  267,  433 
Herzog,    R.    O.,    enzymes,    41;     histidin, 

160;    lactic   acid,    582,    616;     oxydase 

reactions,  875 
Hess,  K.,  297 
Hess,  L.,  43 
Heubner,  O.,  25S,  569 
Heuss,  E.,  847 
Hewlett,  A.  W.,  541 
Hewlett,  R.  T.,  97 
Hewson,    W.,  6,  313 
Heyl,  F.  W\,  185 
Hevmann,  F.,  626 
Heynsius,  A.,  128,  432-434 
Hiestand,  O.,  245 
Hilbert,  P.,  779 
Hildebrandt,    H.,   oxalic   acid,   716,    773; 

amino-benzoic  acids,  783;   toluols,  783; 

glucuronic  acid  conjugation,  785,  786 
Hildebrandt,  P.,  antiemulsin,  64;    mam- 
mary   glands,     644,     668;     glucuronic 

acids,  conjugation,  750 
Hildebrandt,  W.,  267,  744 
Hildesheim,  932 
Hildesheimer,  A.,  726 
ililger,  171 
Hilger,  A.,  579 
Hill,  A.  V.,  277,  278 
ililler,  E..  554 
Hirsch,  Rahel.,  glycolysis,  408;    hippuric 

acid,   721;    amino-acids  in  urine,    756 
Hirsch,  Paul,  722,  912 
Hirschberg,  A.,  307 
Hirschfeld,  E.,  485 

Hirschfeld,  F.,  work  and  nitrogen  elimina- 
tion,    595;  uric    acid,     700;     acetone 

bodies,  819;    protein  catabolism,  903, 

915,  934;  daily  ration,  939 
Herschfeld,  H.,  791 
Hirsfhl,  J.  A.,  806 
Hirschler.  A.,  517.  518,  519,  724 
Hirechstein,  L.,  700,  756 
His,  W.,  550 

Hifl,  \Y.  (jr),  193.  707,  787 
Hlasiwetz,  H..  146 
Hochhaus,  H.,  340 
Hober,    R.,    osmotic    pressure,    13.    15; 

precipitation    properties    of    ions,    25; 

alkalinity  of  blood,  310,  311;    absorp- 


tion, 542;  permeability,  588;  urine 
acidity,  677 

Hone,  J.,  800 

Hoernes,  Ph.,  78 

Horth,  F.,  582 

v.  Hoesslin,  H.,  909-911,  917 

Hoyrup,  M.,  161 

Hofbauer,  L.,  456 

van't  Hoff,  J.,  osmotic  pressure,  2,  3; 
catalysis,  33,  35;  glucosides,  59 

Hoffman,  Ch.,  152 

Hoffmann,  A.,  722 

Hoffmann,  F.  A.,  transudates,  354,  357, 
361,  362;  sugar  in  blood,  398;  glucos- 
curia,  400 

Hoffmann,  J.,  675 

Hoffmann,  P.,  770 

Hofmann,  tyrosin  test,  153 

Hofmann,  Fr.,  559-561 

v.  Hofmann,  Karl,  621 

Hofmann,  K.  B.,  116,  124 

Hofmeister,  F.,  gelatin,  31;  cell  enzymes, 
44;  protein  nitrogen,  77,  82;  amino- 
acids,  binding  of  85;  removal  of  pro- 
teins, 102;  collagen  and  gelatin,  118, 
120;  albumoses  and  peptones,  133,  136, 
139;  grouping  of  proteins,  159;  serum- 
globulins,  259,  261;  pus,  364;  actions 
of  stomach,  479,  480;  protein  absorp-^ 
tion,  527,  528;  assimilation  limit,  533; 
blood  serum  and  earthy  phosphates, 
557;  ovalbumin  and  protein  crystalliza- 
tion, 633,  634;  urea  formation,  684; 
creatinine,  694;  protein  in  urine,  793; 
lactose  in  urine,  815 

Hofmeister,  V.,  4S0,  510 

Hohlweg,  H.,  267,  740,  741 

Holde,  D.,  233 

Hollinger,  A.,  328 

Holmgren,  E.  S.,  452,  806 

Holmgren,  Fr.,  852,  866 

Holmgren,  I.,  489,  566,  569 

Holobut,  Th.,  696 

v.  Hoist,  G.,  169,  354,  362 

Holsti,  O.,  903 

Honore,  Ch.,  526 

v.  Hoogenhuyze,  C.  J.,  creatine  and 
creatinine,  573,  594,  691-694 

Hooker,  D.,  351 

Hooper,  C.  W.,  442 

Hopkins,  F.  G.,  halogen  proteins,  82; 
protein  reaction,  100;  tryptophan,  155- 
158;  protein  crystallization,  263,  633, 
634;  lactic  acid  formation,  533;  uric 
acid,  699,  710,  711,  843;  urobilin,  743, 
746;  Hence-Jones  protein,  792;  butter- 
flies, S4M 

Hoppo-Seyler,  F.,  oxydation,  41;  ovovi- 
tellin, 105;  collagen,  118;  proteins,. 
167;  aucleins,  175;  xanthin,  189; 
lecithin,  243-245;  blood  plasma,  269; 
blood  corpuscles,  274,  304,  blood  pig- 
ments, 275-278,  281,  283,  286,  287- 
290,  293-302;    urobilinoids,  295,   743; 


INDEX   OF  AUTHORri 


959 


glycogen,  307,  390;  blood  analysis,  I 
328;  chyle,  347;  pericardial  fluid,  366; 
pus,  303-365;  strumacvstica,  374; 
bile,  435,  430,  440;  excretin,  523; 
cartilage,  549,  550;  bones  and  teeth, 
553,  558;  lactic  acids,  583,  585,  503, 
748;  retina,  015;  ovovitellin,  028; 
milk,  047,  04N,  666;  bile  acids  in  urine, 
800;  inositc,  S17;  chitin,  839;  sebum, 
844;  skin  breathing,  M0;  respiration 
apparatus,  80S;  oxydation,  871 

Hoppe-Seyler,  G.,  blood  pigment  deter- 
mination, 302;  phenol  elimination, 
725;  indoxvl,  728-732,  urobilin,  744, 
745,  747 

Hop  wood,  A.,  87 

Horbaczewski,  J.,  keratin,  112;  elastin, 
116,  117;  purine  bases,  188,  189; 
uric  acid,  373,  098,  700-702;  urostealith, 
834,  S35;  metabolism,  904 

Hornborg,  A.  J.,  464,  465 

Honre,  R.  M.,  251 

Horodynski,  W.,  334,  336 

Horowitz,  L.  M.,  520 

Horton,  E.,  59 

Hoshiai.  Z.,  787 

Hougardv,  A.,  261 

Howe,  P.  E.,  892 

Howel,  W.  H.,  257,  266,  319,  322 

Hrvntsehak,  Th.,  723 

Huber,  A.,  251,  255 

Hudson,  C.  S.,  55,  57,  199 

v.  Hiibl,  236,  238 

Huebner,  R.,  159 

Hufher,  G.,  leucin,  142;  blood  pigments, 
277-279,  283-286,  289;  spectrophoto- 
metry, 302,  303;  bile,  420;  gases 
of  bird  egg,  636;  urea  determination, 
690;  oxygen  tension,  859,  S61,  862; 
air  bladder,  867 

Hurthle,  K.,  264 

Huaounenq,  L.,  biliverdin,  434:  ha?mato- 
gen,  629;  clupervin,  636;  ash  of  milk, 
and  of  child,  666 

Huiskamp,  VV.,  fibrinogen  and  fibrin  for- 
mation, 253,  254,  256,  258.  320;  nucleo- 
protein  in  blood,  258;  thymus  histone. 
366.  367 

Hulett.  G.  A.,  29 

Hultgren,  E.  O.,  utilization  of  nutrtives, 
531,535,542,  543;  diets,  9L  2,  936 

Hummelberger.  F.,  126 

Humnicki,  V.,  448 

Hundeshagen,  F.,  122,  242 

Hunter,  A.,  100.  Ill,  134,  758 

Hupfer.  Fr.,  721 

Huppert.  H.,  Schutze's  rule.  57;  diges- 
tion products,  133:  elycopen,  307,  391; 
bile  pigment  reactions,  432;  pepsin  de- 
termination. 4(10:  Mesh,  601;  urea,  686; 
uroleucinic  acid,  739;  urine  albumin, 
794;   laiose,  814;   acetone,  825 

Hurtley,  W.  H.,  287,  739 

Hurwitz,  S.  H..  253 


Husson,  071 
Hutchinson,  Hob.,  553 
Hybbinette,  8.,  748 

Ibrahim,  J.,  497 

Ide,  578 

Ignatowski,  A.,  827 

Inagaki,  ('.,  scrum  albumin,  262;  serum- 
globulin,  270;  blood  corpuscles,  341; 
protein  absorption,  528;  muscle  rigor, 
590 

Inoko,  Y.,  277 

Inouye,  K.,  179,  574,  748,  749 

Inove,  Z.,  470 

Irisawa,  T.,  334,  749 

Ishihara,  H.,  423 

Ito,  M.,  791 

Iversen,  A.,  621,  623 

Iwanoff,  763 

Iwanoff,  L.,  182,  206,  508 

Izer,  G.,  autolysis,  43;   uric  acid,  704-706 

Jaarswelde,  G.  J.,  723 

Jablonsky,  J.,  499 

Jackson,  H.  C.,  739 

Jacobs,  W.  A.,  serine,  145;  nucleic  acid, 
178-185;  ribose,  211,  glucothionic 
acid,  369;  sphingosin,  611;  cerebronic 
acid,  611 

Jacobsen,  A.,  331 

Jacobsen,  C.  A.,  673 

Jacobsen,  O.,  437,  438,  779 

Jacoby,  M.,  autolysis,  42,  45,  869;  phos- 
phorous poisoning,  blood,  253,  255, 
256;  pepsin  determination,  470;  tryp- 
sin determination,  506;  fertilization, 
640;  uric  acid  demolition,  706 

Jacubowitsch,  458 

Jaeckle,  H..  233,  235,  559 

Jaderholm.  A.,  285,  289 

Jarger.  A.,  867.  843 

Jaha,  M.  E.,  936,  939 

JaO'c.  M.,  ornithine  and  ornithuric  acid, 
162,  7S3;  bile  pigments.  432,  433; 
urobilin,  521;  creatine  and  creatinine, 
574,  575,  693,  695;  phenylsemiear- 
bazide,  687:  urethane,  692;  uric 
acid,  703,  704,  708;  indican  in  urine, 
727-730;  kyenuric  acid,  739;  urobilin, 
740.  741,  744,  740;  conjugated  glu- 
curonic acids,  751,  786;  behavior  of 
organic  substances,  778,  779,  783,  786; 
furfurol,  7S4;  thiophene,  784;  guani- 
dine  acetic  acid,  787 

de  Jager,  L.,  675,  677,  734,  756,  769 

Jahnson-Blohm,  G.,  03 

Jakowsky,  M.,  513 

v.  Jaksch,  R..  blood  alkalinitv,  309; 
urea,  333;  brain,  005;  volatile  fatty 
acids,  748;  melanin,  799;  pentoseuria, 
816;  acetone,  818 

Jalowetz,  E.,  226 

Jamieson,  G.  S.,  123 

Janney,  N.,  768 


960 


INDEX   OF  AUTHORS 


Jansen,  B.  C.  P.,  491,  581 

Jappelli,  G.,  453 

Jaquet,  A.,  277 

Jastrowitz,  H.,  469,  716,  757 

Jastrowitz,  M.,  208,  815 

Jelinek,  J.,  407,  583 

Jensen,  P.,  581,  5S7 

Jerome,  W.,  Smith,  753 

Jerusalem,  E.,  nucleic  acids,  184;  lactic 
acid,48S;  melanins,  841-843 

Jesner,  S.,  617 

Jess,  A.,  619 

Jessen-Hansen,  H.,  165,  809 

Joachim,  Jul.,  259,  353,  357,  359 

Jochmann,  G.,  307 

Jodlbauer,    A.,  50,  552 

Johansson,  F.,  757 

Johansson,  J.,  E.,  serumalbumin,  262; 
tissue  and  gas  metabolism,  597,  898, 
927-931,  936;  digestion  work  and 
specific  dynamic  action,  930 

Johnson,  St",  691,  694 

Johnson,  T.  B.,  protein  .  sulphur,  79; 
thiopolypeptides,  87;  nucleic  acid,  185; 
cvtosine,  194 

John,  S.,  376 

Jolles,  A.,  pentoses,  209,  210,  816;  bile 
pigments,  431;  milk,  664;  urine 
acidity,  677;  urinometer,  679;  uric 
acid,  711;  urobilin,  743;  albumin  in 
urine,  787;  nucleohistone,  795;  fructose 
determination,  814 

Joly,  670 

Jolvet,  851 

Jones,  D.  B.,  84,  106,  107 

Jones,  W.,  autolysis,  44;  nucleopro- 
teids,  175;  nucleic  acids,  182,  183; 
thymine,  195;  purin  substances  and 
their  enzymes,  368,  371,  373,  701-703, 
706 

Jonescu,  D.,  65 

de  Jonge,  D.,  846,  847 

Jornara,  D.,  847_ 

Josephsohn,  A.,  739 

Junger,  E.,  427 

Jureensen,  E.,  20,  97 

Jungfleisch,  E.,  27,  585 

Junkersdorf,  P.,  413 

Juschtschenko,  A.,  373 

Just,  L,  339 

Justus,  J.,  71 

Juvalta,  X.,  778 

Kaas,  K.,  78,  634 
3\ahn,  R.,  402 
Kalb,  <",.,  918 
Kalbcrlah,  It..  585,818 
Kalmus,  E.,  2X<) 
Kanitz.  A..  506 
Kapfberger,  G.,  53 
Kareff,  N.,  253 
Kashiwabara,  M.,  710 
Kast,    A.,    intestinal    putrefaction,    519; 
urinary   sulphur,    752;     chlorine  excre- 


tion, 758;  halogen  substituted  methane, 
777;  perspiration,  848 

Kastle,  J.  H.,  56,  59 

Kato,  Kan,  636 

Katsuyama,  K.,  161,  334,  582,  896 

Katz,  A.,  744 

Katz,  J.,  598,  599 

Katzenstein,  A.,  926,  927 

Kauder,  G.,  261 

Kaufmann,  M.,  glycogen,  397;  sugar  of 
blood,  398;  sugar  formation,  412; 
fat  formation,  562;  urea,  572;  sugar 
utilization,  592;  lactose,  670;  urea 
formation,  684;  metabolism  experi- 
ments, 890 

Kauffmann,  M.  (Frankfurt),  247,  360,  713 

Kaufmann,  Martin,  894,  912 

Kaup,  J.,  595 

Kausch,  W.,  395 

Kautzsch,  K.j  polypeptides,  87;  glu- 
tamic acid,  147;  proline,  154;  adrenalin 
379;  digestion,  484,  513 

Kaznelson,  H.,  464 

Keller,  A.,  757 

Keller,  Fr.,  525 

Keller,  W.,  783 

Kellner,  ().,  utilization  of  nutriments, 
531;  protein  catabolism  in  work,  595: 
asparagin,  nutrition  value,  912;  diets, 
932 

Kelly,  A.,  839 

Kempe,  M.,  157,  158 

Kennawav,  E.  L.,  83,  109,  700,  715 

Kendall,  A.  J.,  520 

Kermauner,  F.,  521 

Kerner,  G.,  694 

Kiermaver,  211 

Kiesel,  A.,  247 

Kikkoji,  T.,  640 

Kiliani,  H.,  197 

Kirchmann,  J.,  911 

Kirk,  R.,  739 

Kirschbaum,  P.,  613 

Kistermann,  C.,  806 

Kitagawa,  F.,  608,  611 

Kittsteiner,  C.,  847 

Kjeldahl,  J.,  methods  for  nitrogen  deter- 
mination, 687,  688 

Klages,  A.,  427 

Klatte,  F.,  205 

v.  Klaveren,  H.  K.  L.,  288 

Klecki,  K.,  521 

Klein,  \\ .,  675 

Kleine,  F.,  752.  767 

Kleiner,  1.  S.,  717 

Klemensiewicz,  R.,  476,  477 

Klemperer,  G.,  urochrom,  740,  741; 
oxalic  acid,  773;  protein  metabolism,. 
903,  915,  934 

af  Klercker,  O.,  691,  692,  694,  815 

Klingemann,  F.,  670 

Klinuemann,  W.,  484 

Klingenberg,  K.,  779 


INDEX  OF   AUTHORS 


961 


K'ug,  F.,  tryptophan,  155;  pepsin,  466, 
472;  trypsin,  ">02,  503;  phosphoric 
acid  excretion,  762 

v.  Knafil-I.cn/,  E.„  124,  .390 

Knapp,  K  .  sugar  test,  214;  sugar  deter- 
mination, sos.  81 1 

v.    Knieriem,    W.,    cellulose,     510;      urea 

formation,  682j    uric   acid   formation,  704 

Knopfclmaclicr,  \\ '.,  233,  559 

Knoop,  1'.,  liistidin,  159,  160;  methyl- 
imidasol,  201;  demolition  of  fatty 
acids,  774,  781;  synthesis  of  amino 
acids,  775,  786;  demolition  of  aromatic 
substances,  779,  781 

Knop,  W.,  690 

Knorr,  L.,  297 

Kobert,  H.  W.,  276 

Kobert,  R.,  cyanmethemoglobin,  285; 
iron  in  urine,  770;  melanins,  841 

Kobrak,  E.,  662 

Koch,  \\\,  lecithins,  241-245;  cephalin, 
248;  brain  analyses,  605,  607,  609, 
612-614;  milk,  663 

Kocher,  Th.,  3(5 

Kochs,  \\\,  722 

Kochmann,  M.,  902 

Kobner,  440,  441 

Koefoed,  E.,  647 

Kohler,  A.,  600,  892,  912 

Koelichen,  K.,  35 

Koelker,  A.  H.,  455 

Konig,  J.,  599,  657-659 

Koppe,  H.,  blood  corpuscles,  7,  8,  273, 
305,  326;  hydrochloric  acid  of  stomach, 
477 

y.  Korosy,  K.,  digestion,  484,  513; 
parenternally  introduced  protein,  525; 
digestion  blood,  527,  530 

Koster,  H.,  650 

Koettgen,  E.,  616 

Kohler,  R.,  708 

Kohlrauch,  A.,  776 

Kojo,  K.,  795 

Kolisch,  R.,  698,  795 

Kondo,  K.,  indol  and  skatol,  159;  lactic 
acid,  333,  585;  eholesterin,  385;  chon- 
droitin  sulphuric  acid,  548,  549;  urinary 
phosphorous,  757 

Koraen,  G.,  598,  929 

Kordnvi,  A.,  13,  311,  351 

Korkunoff,  A.,  910 

Korn,  A..  469 

Korndofer,  G.,  572 

Korowin,  500 

Kossel,  A.,  protein  nitrogen,  77,  78; 
nitration  of  protamines,  83;  arginase, 
89,  161,  574.  681,  682;  histones,  107- 
109;  protamines,  109-111,  137:  pro- 
tein hydrolysis,  122,  141,  145,  154; 
histidin,  159,  1 1 '»( ) :  hexone  bases,  161, 
164;  arginine,  161,  162;  agmatine,  162; 
ornithin,  163;  lysin,  163;  nucleopro- 
teins,  174;  nucleic  acids,  179,  185; 
purin    bases.    186,    187,    190-193,    367, 


386,  495;  pyrimidine  bases,  194,  195; 
primary  and  secondary  cell  constituents, 
240;  hemoglobin,  277;  nucleohistone, 
307,  366;  blood  plates,  308;  pus,  304; 
protagon,  606,  007,  610;  cerebrosides, 
607,  610;  ichthulin,  630 

Kossler,  A.,  725 

Kostin,  S.,  2S7 

Kostvtschew,  S.,  184,  Is.", 

Kotake,  Y.,  170.  393,  394,  711,  780 

Kowalewski,  K.,  132,  185,  704,  767 

Kowarski,  A.,  572 

Kraft,  F.,  17 

Kramm,  W.,  695 

Kranenburg,  W.  R.  H.,  476 

Kraske,  B.,  333 

Krasnosselsky,  T.,  369 

Kratter,  Jul.,  560 

Kraus,  Fr.,  384,  410,  411,  583 

Krause,  R.  A.,  692 

Krauss,  E.,  730 

Krauze,  L.,  83 

Krawkow,  X.  P.,  amyloid,  171,  172; 
chitin,  S39.  S40 

Kreglinger,  G.,  341 

Krehl,  L.,  795 

Kreis,  H.,  233 

Kresteff,  S.,  477 

Kreuzhage,  C,  595 

Krieger,  H.,  261,  263,  763 

Krimberg,  R.,  575-578 

Kristeller,  L.,  692 

Krober,  E.,  209 

Kronig,  B.,  71 

Krogh,  A.,  oxygen  in  blood,  859,  862,  864, 
867;  microtonometer,  861,  864;  car- 
bon dioxide  tension,  864;  metabolism 
experiments,  868,  881 

Krogh,  M.,  690,  862,  864,  866 

Kronecker,  F.,  723 

Krshyschkowsky,  K.,  463 

Kruger,  A.,  79,  120,  230 

Kruger,  Fr.,  modification  of  proteins,  97; 
haemoglobin,  280;  leucocytes,  305; 
blood,  335,  336;  spleen,  371;  liver, 
388;  sulphocyanides  in  saliva,  455; 
milk,  654 

Kruger,  M.,  purin  bases,  186,  188;  in 
feces,  520;  in  urine,  711-715;  ammonia, 
768 

Kruger,  Th.  R.,  136,  578,  598 

Krug,  B.,  918 

Krukenberg,  F.  C.  W.,  keratinalbumoses 
113;  skeletins,  121;  cornein.  122 
cornicrystallin,  123;  hyalogens,  170 
171;  lipochrome,  267;  hsemoerythrin 
303.  304;  muscle  extractives,  572,  577 
bird  egg,  630;  uroostealith,  834-5 
bird  feathers,  843 

Krumbholz,  C.  J.,  536 

Krummacher,  ().,  sugar  formation,  412; 
work,  595;  calorific  power,  889;  protein 
metabolism,  909;  nutritive  value  of 
gelatin,  911 


962 


INDEX  OF  AUTHORS 


Krzemecki,  A.,  82 

Kudo,  T.,  476,  479,  506 

Kiibel,  F.,  457 

Kiihling,  ().,  784,  785 

Kuhne,  W.,  enzymes,  40;  neurokeratin, 
112,  605,  614;  gelatin,  119;  albumoses 
and  peptones,  128,  129,  132,  135-139 
paraglobulin,  258;  luematoidin,  301 
glycogen,  390;  gastric  digestion,  472 
485;  pancreas  and  its  enzymes,  500 
502,  503,  508,  509;  fat  emulsion,  536 
muscles,  566-568,  589;  smooth  mucles 
602;  pigments  of  the  eye,  615,  616 
corpora  lutea,  623 

Kiilz,  C,  355 

Kiilz,  E.,  cystine,  147;  pentoses,  208,  815; 
isomaltose,  225;  glycogen,  390-394, 
397,  592;  diabetes,  400,  403;  saliva, 
454,  456;  gastric  juice,  476,  477; 
pancreatic  diastase,  500;  gases  of  milk, 
664;  conjugated  glucuronic  acids,  777, 
785;  oxybutyric  acid,  826 

Kiilz,  R.,  283,  857 

Kueny,  L.,  215 

Kiister,  F.  W.,  28 

Kuster,  \V.,  blood  pigments,  283,  289- 
298,  430;  bile  pigments,  425,  428-431, 
433,  434,  429 

Kiittner,  S.,  652 

Kuhn,  617 

Kuliabko,  A.,  494,  691 

Kullberg,  S.,  205 

Kumagai,  T.,  737 

Kumagawa,  M.,  fat  determination,  238; 
fat  formation,  562;  sugar  determination, 
809;  protein  metabolism,  903,  915,  934 

Kunkel,  A.  J.,  arsenic,  72;  carbon 
monoxide  blood  test,  287;  iron  prep- 
arations, 340;  bile,  435,  443;  iron  in 
urine,  770 

Kuprianow,  J.,  582 

Kurajeff,  D.,  coagulose,  58,  135;  halogen 
protein,  83;  protamine,  109;  trypto- 
phan, 155 

Kurbatoff,  D.,  237 

Kurpjuweit,  O.,  520 

Kusmine,  K.,  351 

Kusumoto,  Ch.,  448,  449 

Kutscher,  Fr.,  protein  nitrogen,  77 
gelatin,  83;  proteolysis,  106,  107,  122 
146;  histone,  107,  108;  protamine,  109 
digestion  products,  132;  histidine,  159 
160;  arginine,  161,  162;  agmatin,  162 
lysine,  163,  164;  hexone  bases,  164 
aporrhegmen,  166;  cytosin,  194 
thymin,  368;  erepsin,  493;  guanidine 
507;  choline,  507;  intestinal  digestion 
513;  absorption,  527,  528;  bases  of 
meat  extract,  575,  576;  methylguani- 
dine,  60S;  urin:iry  bases,  757,  826,  828 

Kuwsehinski,  P.,  499 
•  Kyes,  P.,  241 

Laache,  S.,  342 


Ladenburg,  A.,  621 
Laidlaw,  P.  P.,  301,  380,  843 
Laitinen,  T.,  309 
Lambling,  E.,  680 
Lampe,  A.,  912 
Lampel,  H.,  78,  126 
Lanceraux,  406 
Landau,  A.,  361 
Landauer,  A.,  518,  899,  920 

Landergren,  E.,  utilization  of  nutriments, 
531,  535;  metabolism,  894,  903,  914; 
diets,  932,  935 

Landois,  L.,  275,  343 

Landolt,  H.,  841 

Landsteiner,  K.,  69 

Landwehr,  H.,  749 

Lane-Clavpton,  J.  E.,  43 

Lang,  G.;  484,  793 

Lang,  J.,  151,  349 

Lang,  S.,  703,  704,  776 

de  Lange,  C,  242,  664 

Lange,  F.,  818,  819 

Langer,  L.,  235 

Langgaard,  A.,  660 

Langhaus,  A.,  230 

Langhaus,  Th.,  301 

Langbeld,  K.,  SI,  426 

Langley,  J.  N.,  457,  459,  477 

Langstein,  L.,  carbohydrates  in  proteins, 
84,  168,  262;  digestion  products,  132; 
skatosin,  159;  fibrin  and  leucocytes, 
253;  blood  globulin,  260;  rest  nitro- 
gen in  blood,  267;  serumprotein,  270; 
deamidation,  410,  411,  775;  lactic  acid, 
583;  ovoglobulin  633;  ovalbumin,  633, 
634;  ovomucoid,  634;  casein,  662; 
alcaptonuria,  735,  736,  739;  C:N 
quotient,  772,  884;  lactose  in  urine,  815 

Lankester,  E.  R.,  303,  304 

Lannois,  E.,  533,  846 

Lapicque,  L.,  371,  387,  388,  936 

Lappe,  J.,  492 

Laptschinsky,  M.,  619 

Laqueur,  E.,  autolysis,  43;  lipase,  476; 
casein  and  rennin  coagulation,  648-651 

Laqueur,  J.,  340 

Larin,  A.  M.,  469 

Larsson,  K.  O.,  759 

Lassaigne,  J.  L.,  717 

Lassar,  O.,  309 

Lassar-Cohn,  422-425,  427,  435 

Latarjet,  A.,  464 

Latschenberger,  J.,  bile  pigments,  441, 
442;  iron  in  bile  and  liver,  443;  pro- 
tein absorption,  525 

Latschinoff,  P.,  422-426 

Lattes,  L.,  822 

Laulanio,  F.,  563,  597,  927 

Lauritzen,  M.,  769 

de  Laval,  G.,  656 

Laves,  E.,  660 

Laves,  M.,  397,  403,  404 

Laveson,  H.,  479 

Lavoisier,  M.,  926 


INDEX   OF  AUTHORS 


963 


Lawes,  563 

Lawrow,  D.,  roaguk>6e,  58,  135;  histone, 

ln7,    1(IS;   digestion   products,    132)    his- 

tidine,  160;  blood  pigments,  288; 
conjugated  glucuronic  acids,  7s.", 

Laxa,  <>..  649,  651 

Lazarus-Barlow,  W.  8.,  351 

Lea,  Sh.,  65 

Leathes,  J.  B.,  lymph,  13;  autolysis,  44; 
liver  fat,  384,  385;  protein  absorption, 
627j    ovarial  fluid,  626;    uric  acid,  700 

Leavenworth,  Ch.,  78,  84,  L63 

Lebedeff,  A.,»384,  660,  501,  009 

v.  Lebedew,  A.,  205 

Leclerc,  A.,  848 

Leconte,  P.,  403 

Ledderhose,  G.,  218,  839 

Ledoux,  A.,  324 

Leers,  O.,  285 

van  Leersum,  E.  C,  209,  221 

Lefevre,  K.  I'.,  222 

Lefmann,  G.,  092 

Legal,  E.,  158,  823 

Lenmann,  C,  fat  formation,  503;  meta- 
bolism in  hunger,  893;  in  work,  920, 
927;  asparagin,  food  value,  912 

Lehmann,  C.  G.,  458,  632 

Lehmann,  Fr.,  849 

Lehmann.  K.  B.,  288,  560 

Lehndorff.  H.,  300 

Leichtenstern,  O.,  338,  339 

Leick,  559 

Lemaire,  F.,  749,  815 

Lenk,  E.,  423,  589,  590 

Leo,  H.,  liver  fat,  384,  501;  diabetes, 
404;  acidity  in  gastric  juice,  4S9; 
laiose,  814;  nitrogen  deficit,  881 

Lepage,  L.,  498,  499 

Lepine,  R.,  glucuronic  acids,  221,  331; 
pentoses,  264;  sugar  in  blood,  264,  329, 
330;  glycolysis,  332,  333,  34G,  407, 
408;  glycogen,  393.  phlorhizin-diabetes, 
400;  absorption,  533;  urinary  sulphur, 
752,  753;  urinary  phosphorous,  757; 
urinary  poisons,  758;  urine  maltose,  814 

Leponois,  E.,  758 

Lerch,  019 

Lesem,  W.,  606,  607 

Lesnik,  M.,  778,  779 

Lesser,  E.  J.,  909-911,  917 

Lesser,  K.  A.,  350 

Letsche,  E.,  blood  serum,  264,  266; 
methaemofdobin,  283;  bile  acids,  419, 
420,  423 

Leube,  W.,  787,  848 

Leuchs,  H.,  serine,  145;  arabinose,  201; 
glucosamine,  218;  oxyprolin,  155 

Levene,  P.  A.,  autolysis,  44,  620;  poly- 
peptides, 89  ;  nucleoalbumin,  104; 
albumin  hvdrolysis,  106,  107,  119,  134, 
141,  143,  144,  146;  plasteins,  135; 
tendon-mucin,  168,  544;  nucleic  acids, 
178-185,  643;  guanine,  190;  pyrimi- 
dine  bases,  194,  195,  507,   ribose;  211; 


ghicothionic  acids,  307,  369,  387,  547, 
043;  spleen,  369;  liver,  383;  phlor- 
izin diabetes,  400;  glycolysis,  408, 
333;  trypsin,  502,  503;  brain  protein, 
604;  cerebroside,  609,  611;  Bphingosin, 
611;  cerebronic  acid,  011;  ichthulin, 
630;  urea,  082,  089  ;  creatin  and 
creatinine,  692;  lactic  acid,  583 

Levi-Malvano,  M.,  235,  230 

Levison,  L.,  470 

Levites,  S.,  78,  449-450,  476,  514 

Levy,  A.  G.,  309 

Levy,  H.,  784 

Levy,  Ludw.,  571 

Levy,  M.,  557 

Lewandowskv,  M.,  525 

Lewin,  Karl,  721,  728 

Lewin,  L.,  blood  pigments,  281,  285,  286, 
288,  289;  hydroquinone,  727;  urobilin, 
745 

Lewinsky,  J.,  269,  270,  721 

Lewis,  D.  H.,  53 

Lewis,  Th.,  602 

Lewy,  B.,  621 

v.  d.  Leyen,  E.,  728 

Lichtwitz,  L.,  66,  749 

Liddle,  L.  M.,  107 

Lieb,  Ch.,  702 

Lieben,  A.,  301,  306,  823 

Liebermann,  C.,  447,  636,  843 

Liebermann,  H.,  754 

Liebermann,  L.,  protein  reaction,  100; 
lecithalbumins,  105;  nucleins,  175, 
176;  hen's  egg,  628,  630,  632,  637, 
638;  kidneys,  673;  Guaiac  test,  796 

Liebermeister,  G.,  258 

v.  Liebig,  J.,  mineral  substances,  7:; 
fat  formation,  563;  work  and  meta- 
bolism, 594,  596;  urea,  686;   diets,  932 

Lieblein,  V.,  361,  684 

Liebrecht,  A.,  82 

Liebreich.  O..  606,  844 

Liechti,  P.,  726 

Liepmann,  W.,  642 

van  Lier,  E.  H.  B.,  545 

van  Lier,  G.  A.,  8 

Lifschutz,  J.,  oleic  acid,  237;  cholesterin, 
447,  449;  isocholesterin,  446;  wool  fat, 
846 

Likhatscheff,  A.,  785 

Lilienfeld,  L.,  nucleo  histone,  108,  307; 
fibrin-ferment  and  blood  coagulation, 
256,  314,  316;  blood  plates,  308; 
thymus,  366,  368 

Lillie,  R.  S.,  colloids,  16,  17,  31;  salt 
action,  73 

v.  Limbeck,  R.,  309,  310 

Limpricht,  H.,  572 

Lindberger,  W.,  506,  517 

Lindemann,  L.,  208 

v.  Linder,  M.,  843 

Linden,  8.  E.,  23,  26 

Lindhard,  J.,  814 

Lindvall,  V.,  112 


964 


INDEX  OF  AUTHORS 


Ling,  A.  R.,  226 

Linn,  K.,  445,  446 

Linnert,  K.,  612,  613 

Linossier,  G.,  675 

Linser,  P.,  844 

Lintner,  C.  J.,  229 

Lipliawskv,  A.,  824,  825 

Lipp,  A.,  152 

Lippich,    Fr.,    metallic   albuminates,   96; 

leucine,     143;  excrements,    521;  urein, 

691 ;  uramino  acids,  786 
v.  Lippmann,  E.  O.,  153 
Lipschiitz,  A.,  902 
Lister,  J.,  313 
Ljubarsky,  E.,  239 
Ljungdahl,  M.,  402 
Lloyd- J  ones,  E.,  308 
Lochhead,  A.  C,  606 
Lochhead,  J.,  640 
Loche,  F.  S.,  72,  592 
Lockemann,  G.,  307 
Locquin,  R.,  144 
Loeb,  A.,  415,  767,  333 
Loeb,  J.,  muscles,  9;  Overton's  theory,  10; 

enzymes,  70;    antagonistic  salt  action, 

72,  73;  artificial  fertilization,  639,  640; 
metabolism,    928;     fundulustrials,    70, 

73,  74 

Loeb,  L.,  256,  319 

Loeb,  W.,  37,  212,  597 

Loebisch,  Wilh.,  643,  668 

Loebisch,  W.  F.,  434,  544 

Lohlein,  W.,  469,  505,  506 

Loning,  H.,  611 

Lonnberg,  J.,  549,  673 

Lonnqvist,  B.,  462 

Loeper,  M.,  265 

Lorcher,  G.,  474 

Loeschcke,  K.,  392 

Lotsch,  E.,  484 

Loevenhart,  A.  S.,  enzymes,  56,  59,  71, 
501,  502,  563 

Loew,  O.,  protein,  78,  82,  97,  126;  sugar 
synthesis,  212 

Lowe,  S.,  10 

Lowenthal,  S.,  43 

Loewenthal,  W.,  511 

Loewi,  O.,  phlorhizin-diabetes,  400;  sugar 
formation,  412;  protein  synthesis,  529; 

,    urea    formation,    682;  allantoin,    718; 

'  conjugated  glucuronic  acids,  750;  phos- 
phorous metabolism,  761 

Loewit,  M.,  314 

Loewy,  4..,  diamines,  163;  blood  alkalin- 
ity, 310;  high  altitudes,  341,  929; 
liver  nitrogen,  387;  work  and  meta- 
I  bolism,  595;  acid  action,  676;  amino- 
acids  in  urine,  757;  cystinuria,  827; 
gases  of  blood,  851,  852,  859,  861,  865; 

I  alveolar  air,  860,  863,  865;  metabol- 
ism, 888,  925-929;  maintenance  value, 
s',17,  SOS 

Loewy,  E.,  839 

Lohmann,  A.,  lysine,  164;    choline,  246, 


247,  379,  507;  methylguanidine,  698; 
urinary  bases,  757 

Lohnstein,  Th.,  679,  804,  813 

Lohrisch,  H.,  510 

Lombardi,  M.,  96 

Lombroso,  U.,  532,  535,  536,  539,  540 

London,  E.  S.,  enzymes,  53;  nucleic 
acids,  472;  gastric  lipase,  476;  diges- 
tion, 482-485,  513,  514;  pancreatic 
juice,  498;  Eck's  fistula,  529,  702; 
small  intestines,  541;  creatinine,  694; 
nuclein  metabolism,  702,  705;  starva- 
tion blood,  896 

Long,  J.  H.,  lecithin,  244,  245;  casein, 
648,  649;  urine  nitrogen,  680;  urinary 
coefficient,  771;  elimination  of  alkali 
earths,  769 

Longcope,  W.  T.,  43 

L6pez-Su£rez,  J.,  476 

Lorrain-Smith,  J.,  343 

Losev,  G.,  29 

Lossen,  F.,  83 

Lossen,  J.,  325 

Lottermoser,  A.,  19,  23 

Luchsinger,  B.,  394,  847 

Luciani,  L.,  339,  892 

Luckhardt,  A.  B.,  348 

Ludwig,  C.,  bile,  441;  gastric  digestion, 
485;  pancreatic  juice,  495;  absorption 
of  proteins,  526,  527;  of  sugar,  534; 
gases  of  blood,  850,  851 

Ludwig,  E.,  239,  627,  709,  710 

Lucke,  A.,  hyalin,  171,  840;  pus,  365; 
benzoic  acid  reaction,  722 

Liidecke,  T.,  243 

Liidecke,  K.,  247 

Luthje,  H.,  sugar  formation,  408,  410, 
412;  nitrogen  retention,  530;  oxalic 
acid,  715 

Lukjanow,  S.,  415,  896 

Lukomnik,  I.,  136 

Lummert,  W.,  384,  563 

Lundsgaard,  Chr.,  75,  310 

Lunin,  N.,  900 

Lusk,  Gr.,  phlorhizin-diabetes,  399,  400, 
407,  409,  412,  413;  lactose  in  intestines, 
532;  in  urine,  815 

Lussana,  F.,  398 

Luther,  E.,  808 

Luzzatto,  A.,  815 

Lyman,  I.  F.,  702 

Lyon,  E.  P.,  73 

Lyttkens,  H.,  265,  329,  332 

Maas,  O.,  126 

Maase,  C.,  774,  779,  780,  822 

Macadam,  J.,  595 

Macallum,  A.  B.,  271,  340,  586,  769 

Maccallum,  A.,  (jr.),  711 

MacCallum,  J.  B.,  490 

McClerulen,  J.  F.,  630 

McClendon,  73 

MacCollmn,  E.  V.,  902,  903 

McCrudden,  F.  H.,  557 


INDEX  OF  AUTHORS 


«J65 


Macfadven,  A.,  41,  512 

v.  Mach,  W.j  703 

Markay,  J.  C.  EL,  MM) 

Markic.  W.  ( '..  573,  57  1 

MacLean,  H..  243,  247 

Maclean,  H.,  407,  583 

Macleod,  J.,  glycoecuria,  402;  bone  mar- 
row, 553;  phosphocarnic  acid,  578, 
593;  carbamic  acid,  683,  691 

MacMunn,  Ch.,  A.,  ha-matoporphyrin, 
295,  797,  843;  echinochrom,  301; 
cholohu'inatin.  435;  myohaematin, 
571;  urobilinoid,  743;  tetronerythrin, 
843 

Madsen,  Th.,  50,  57,  449-450 

Mamianini,  G.,  159 

Magne,  H.,  670 

Magnier.  770 

Magnus-Alsleben,  E.,  772 

Magnus,  G.,  S50 

Magnus,  R.,  52,  524 

Magnus  Lew,  A.,  spleen,  372;  thyroid 
gaud,  374',  376;  liver,  382,  388,  389; 
diabetes,  413;  salivary  glands,  452; 
pancreas,  495 ;  analyses  of  muscles,  599, 
600;  of  brain  tissue,  614;  kidneys,  673; 
hippuric  acid,  721,  722;  fatty  acids 
in  urine,  748;  benzoicglucuronic  acid, 
751;  Bence-Jones'  protein,  792;  ace- 
tone bodies,  818,  820,  821,  826;  respira- 
tion, 869;  metabolism,  8S9,  923-925, 
929,  897,  898 

Maignon,  ¥.,  581 

Maillard,  L.  C,  creatinine,  693;  indoxyl 
sulphuric  acid,  727,  730;  urinary  sul- 
phur, 752;  urinary  phosphorous,  762; 
ammonia,  766 

Maillard,  M.  L.,  71 

Majert,  W.,  621 

Makris,  C,  660 

Malcolm,  J.,  761 

Malengreau,  F.,  242,  366,  367 

Malenuck,  W.  D.,  109,  110 

Malfatti,  H.,  urine,  purin  bases,  715, 
amino-acids,  756;  ammonia,  768,  769; 
phosphorous  elimination,  788;  fruc- 
tose determination,  814 

Mall,  F.,  121 

Mallevre,  A.,  510 

Maly,  R.,  oxyproteic  acids,  82,  83; 
peptones,  137;  bile  pigments,  429, 
431-433,  743;  saliva,  453;  hydro- 
chloric acid  secretion,  477;  putrefac- 
tion, 517;  luteins,  631 

Manasse,  P.,  385 

Manche,  M.,  592 

Manchot,  \Y.,  279,  286,  298 

Mancini,  St.,  208,  267,  740,  741 

Mandel,  J.  A.,  glutamic  acid.  147;  nucleic 
acids,  179,  643;  adenine-hexose  com- 
pound, 180;  guanylic  acid,  183;  naph- 
thoresorcin  reaction,  223;  glucothionic 
acids,  307,  369,  387,  643,  673;  spleen, 
369;  liver,  383;  mammary  glands,  668; 


reno-sulphuric  acid,  673;  urinary  phos 
phorouB,  757 

Mandel,  H.,  107 

Mandelstaiiiin.  B.,  415 

Mangold.  E.,  390,  894 

Manicardi,  ( '..  578 

Mann.  8.,  612,  613 

Manning,  T.  D.,  191 

Mansfeld,  G.,  265,  360,  377 

Maquenne,    L,    starch,    227,    229,    230 

cellose,  231:    protein  assimilation,  525 

sugar    absorption,    534;     iuosite,    579. 

sarcosin,     776;    digestion     work,     929! 

930 
Marcet,  188,  523 
Marchetti,  < ...  560 
Marchlewaki,  1...  leaf  and  blood  pigments, 

276,     277,     296,     297;      hajmin,     292; 

cholohu'inatin  and  bilipurpuriu,435 
Marcus,  E.,  259 
Marcuse,  \Y.,  592,  593 
Mares,  ¥.,  700,  702 
Marfori,  P.,  773 
Margulies,  806 
Maiie.  P.  L.,  307,  364 
Marino-Zucco,  246 
Mark,  H.,  385 
Markewicz,  M.,  767 
Marquardsen,  E.,  519 
Marshall,  J.,  735 
Martin,   C.   J.,   fibrin   ferment,   57,   256; 

toxin-antitoxin  combinations,  68;  blood 

coagulation,  323 
Martin,  S.  H.,  510 
Martz,  846 
Marum,  A.,  820 
Marx,  A.,  756,  818,  822 
Marxer,  A.,  406 
Maschke,  O.,  94,  695 
Masius.  J.  B.,  429,  521 
Massen,  V.,  683 
Masuyama,  M.,  628 
Mathews,    A  ,   arbacin,    108;    protamine, 

109,  623;     lysine,    163;     nucleic   acids, 

184;  fibrinogen,  252,  253 
Mathieu,  E.,  851 
Matthes,  M.,  519,  795 
Mattili,  H.  A.,  892 
Mauthner,  J.,  149,  445,  814,  912 
Mawas,  I.,  253 
Maximowitsrh,  S.,  261,  262 
May,  R.,  208 

Mayeda,  M..  172,  173,  462 
Mayer,  Arthur,  770 
Mayer,  A.,  L05 
Mayer,  E.  W..  445.  448 
Maver,  J.,  920 
Mayer,  L..  532,  540 
Mayer.  Mart.,  253.  270 
Mayer,  P.,  isoserin,  145;   cystin,  147-149; 

mannoses,  201,  203;    glucuronic  acids, 

221,    222;     lecithin,    244;     conjugated 

glucuronic   acids,    331,    749,    750,   817; 

phosphatides,   385;    deamidation,   410; 


966 


INDEX  OF  AUTHORS 


1      inosite,  579;    oxalic  acid,  716;    mdican, 

728;  skatoxyl-glucuronic  acid,  732 
Mayo-Robson,  A.  VY.,  414 
Mays,  K.,  502,  503,  616 
Mazurkiewicz   \Y..  499 
Meara,  F.  S.,  131 
Medigreceanu,  Fl.,  182,  277,  481 
Meek,  W.  I.,  253 
Mehu,  C,  351,  745,  746 
Meigs,  E.  B.,  589,  590,  603 
Meikere,     G.,     inosite,     579,     580,     818; 

urinary  chlorine  compounds,  758 
Meinert.'C.  A.,  531,  932 
Meinertz,  J.,  385 

Meisenheimer,  J.,  41,  205,  207,  213 
Meissl,  E.,  563 
Meissl,  Th.,  642 
Meissner,     F.,    digestion    products,   471, 

472;     urea  formatiom,   684;   allantoin, 

716,  717;  hippuric  acid,  720 
Meister,  V.,  684 
Mellanby,  E.,  573,  574 
Mellanby,  J.,  serum  proteins,    259,  322; 

peptone-blood,  325;    creatine  and  crea- 
tinine, 692,  694 
Mendel,  Lafayette  B.,  enzymes,  52,  53; 

lymph    formation,    351;     saliva,    454; 

trypsinogen,   496;    protein  absorption, 

525;   creatine,  693;  uric  acid,  702,  705; 

allantoin,     717;  kynurenic    acid,     740; 

artificial  feeding,  904,  906 
Mendes  de  Leon,  M.  A.,  662 
Menozzi,  A.,  449-450 
Menschutkins,  35 
Menzies,  J.  A.,  285 
de  Merejkowski,  C,  843 
v.  Mering,  J.,  urochloralic  acid,  221,  777; 

sugar  in  blood,  264;   blood  from  portal 

vein,  336,  532;  glycogen,  394;  phlorizin 

diabetes,  399,  400;  pancreatic  diabetes, 

404;  amylolysis,  456,  500 
Merunowicz,  J.,  293 
Mesermitzki,  628 
Messinger,  J.,  825 
Messner,  E.,  529 
Mester,  Br.,  519,  732,  752 
Mett,  S.,  469 
Meyer-Betz,  Fr.,  haematoporphyrin,  295; 

bilirubin,    429,    744;  urobilinoids,    743; 

urobilinogen,  744,  746,  747 
Meyer,  C,  388 
v.  Meyer,  E.,  23 
Meyer,  E.,  440 
Meyer,  Erich,  735,  739,  783 
Meyer,  G.,  531 
Meyer,  G.  M.,  amino-acids  in  blood,  266; 

glucose,    408,    333;     urea,    682,    689; 

lactic  acid,  583 
Meyer,  H.,  221,  703,  704 
Meyer,  Hans,  267 
de  Meyer,  J.,  408 
Meyer,  K.,  64 
Meyer,  Kurt,  32,  396 
Meyer,  Lothar,  860 


Meyer,  P.,  425,  429 

Meyer-Wedell,  L.,  385 

Michaelis,  H.,  265 

Michaelis,  L.,  cataphoresis,  20;  colloid 
envelopment,  23;  adsorption,  49-51, 
97,  102;  reaction  of  blood,  75;  coagula- 
tion of  proteins  by  heat,  20,  97;  albu- 
moses,  134;  sugar-of  blood,  264,  235, 
328,  329;  butyrinases,  265;  protein 
absorption,  525;  milk,  657 

Michaud,  L.,  401,  818,  822,  917 

Michel,  A.,  261,  262,  263 

Micheli,  F.,  139 

Micko,  K,  521,  578 

v.  Middendorff,  M.,  336 

Mieg,  W.,  631 

Miescher,  F.,  protamines,  109-111;  nu- 
clein,  175;  nucleic  acid,  179;  pus,  364, 
365;  spermatozoa,  622,  623;  salmon 
metabolism,  902 

Miethe,  A.,  281,  285,  286,  289 

Migay,  Th.,  481 

Miller,  I.  R.,  701-703,  706 

Millon,  M.  E.,  99,  653 

Mills,  W.,  715 

Milroy,  J.  A.,  290 

Milroy,  T.  H.,  175,  761 

Minami,  D.,  501 

Minkowski,  O.,  blood  alkalinity,  309; 
ascitic  fluid,  358;  glycogen,  397; 
sugar  of  blood,  293;  phlorhizin- 
diabetes,  399,  400;  pancreatic  diabetes, 
403-406;  duodenal  diabetes,  405;  bile 
pigment,  442,  443;  pancreas  and 
absorption,  532;  fat  absorption,  535, 
539,  540;  lactic  acid,  582,  749;  uric 
acid,  703-705;  allantoin,  717;  his- 
tozym,  723;  blood  in  diabetes,  856 

Mitchell,  P.  H.,  702 

Mitjukoff,  K.,  626 

Mittelbach,  F.,  254,  739,  793 

v.  Mituch,  A.,  637 

Miura,  K.,  264,  393,  492 

Miyamota,  S.,  467 

Moeckel,  K.,  265 

Mollenberg,  R.,  338 

Moeller,  J.,  521 

Moller,  S'.,  825 

Morner,  C.,  Th.,  albumoid,  114,  546.  549, 
617;  gelatin,  118,  120,  546;  ichthy- 
lepidin,  121;  gorgonin  and  pennatulin, 
122;  cornicrystalline,  123;  proteins 
of  anthozoa,  123;  tyrosin  test,  154; 
membranin,  171,  550,  617;  fructose, 
217;  vitreous  humor,  545,  616;  car- 
tilage tissue,  546-550;  cornea,  550; 
mucoid,  545,  550,  551;  bones,  555; 
crystalline  lense,  617,  618;  sugar 
in  egg-white,  632;  ovomucoid,  634, 
635;  perca  globulin,  636;  homogentisic 
acid,  738,  chlorine  determination, 
760;  gallic  and  tannic  acid,  785;  cal- 
cium diphosphate  in  urine  sediment, 
831 


INDEX  OF  AUTHORS 


967 


Morner,  K.  A.  H.,  sulphur  of  proteins, 
79;  cystine  and  cystein,  100,  113,  114, 
147-149;  thiolactic  acid,  79,  113,  151; 
albuminate,  120,  509;  pyro-racemic 
acid,  150;  proteins  of  serum,  '259,  200, 
262;  lucrum,  292,  293;  blister  fluid, 
301;  hydrochloric  acid  determination, 
489;  chondroitin  sulphuric  acid,  547, 
073;  muscle  pigments,  571  •  urinary 
nitrogen,  080,  085;  urea  determina- 
tion, 688,  689j  fatty  acids  in  urine, 
7  IS;  urinary  nubecula,  757;  acetanilid, 
779;  albumin  in  urine,  787;  nucleo- 
albumin  of  urine,  794;  melanins,  799, 
841 ;  bile  acids  in  urine,  800 

Mohr,  Fr.,  488,  759 

Mohr,  L.,  diabetes  and  sugar  formation, 
400,   412,   413;    purines   in  urine,  713 

Mohr,  P.,  112 

Moitessier,  J.,  287,  758,  792,  793 

Moleschott,  J.,  932 

Molisch,  H.,  215 

Moll,  L.,  103 

Monari,  A.,  593,  594,  098 

Monod,  O.,  054 

Moor,  Ovid.,  090,  091 

Moore,  J.,  213 

Moore,  B.,  theory  of  Overton,  10;  adsor- 
pates,  12,  29;  colloids,  10;  glycoseuria, 
402;  bile  and  fatty  acids,  511,  530; 
intestinal  contents,  519;  fat  synthesis, 
535;  fat  emulsion,  530 

Mooser,  YV.,  720 

Moraczewski,  W.,  excrements,  521;  heart 
muscle,  599;  pseudonuclein,  051;  in- 
dican  of  urine,  728,  729 

Morat,  J.,  592 

Morawitz,  P.,  fibrin  and  leucocytes,  147; 
serum  proteins,  270;  blood  coagula- 
tion,  314,  315,  318-321,  324,  325; 
detection   of  albumoses   in   urine,    792 

Morax,  V.,  724 

Moreau,  A.,  807 

Moreau,  J.,  229,  230 

Morel,  A.,  fibrinogen,  252,  253;  serum 
lipase,  205;  blood  coagulation,  234; 
glycolysis,  332;  hsematogen,  029;  milk, 
054 

Morel,  I.,  077 

Morel,  L.,  501 

Moreschi,  A.,  449 

Morgen,  A.,  007 

Morgenroth,  J.,  04,  08 

Mori,  Y.,  531,  932 

Moriggia,  A.,  847 

Morishima,  K.,  382 

Moritz,  479 

Moritz,  F.,  354,  400,  401,  075 

Moriya,  G.,  582,  606 

Morkowin,  N.,  109 

Morochowetz,  L.,  540 

Morris,  G.  H.,  41,  220,  229 

Morse,  H.  N.,  3 

Moruzzi,  G.,  247 


Moscatelli,  R.,  allantoin,  355,  359;  lactic 
acid,  593, 748,  749 

Moscati,  <i.,  starch  assimilation,  532; 
glycogen,     581,     591;     placenta,    040 

Mosen,   l;.,  308 

Mosse,  ML,  pseudochylous  appearance, 
358;  sugar  in  blood,  398;  hydrochloric 
•  acid  in  stomach,  470;  ethereal  sulphuric 
acids,  7l'  I 

Mott,  F.  W.,  choline  in  blood,  240;  in 
cerebrospinal  fluid,  300;  diseases  of  the 
nervous  system,  (>14 

Mottram,  \\ .  H.,  384,  385 

Mouton,  H.,  65 

Muhle,  P.,  130 

Miihsam,  J.,  335 

Miiller,  A.,  15,  23 

Midler,  Alb.,  489 

Miiller,  Eduard,  307 

Muller,  Erich,  510 

Midler,  Ernst,  419,  421,  423 

Muller,    Fr.,  130 

Muller,  Franz,  377,  378,  888,  920,  929 

Muller,  Friedrich,  autolysis  of  pneu- 
monic infiltrations,  45,  304,  809;  glu- 
cosamine from  proteins,  83,  84,  108, 
109,  170,  620;  high  altitudes,  341; 
starvation  (indican)  510;  fat  absorp- 
tion, 538,  539;  ethereal  sulphuric 
acids,  724;  urobilin,  744;  sulphur  of 
urine,  752,  753;  aniline,  779;  acetone 
bodies,  818;  feces  nitrogen,  831 

Muller,  Joh.,  580,  592,  028 

Muller,  Jul.,  727 

Muller,  Max,  000,  721 

Muller,  Martin,  578 

Muller,  Paul,  448,  521 

Muller,  Paul,  Th.,  252,  253,  270,  553 

Muller,  W.,  009,  01 0 

Muntz,  A.,  669 

Miinzer,  E.,  682,  685 

Miither,  A.,  210 

Muirhead,  A.,  083 

Mulder,  G.  J.,  Ill 

Munk,  I.,  chyle  and  lymph,  340-349; 
sulphocyanides,  455,  450,  751;  in- 
testinal contents,  519;  absorption  of 
protein,  525,  520;  of  sugar,  534;  of  fat, 
535,  537,  538;  fat  synthesis  and  fat 
formation,  500,  503;  work  and  meta- 
bolism, 595;  smooth  muscles,  002; 
milk,  055,  050;  urea,  082;  phenol 
elimination,  725;  phosphoric  acid  elim- 
ination, 702;  bile  pigment  reaction,  801 ; 
starvation  metabolism,  895,  890; 
nutritive  value  of  gelatin,  911;  of 
albumoses,  912;  of  asparagin,  912; 
protein  requirement.  915;  water  and 
metabolism,  920 

Murlin,  J.  R.,  911 

Murray,  Fr.  \V..  500 

Murschhauser.  H.,  923 

Musculus,  F.,  amylolysis,  229,  450,  500; 
urochloralic  acid,  777;  urease,  829 


968 


INDEX  OF  AUTHORS 


Myers,  V.,  195,  692,  693 
Mygge,  J.,  793 

Mvlius,  F.,  starch  iodide,  228;  bile  acids, 
419,  422-425 

v.  NagelilC,  227 

Nageli,  K.,  5 

Nageli,  O.,  676,  677 

Nagano,  J.,  491,  532,  533 

Nakaseko,  R.,  393 

Nakayama,  M.,  182,  493,  801 

van  Name,  W.  G.,  118,  121 

Nasse,  H.,  muscle  experiments,  9;  blood, 
336,337;  lymph,  349;  spleen,  371 

Nasse,  O.,  proteins,  78,  99,  glutin,  120; 
dextrins,  230;  glycolysis,  332;  gly- 
cogen, 390,  391,  590-592;  saliva,  456; 
457;  musculin,  568,  570;  oxidations,  877 

Naunyn,  B.,  glycogen,  394;  bile  pig- 
ments and  liver,  442,  443;  demolition 
of  aromatic  substances,  778,  779 

Nawratzki,  360 

Nebelthau,  E.,  393,  799 

Necker,  F.,  791 

Nef,  J.  U.,  213,  214 

Neilson,  C.  H.,  37,  53,  454 

Neimann,  W.,  glucuronic  acids,  221,  222, 
750,  751 

Nelson,  L.,  109,  110 

Nencki,  L.,  779 

Nencki,  M.,  protein  sulphur,  78;  trypto- 
phan, 155;  skatol  acetic  acid,  155; 
indol,  157;  blood  pigments,  276,  277, 
280,  291-295;  haematoporphyrin,  295; 
diabetes,  403;  gastric  juice,  465-467, 
476,  477,  485;  enzymes  of  the  stomach, 
475;cleavage  of  esters,  501;  intestinal 
digestion,  511,  512;  intestinal  put- 
refaction, 514;  reaction  in  intestine, 
519;  ammonia,  528,  683,  768;  urea 
572,  682-684;  carbamic  acid,  683 
nrosein,  733,  740;  urobilinoids,  743, 
demolition  of  aromatic  substances, 
778,  780,  785,  melanins,  841 

Neppi,  B.,  505 

Nerking,  J.,  lecithin,  241,  244;  glycogen, 
392;     bone    marrow,    553;     milk,    663 

Nernst,  \Y\,  permeability  of  membranes, 
9;  division  rule,  27;  diffusion,  36; 
toxin-antotixin  reaction,  69;  gas  chains, 
74,  272 

Nerseasoff,  N.,  744 

Neubauer,  C,  creatin,  575;  creatinine, 
691;  ammonia,  766 

Neubauer,  E.,  245,  404 

Neubauer,  O.,  protein  reaction,  100; 
acids  of  alcaptonurics,  736,  738,  739; 
demolition  of  amino-acids,  775,  779, 
780,  786;  conjugation  of  glucuronic 
acid,  777;    acetone  formation,  780,  818, 

Neuberg,  C,  autolysis,  43;  putrefactive 
products  of  proteins,  82;  gelatin,  83; 
artificial     phosphoproteins,     105;      iso- 


leuctne,  144;  isoserine,  145;  oxyamino 
succinic  acid,    147;     cystine,    147-149 
proline,    154;     tryptophan,    157,    158 
diamine  formation,  163;    amyloid,  171 
172;    nucleic  acids,  178,  179,  182,  183 
sugar  demolition,  200;    mannoses,  201 
203;     pentoses,      203,     208-211,    815 
sugar-free  fermentation,  206;    glucose 
215;  galactose,  216;  laevulose,  217,  218 
355;      glucoseamine,     219,     626,     629 
aminoaldehyde,    219,    220;     glucuronic 
acids,  221,  223,   749-751,  817;    amino- 
acids  in  blood,  267;    glucothionic  acid, 
369,  673;    melanin,  380;    softening  of 
liver,  386,  387;  glycogen,  393;  deamdia- 
tion,    410,  411;    cholesterin,   445,    447; 
chondrosin,    548;    inosite,    579;     lactic 
acid,  583;    lactose,  655;    renosulphuric 
acid,  673;  urine,   678;  heteroxan thine, 
713;    phenol  determination,    725,   726; 
skatoxyl-glucuronic  acid,  732;    amino- 
acids     in   urine,    756;    mineral    meta- 
bolism,    758,    769;    tataric  acid,    774; 
phenylhydrazine  test,  806;  acetone  818; 
cystinuria,   827;   tyrosinase,  843;  min- 
eral metabolism,  899,  901,  902 

Neumann,  Alb.,  nucleic  acids,  179,  184, 
185;  pyrimidine  bases,  194,  195;  orcin 
test,  209;  iron  in  urine,  770;  phenyl- 
hydrazine  test,  806 

Neumann,  E.,  443 

Neumann,  Jul.,  338 

Neumann,  O.,  921,  934 

Neumann,  R.,  920 

Neumann,  Walt,  136 

Neumeister,  R.,  keratins,  112;  albumoses 
and  peptones,  128-131;  tryptophan, 
155;  dextrins,  230;  glycogen,  391; 
protein  assimilation,  525;  ovomucoid, 
634 

Neusser,  E.,  797 

Nickles,  J.,  635 

Nicloux,  M.,  56,  264 

Nicolaier,  A.,  710 

Niemann,  A.,  532,  539 

Nierenstein,  E.,  469 

Nilson,  G.,  228 

Nilson,  L.  F.,  658 

Nishi,  M.,  404 

Njegovan,  VI.,  245 

Le  Nobel,  C.,  295,  743,  797 

Noel-Paton,  D.,  lymph,  349;  liver,  384, 
385;  glycogen,  sugar  formation,  399; 
bile,  414,  438;  creatine  metabolism, 
573-575;  lactose.  670 

Noguchi,  H.,  449-450 

Nogueira,  A.,  673 

Nolf,  P.,  osmotic  pressure,  13;  fibrinogen 
252,  253;  fibrinolysis,  255,  256;  albu- 
moses in  blood,  263:  blood  coagulation, 
318,  319-322,  324,  325,  320;  saliva, 
452;  absorption,  526,  527;  carbamic 
acid,  683,  691 

Noll,  A., 184,  614,  535 


INDEX   OF  AUTHORS 


9G9 


v.  Norden,  C,  spectrophotometry,  302; 
diabetes,  401,  404,  405,  412;  liver 
and  urinary  nitrogen,  685;  ethereal 
sulphuric  acids,  724;  albumin  in  urine, 
787;  acetone  bodies,  818;  metabolism, 

915,  *r >  1  * > 

v.  Noorden,  K.,  333 
Nothwang,  Fr.,  899 
Noskin,  J.,  375 

Novi,  J.,  435,  459 
Now.  V.,  126 
Nowak,  .!..  con,  808,  881 
Nuremberg,  A.,  370 
Niirenbenr,  A.,  56 
Nussbaum,  M.,  860,  864,  865 
Nuttal,  C.  516 
Nylander,  E.,  214 
Nylen,  S.,  457.. 

Obermayer,  Fr.,  protein  pieeipitation, 
102;  globulins,  103;  bile  pigments, 
434,  801,  802;  indican  detection,  729, 
730 

Oberniuller,  K.,  445,  446 

Odake,  8.,  905,  906 

Oddi,  R.,  511,  675 

Odenius,  R.,  643 

Oertel,  H.,  757 

Oertmann,  E.,  41 

Oerum,  H.  P.,  (sr.),  627,  911 

Oerum,  H.  P.  T.  (jr.),  342,  437,  730 

Oesterberg,  E.,  680,  693 

Offer,  Th.  R.,  pentose-amine,  219;  gly- 
cogen, 396;    chitin,   839;    alcohol,   921 

Offringa,  J.,  282 

Ofner,  R.,  217,  218 

Ogata,  M.,  485 

Ohta,  K.,  774 

Oidtmann,  H.,  366,  368,  870 

Oker-Blom,  M.,  8,  377 

Okunew,  W.,  135 

Olinger,  J.,  529 

Oliver,  G.,  379 

Ollendorff,  G.,  203 

Olsavszky,  V.,  762 

Omeliansky,  V.,  510 

Omi,  K.,  492,  526 

van  Oondt,  401 

Opie,  E.  L.,  307 

Oppenheimer,  C.,  serumalbumin,  261; 
parenteral  protein  assimilation,  525; 
respiration,  868;  oxydation  enzymes, 
875;  nitrogen  elimination,  881;  sur- 
face rule,  923 

Oppenheimer,  Max,  583 

Oppenheimer,  8.,  583 

Oppler,  B.,  329,  513 

Orbdn,  R.,  492 

Orgler,  A.,  acetone,  83,  818;  chondrosin, 
548;  uric  acid,  638 

Orndorff,  W.  R.,  428,  430,  431 

Orton,  K.,  738,  739 

Osborne,  Th.  B.,  proteins,  77-79,  84, 
104,    106-108,    163;    polypeptides,   89; 


nucleic  acids,  179,  186;  ovovitellin, 
628,  620;  protein  of  white  of  egg,  633, 
634;     calorimetry,    886;     phosphorous 

metabolism,  903;  artificial  nutrition. 
904,  '.toe, 

Osborne,  W.  A.,  581 

Ostertag,  659 

Ostwald,  Willi.,  28,  29;  catalvsis,  33,  35, 
36 

Ostwald,  Wo.,  15,  31,  279 

Ostwald,  A.,  halogen  protein,  82;  thy- 
roid glands,  373,  375,  376;  di-iodotyrosin, 
508;  urinary  globulin,  701 

Otori,  J.,  mucin,  169;  transudates,  355; 
guanidine,  507,  574;  pseudomucin, 
626 

Ott,  A.,  769 

Otte,  P.,  486 

Otto,  J.  G.,  sugar  of  blood,  265;  blood 
pigments,  277,  283,  284.  302;  blood, 
328,  329,  335,  338,  339,  532;  skatoxyl- 
sulphuric  acid,  732;  sugar  determina- 
tion, 811 

Overton,  E.,  plasmolysis,  6,  8;  muscle 
experiments,  9,  588;  amphibians,  9; 
theory  of  permeability,  9-11 

Owen,  Rees,  347 

Paal,  C.,  proteins,  78;    gelatin  peptones, 

120;    alkali  albuminate,  126;    peptone, 

130,  131,  137 
Pacchioni,  D.,  381 
Pachon,     V.,     blood     coagulation,     324; 

stomach  extirpation,  485,  486;  trypsino- 

gen,  496 
Paderi,  332 
Padtterg,  J.  H.,  838 
Pages,   C,   blood   coagulation,   251,   316, 

317;  rennin  action,  650;  milk,  667 
Pagniez,  Ph.,  314,  315 
Paigkull,  L.,  exudates,  353,  354,  357,  358, 

bile,  417 
Painter,  H.  M.,  130,  457 
Panek,  K.,  753,  754,  763 
Panella,  A.,  266,  578,  602 
Panormoff,  A.,  581,  633,  635 
Panum,  P.,  serumcasein,  258;    starvation 

blood,  338,  339,  896;    transfusion,  340, 

344;    amount  of  blood,  343;    nitrogen 

elimination,    910,    nutritive    value    of 

gelatin,  911 
Panzer,   Th.,    proteins,   83;    chylus,   347, 

cerebrospinal    fluid,     360;      bile,     423; 

colloid,  625,  626 
Pappenhusen,  Th.,  484 
Paraschtschuk,  S.,  669 
Parastschuk,  S.  W.,  474 
Parcus,  E.,  610 
Parke,  J.  L.,  632,  635 
Parker,  W.  H.,  16,  536 
Parmentier,  E.,  660 
Parnas,  J.,  248,  583 
Partridge,  C.  L.,  371,  702 
Paschutin,  V.,  350,  492 


970 


INDEX  OF  AUTHORS 


Pascucci,  O.,  273,  274 

Pascheles,  W.,  776 

Pasqualis,  G.,  749 

Pasteur,  L.,  40,  41,  516 

Patein,  G.,  808 

Patten,  A.  J.,  79,  149,  160 

Patten,  J.  B.,  457 

Paul,  Th.,  71,  707 

Pauli,  \V.,  colloids,  17,  18,  20,  25,  30;- 
proteins,  95,  97;  gelatin,  31,  119 

Pauly,  H.,  histidin,  82, 159-161,  adrenalin, 
379 

Pautz,  W.,  361,  492,  617 

Pavy,  F.  W..  carbohydrate  groups  in 
proteins,  84,  396;  isomaltose.  264; 
glycogen,  392,  398;  sugar  in  blood  and 
diabetes,  400;  self-digestion  of  stomach, 
486;  work  and  metabolism,  595;  sugar 
determination,  808,  809 

Pawlow,  J.  P.,  secretion  of  enzymes,  52, 
53;  Schutze's  rule,  57;  bile  fistula,  414; 
saliva,  454;  stomach  and  gastric  juice, 
461-463,  465,  468;  stomach  enzymes, 
474;  pyloric  reflex,  481;  intestinal 
juice,  490;  pancreatic  juice  and  enzymes 
of  pancreas,  49.5-498,  499,  501;  diges- 
tion in  intestines,  513;  ammonia  in 
blood,  683;  Eck's  fistula,  684 

Payer,  A.,  338 

Pearce,  R.  G.,  399 

Peiper,  G.,  309 

Pierce,  G.,  71 

Peju,  P.,  253 

Pekelharing,  C.  A.,  cataphoresis,  50,  51; 
fibrin  ferment  and  blood  coagulation, 
256,  257,  316,  317,  319,  320;  nucleo- 
proteins,  258,  569;  stomach  enzymes, 
466-468;  creatin  and  creatinine,  594, 
692,  693 

Penny,  E.,  725 

Penzoldt,  F.,  402,  824 

Pernou,  M.,  371 

Perrin,  J.,  15,  20 

I  eschek,  E.,  912 

I  etersen,  P.,  599 

Petit,  A.,  758 

Petrowa,  M.,  416 

Petry,  E.,  651 

v.  Pettenkofer,  M.,  bile  acid  test,  418; 
fat  formation,  560,  561;  work  and 
metabolism,  594,  596,  597;  respira- 
tion apparatus,  868,  869;  metabolism 
experiments,  879,  881,  908,  926;  diets, 
932 

Pittibone,  C.,  690 

I'd  rone,  A.,  315 

Petzseh,  E.,  902 

Pfaff,  P.,  414 

Pfannenstiel,  J.,  620 

Pfaundler,  M.,  132,  133,  681 

Pfeffer,  W.,  2,  3 

Pfeiffer,  E.,  662,  665 

Pfeiffer,  I,.,  841 

Pfeiffer,  Th.,  918 


Pfeiffer,  Wilh.,  705 

Pfleidener,  470,  471 

Pfluger,.  E.,  oxydations,  41;  life  of  cells, 
45;  carbonic  acid  of  lymph,  347;  gly- 
cogen, 390,  392,  393,  394,  413,  549; 
diabetes  and  sugar  formation,  405,  408, 
412,  413;  duodenum  and  diabetes,  405; 
gases  of  saliva,  453,  857;  bile  and  fatty 
acids,  511,  536;  fat  formation,  561, 
562;  muscle  metabolism,  591,  595, 
597;  gases  of  milk,  657,  857;  urinary 
nitrogen,  680;  urea  determination,  690; 
sugar  tests,  803,  805;  sugar  determina- 
tion, 811;  gases  of  blood  and  respira- 
tion, 850,  851,  854,  858,  866-868; 
N:C  quotient  in  urine,  884;  protein 
metabolism,  904,  9/)8,  909;  external 
temperature  and  metabolism,  929; 
protein  allowance,  934 

Phisalix,  C.,  847 

Picard,  J.,  616,  617 

Piccard,  109 

Piccolo,  G.,  301,  623 

Pick,  A.,  470 

Pick,  E.  P.,  albumoses  and  peptones,  131, 
133-135,  139;  serumglobulins,  259; 
peptozym,  325;  thyreoidea  and  adren- 
alin-glycosuria,  376 

Pick,  F.,*399 

Pickardt,  M.,  355,  549,  550 

Pickering,  J.  W.,  85,  86,  323 

Picton,  H.,  22,  26 

Pierallini,  G.,  773 

Piettre,  M.,  stroma  of  blood  corpuscles, 
275;  blood  pigment,  281,  285;  hsemin, 
292;  hyoglycocholic  acid,  421;  melanin, 
842 

Pigeand,  J.,  354 

Pighini,  G.,  613 

Pilotv,  O.,  blood  and  blood  pigments, 
220,  277,  290,  292-298,  295,  297; 
bile  pigments,  429,  conjugated  glu- 
curonic acids,  751 

Pilz,  O.,  137 

Pilzecker,  A.,  440 

Pimenow,  P.,  462 

Pincussohn,  L.,  23 

Pineles,  Fr.,  376 

Pinkus,  S.  N.,  proteins  and  their  crystal- 
lization, 82,  95,  263,  633,  634 

Piontkowski,  L.  F.,  463 

Piria,  153 

Planer,  J.,  486 

Plattner,  E.,  418 

Plaut,  M.,  756 

Playfair,  932 

Pletnew,  D.,  493,  532 

Plimmer,  R.  H.,  Aders,  enzymes,  53; 
gelatin  hydrolysis,  119;  nucleopro- 
teins,  174,' 630;  livetin,  629;  ichthulin, 
630;  hatching  of  the  egg,  637;  casein 
digestion,   652;    uric  acid,  702 

P16sz,  P.,  blood  corpuscles,  274;  liver 
382,      383;      urinary     pigments,      740; 


INDEX   OF  AUTHORS 


971 


proteid  in  urine,  787,  albumoses, 
nutrition  value,  912 

Poda,  H.,  521 

Poduschka,  P.,  717 

Poehl,  A.,  517,  021 

Pohl,  J.,  dextrin,  230;  globulin  determina- 
tion, 261,  793;  liver,  383;  acid  poison- 
ing, 676;  urea,  682;  allantoin,  716,  717; 
oxalic  acid,  773;  demolition  of  fatty 
acids,  774;  phthalic  acid,  778 

Poleck,  631 

Policard,  A.,  324 

Polimanti,  O.,  561 

Politis,  G.,  912 

Pollak,  H.,  105 

Pollak,  L.,  505,  776 

Pollitzer,  S.,  912 

Polwzowa,  W.,  482,  514 

Ponfick,  E.,  343,  344 

Ponamarevv,  489,  490 

Pons,  Ch.,  364,  548,  757 

Popel,  \\\,  339 

Popielska,  Helene,  463 

Popielski,  L.,  enzymes,  53,  324;  saliva, 
454;  pancreatic  juice,  496,  498 

Popowsky,  N.,  158 

Popper,  H.,  glvcogen,  395;  bile  pigments, 
434,  801,  802;  pancreatic  juice,  500, 
509 

Porcher,  Ch.,  lactose,  670;  indican  of 
urine,  728-732;  skatol  red  and  uroro- 
sein,  733;  uroerythrin,  748;  phthalic 
acid,  778 

Porges,  O.,  245,  259 

Porteret,  E.,  393 

Portier,  P.,  407,  532 

Posner,  C,  620,  621,  787 

Posner,  E.  R.,  169,  472,  606 

Posselt,  L.,  122 

Posternak,  S.,  578,  579 

Pottevin,  H.,  59,  226 

Pouchet,  A.  G.,  carnine,  577,  711; 
urinarv  poisons,  757;  lungs,  870 

Poulet,  V.,  869 

Poulsen,  W.,  550 

Poulsson,  E..  572 

Pozerski,  E.,  65,  498 

Pozzi-Escot,  E.,  877 

Prausnitz,  W.,  400.  521,  894 

Prayon,  I.,  693 

Pregyl,  Fr.,  keratins,  112,  114;  koilin, 
115,  124;  carbon  monoxy  haemoglobin, 
290;  dehydrocholan,  418;  bile  acids, 
423,  426;  intestinal  juice,  490,  491; 
colloid,  626;  ovalbumin,  634;  oxypro- 
teic  acid,  753;  polypeptides  in  urine. 
756;  €:N  quotient,  772 

Presch,  W.,  752,  753 

Preti,  L.,  64,  71,  706 

Preusse,  C,  phenols  in  urine,  725-727; 
behavior  of  aromatic  substances  in 
animal  body,  778,  779,  786 

Prevost,  J.  L.,  541,  684 

Prever,  W.,  288,  642 


Preysz,  K.,  762 

Pribram,  E.,  778 

Pribram,  H.,  106 

Pribram,  R.,  766 

Pridgent,  G.,  242 

Pringle,  H.,  histopeptone  ,109;  prota- 
mines, 109,110;  absorption,  525,  528, 
530;   blood  coagulation,  320 

Pringsheim,  H.,  230,  510 

Pristley,  J.  H.,  37 

Prochownik,  L.,  642,  643 

Proscher,  F.,  431,  659,  667 

Profitlich,  W.,  389 

Prutz,  W.,  728 

Prym,  O.,  372,  496 

Przibram,  H.,  571 

Pugliese,  A.,  319,  476 

Pulvermacher,  G.,  212 

Pupkin,  Z.,  309 

Pyman,  F.  L„  160 

Quadiarello,  G.,  675 
Quevenne,  Th.,  348,  349 
Quincke,  G.,  23,  64(5 
Quincke,  H.,  301,  340 
Quinquand,  Ch.,  urea,  333,  335;    muscle 
work,   592;    fatty  acids  in   urine,    773 
Quinton,  R.,  13 

Raaschou,  C.  A.,  183 

Rabinowitsch,  A.  G.,  484 

Rachford,  B.  K.,  501,  506,  511 

Radenhausen,  P.,  646 

Radziejewski,  8..  559,  560 

Radzikowski,  C,  462 

Raehlmann,  E.,  616,  617 

Raineri,  G.,  642 

Rakoczv,  A.,  475 

Ramsden,  W.,  30,  97 

Ranc,  A.,  267 

Ranke,  H.,  700 

Ranke,  J.,  344 

Ransom,  H.,  449 

Raper,  H.  8.,  136 

Rapp,  R.,  41 

Raske,  K.,  140,  148 

Rauchwerger,  D.,  445,  447 

Raudnitz,  R.  W.,  644,  649 

Reach,  F.,  381,  466,  597 

Reale,  E.,  716,  752 

Reemlin,  E.  B.,  513 

Reese,  H.,  756 

Regnault,  H.  V..  849,  868,  881 

Reh,  A.,  104,  366,  652 

Rehfuss,  M.,  804 

Rehn,  E.,  253 

Reich,  M.,  770 

Reich.  O.,  771 

Reichel,  H.,  650 

Reich-Herzberge,  F.,  508 

Reid,  E.  W.,  16 

v.  Reinbold,  B.,  283,  507,  808,  283 

Reinecke,  558 

Reiset,  J.,  849,  868,  881 


972 


INDEX  OF  AUTHORS 


Reiss,  E.,  260 

Reiss,  W.,  619 

Reitzenstein,  A.,  711 

Rekowski,  L.,  786 

Rennie,  J.,  494 

RenVall,  G.,  769 

Rettger,  I.,  257,  322 

Rettger,  L.  F.,  496 

Reuss,  A.,  355,  357 

Rewald,  B.,  179,  180,  210,  211 

Reye,  W.,  254  , 

Remolds,  J.  E.,  823 

de  Rev-Pailhade,  J.,  877 

Rhodin,  N.  J.,  43 

v.  Rhorer,  L.,  677 

Ribaut,  H.,  877 

Richards,  A.  X.,  albumoids,  116,  117,  118; 
hexone   bases,    164;     saliva,    455,    456 

Richaud,  A.,  599 

Richet,  Ch.,  gastric  juice,  465;  fat  forma- 
tion, 458;  urea,  682;  uric  acid,  706; 
thalassin,  846;  respiration,  869;  sur- 
face rule,  923;  spleen,  372 

Richter,  Max,  621 

Richter,  P.,  873 

Richter,  P.  F.,  osmotic  pressure,  13; 
amino-acids  in  blood,  267;  softening 
of  the  liver,  386,  387,  685 

Rieder,  H.,  881 

Riegel,  464 

Riegel,  M.,  647 

Riegler,  E.,  290 

Riehl,  M.,  869 

Riess,  L.,  748,  749,  780 

Riesser,  O.,  162,  611 

Rinaldi,  U.,  572 

Ringer,  A.  I.,  721 

Ringer,  A.  J.,  412 

Ringer,  L.,  316 

Ringer,  S.,  72 

Ringer,  W.  E.,  50,  51,  675,  677,  708 

Ringstedt,  O.  T.,  675 

Ritter,  A.,  sugar  in  blood,  398;  phlor- 
hizin-diabetes,  400;  fat  absorption, 
535;  urinary  fermentation,  829 

Ritter,  F.,  439,  440,  445,  449 

Rittausen,  H.,  proteins,  94;  leucinimide, 
143;  milk,  655 

Riva,  A.,  740,  748,  799 

Rivalta,  F.,  354 

Roaf,  H.  E.,  lipoid  theory,  10;  adsorpate 
12,  29;  osmotic  pressure  of  protein,  17, 
17;  lecithin,  246;  glycoseuria,  402 

Roberts,  F.,  279 

Roberts,  W.,  509,  793,  812,  813 

Robertson,  T.  B.,  lipoid  theory,  10; 
protein  salts,  95;  crystallized  albumin, 
262;  globulins,  263,  270;  casein,  648, 
649 

Roch,  G.,  790 

Rockwood,  C.  W.,  757 

Rockwood,  D.,  bile  and  fatty  acids,  511, 
536;  intestinal  contents,  519 

Rockwood,  E.  W.,  525 


Rodhe,  E.,  100 

Rodier,  A.,  338 

Roden,  H.,  63 

Roeder,  G.,  194,  195 

Rohmann,    F.,    amvlolvsis   and   diastase, 
225,    265,    346,    456;     glycolysis,    333; 
blood,    335;     glycogen    393,    398;     in- 
testinal   juice,     491,     492;      bile    and 
putrefaction,     518;      absorption,     532- 
534,   538;    muscles,    565,    589;    casein, 
649;     phosphorous    metabolism,     761 
902;     sebum,    844;     wool    fat,    846 
coccygeal   glands   secretion,   846,   847 
oxydase  action,  875;    artificial  feeding, 
904 

Rohrig,  A.,  591,  849 

Rose,  B.,  650 

Rose,  C.,  558 

Rose,  Heinrich,  429,  295 

Rosing,  E.,  877 

Roger,  G.  H.,  381 

Roger,  H.,  457,  510 

Rogozinski,  F.,  136 

Rohde,  A.,  703 

Rokitansky,  P.,  748 

Rona,  P.,  cataphoresis,  20;  enzymes,  56; 
adsorption,  97,  102;  thymuslhistone; 
109;  gelatin,  119;  albumoses,  134; 
sugar  in  blood,  264,  265;  butyrinases 
265;  calcium  in  serum,  269;  sugar 
in  blood,  328,  329;  glucose,  333, 
duodenal,  secretion,  489,  490;  protein 
absorption,  525,  529;  milk,  657;  alcap- 
tonuria,  735,  736 

Ronchese,  A.,  768,  769 

Ronchi,  J.,  849 

Roos,  E.,  376,  761,  806,  807 

Roosen,  O.,  698 

Rorive,  F.,  217 

Rosa,  E.  B.,  869 

Rose,  W.  C.,  692,  693 

Rosemann,  R.,  gastric  juice,  465,  479; 
milk,  670;  nitrogen  elimination,  910; 
alcohol,  921 

Rosenbach,  O.,  749,  800,  827 

Rosenbaum,  A.,  408 

Rosenberg,  Br.,  401 

Rosenberg,  S.,  duodenal  diabetes,  405; 
bile,  415;  pancreatic  juice,  497;  putre- 
faction, 518;  pancreas  and  absorp- 
tion, 532,  535,  539 

Rosenberger,  F.,  579,  816 

Rosenfeld,  B.,  484 

Rosenfeld,  F.,  728,  748,  912 

Rosenfeld,  G.,  fat  and  fat  formation,  384, 
560-562,  669;  uric  acid,  700;  phenyl- 
hydrazine  test,  806;  acetone  bodies,  819 

Rosenfeld,  L.,  136 

Rosenfeld,  M.,  290,  293 

Rosenfeld,  R.,  264 

Rosenheim,  O.,  tryptophane  reaction,  157; 
cerebrospinal  fluid,  360;  pancreatic 
lipase,  502;  muscle-work,  592;  pro- 
tagon,  606-609 


INDEX  OF  AUTHORS 


973 


Rosenheim,  Th.,  915 

Rosenqvist,  E.,  412 

Rosensbein,  A.,  chvle  and  lymph,  347, 
348,  349;  absorption,  520,  534,  537 

Rosenstein,  \V.,  403 

Rosenthal,  J.,  868 

Rosenthaler,  L.,  59,  66 

Rosin,  11.,  fructose,  217,  814;  indican, 
730;  skatol  pigments,  733;  Rosen- 
bach's  urine  test,  827 

Rossi,  O.,  360 

Rost,  K.,  920 

Rost,  Ft.,  265 

Rostoski,  ().,  792,  793 

Roth,  (>.,  207 

Roth,  W.,  351 

Rothberger,  C.  J.,  683 

Rothera,  C.  H.,  77,  148,  824 

Rothmann,  A.,  573,  692 

Rotmann,  F.,  355 

Rotechy,  A..  295 

Roux,  E.,  227 

Rovida,  C.  L.,  hyaline  substance,  274, 
304 

Rovighi,  A.,  517,  724 

Rowland,  S.,  41-43,  371,  571 

Rowntree,  L.  G.,  183,  642,  643 

Rozenblat,  H.,  462 

Rubbrecht,  R.,  270 

Rubner,  M.,  protein  sulphur,  79;  sugar 
test,  215,  806,  815;  utilization  of 
nutiiments,  531,  534,  535,  538;  fat 
formation,  502,  563;  milk,  664;  meta- 
bolism experiments,  879,  S85,  890, 
894,  922,  923,  928,  929;  nitrogen 
of  excrements,  881 ;  heat  of  combus- 
tion, 885,  886,  891,  932:  utilization 
quota,  890,  910;  protein  catabolism, 
910,  914,  916,  918;  specific  dynamic 
action,  917,  930;    surface  rule,  923,  924 

Rubow,  W.,  586 

Rudinger,  ('.,  375,  406 

Riidel,  G.,  707 

Ruff,  O.,  200,  203 

Ruge,  E.,  515 

Rulot,  H.,  255,  256 

Rumpf,  Th.,  412,  767 

Runeberg,  J.  W.,  357 

Ruppel,  W.  G.,  606,  660,  844 

Russel,  653 

Russo,  M.,  839 

Russo,  Ph.,  744 

Rutherford,  A.,  838 

Rutherford,  Th.  A.,  112 

Ryan,  L.  A.,  603 

Rywosch,  D.,  273 

Sabanejew,  A.,  137 

Sabbatani,  S.,  316 

Bachaijin,  328 

Bacharow,  X.,  135 

Sachs,  Fritz,  papain,  65;  pentoses,  210; 
nuclease,  182,  493,  508;  hydrochloric 
acid  secretion,  477;  acetone  formers,  818 


Sachs,  H.,  64 

Sachsse,  K  ,  215,  228 

Sackur.  <>..  648,  649 

Saaikoff,  W.,  Us 

Sagelmann,  A.,  482 

Sahli,  H.,  303,  325,  809 

Saiki,  T.,  603,  748,  749 

Saillet,  hsematoporphyrin,  295,  797,  798; 

•  urobilin  and  urobilinogen,  740,  743, 
744,  740,  747 

Sainsburv,  316 

de  Saint-Martin,  L.,  286 

Saint  Pierre.  C,  867 

Saito,  S..  334,  582 

Salaskin,  S.,  digestion  products,  132; 
plastein,  135;  leucinimid,  14)i:  blood 
alkalinity,  309;  ammonia,  334,  336, 
528,  683;  erepsin,  393;  urea,  682,  684; 
liver  and  acid  formation,  685;  uric 
acid  formation,  703,  704 

Salkowski,  E.,  autolysis,  42;  denaturing 
of  proteins,  97;  pseudonuclein,  104, 
651;  albumoses,  131;  in  urine,  792; 
putrefaction  products,  141,  514;  skatol 
carbonic  acid,  155,  732,  733;  indol,  158; 
pentoses,  208,  209,  815;  glucuronic 
acid,  221;  cerebrospinal  fluid,  360; 
synovin,  302;  liver-proteins,  383,  384; 
glycogen,  393;  eholesteiin,  447:  saliva, 
459;  trypsin,  503;  putrefaction,  517; 
corpse  wax,  500;  flesh,  600;  dermoid 
cyst,  027;  dextrose  in  white  of  egg, 
032;  ovomucoid,  034;  casein,  651, 
652;  urea,  082,  684,  690;  creatinine, 
696;  uric  acid,  705,  709,  710;  purine 
bases,  714;  oxalic  acid,  710,  773; 
allantoin,  716,  717;  hippuric  acid,  720; 
phenaceturic  acid,  723;  ethereal-sul- 
phuric aoi.ls,  724;  indican,  729,  730; 
urobilin,  745;  urine,  fatty  acids,  748, 
829;  carbohydrates,  749,  815;  sulphur 
compounds,  752,  753;  adializable 
urinary  constituents,  757,  795;  urinary 
sulphur  acids,  704;  alkalies,  700; 
demolition  of  various  substances,  770, 
777,  783;  ha^matoporphyrin,  797; 
sugar  tests,  805,  806;  acetone  deter- 
mination, 825;  water  and  metabolism, 
920 

Salkowski,  H.,  141,  514,  720 

Salomon,  Georg,  purin  bases,  187;  glyco- 
gen, 207;  lactic  acid,  334;  urinary  purines, 
711-713 

Salomon,  H.,  410,  818 

Salomon,  W.,  082 

Salomone,  G.,  78 

Salomonsen,  K.  E.,  740,  741 

Samec,  M.,  141 

Sammet,  O.,  825 

Sammis,  J.  L.  936 

Samuely,  F.,  proteolysis,  107;  amino- 
acids  in  urine,  756;  cystine  demoli- 
tion, 776;  melanoids,  843 

Sandgren  J.,  265,  329,  332 


974 


INDEX  OF  AUTHORS 


Sandmeyer,  W.,  pancreatic  diabetes,  405; 

absorption,  532,  539,  540 
Sandstrom,  J.,  374 
Saneyoshi,  S.,  774,  777,  817 
Sasaki,  K.,  757 
Sasaki,  T.,    146,  147,  781,  784 
Sato,  T.,  369 
Satta,  G.,  681 
Sauer,  K.,  402 
Sauerbeck,  E.,  494 
Savare,  M.,  640,  641,  757 
Savory,  H.,  792 
Sawitsch,  W.,  496,  499 
Sawjalow,  W.,  58,  135,  475 
Saxl,  P.,  43,  387,  569 
Scaffidi,  V.,  ferratin,  384;    iron  in  liver, 

387;     purine    bases,    in    muscles,    572, 

594,  603;  uric  acid,  706 
Schade,  H.,  707 
Schafer,  E.,  379 
Schaeffer,  50,  51 
Schaffer,  F.,  78 

Schaffer,  Ph.,  692,  711,  768,  827,  828 
Schalfejeff,  M.,  292,  293 
Schardinger,  F.,  581,  877,  230 
Scheele,  M.  H.,  454 
Scheermesser,  W.,  136 
Scheibe,  A.,  659,  664 
Schemiakine,  A.  J.,  477,  478 
Schenck,  Fr.,  83,  398 
Schenck,  M.,  422,  423 
Schepowalnikoff,  N.  P.,  495 
Scherer,  Fr.,  925 
v.  Scherer,  J.,  lymph,  348;    inosite,  579, 

580;  meta  and  para-albumin,  625 
Scheuer,  M.,  464 
Scheunert,  A.,  gastric  digestion,  479,  482; 

duodenal    secretion,    490;      pancreatic 

stones,  509;  cellulose,  510;  peroxydase, 

874 
Schewket,  O.,  817 
Schierbeck,  N.  P.,  saliva,   457;    gases   of 

stomach,    486;     trypsin    action.     506; 

excrements,  520;     skin  breathing,  849 
Scruff,  A.,  469 
Schiff,  H.,  protein,  78;    biuret  test,  101; 

cholesterin,  448;  urea,  686;  uric  acid,  709 
Schiff,  M.,  spleen,  372;    liver,  381,    sugar 

of  liver,  398;    bile,  416,  541;     charging 

theory,  477,  496 
Schindler,  S.,  368 
Schittenhelm,  A.,  nucleic  acid,  182,  514; 

blood  coagulation,  318,  323;    deamida- 

tion  enzymes,  371;     purine  bases,  520; 

urea  formation,  682;    uric  acid  and  its 

formation,  373,  699-703,  705,  706,  709; 

amino-acids    in    urine,    756,    757,    827; 

ammonia,  767,  768 
Schlapfer,  V.,  912 
Schlatter,  K.,  485,  486 
Sdilcsinger,  A.,  457 
Sfhlcsinger,  W.,  747 
Bchliep,  L„  825 
Sclilosing,  Th.,  768 


Schloessing,  C.  22 

Schlossberger,  J.  E.,  665,  670 

Schlossmann,  A.,  656,  658,  659,  663,  923 

Schmey,  M.,  387,  599 

Schmid,  Jul.,  702,  715 

Schmidt,  Ad.,  513,  523 

Schmidt,  Albr.,  621 

Schmidt,  Alex.,  blood  coagulation,  256, 
257,  305,  314-317,  319,  321-323; 
fibrinoplastic  substances,  258,  319, 
320;  blood  corpuscles,  of  frogs,  375; 
leucocytes,  305,  306;  cell  protein,  306, 
307,  367;  saliva,  453,  454;  gases  of 
blood,  852 

Schmidt,  C,  serum,  270;  blood,  328; 
lymph,  348;  transudates,  353;  saliva, 
458;  mucus  of  the  mouth,  453,  454; 
gastric  juice,  465;  pancreatic  juice, 
499,  500;  bile,  518;  fat  absorption, 
538;  osteomolacia,  555 

Schmidt,  C.  H.  L.,  82 

Schmidt,  C.  W.,  870 

Schmidt,  E.,  572 

Schmidt,  Ernst.,  695 

Schmidt,  Fr.,  818 

Schmidt,  Hub..  101 

Schmidt,  P.,  711 

Schmidt-Muhlheim,  251,  526,  910 

Schmidt-Nielsen,  Signe,  50 

Schmidt-Nielsen,  Sigvae,  rennin,  50,  651; 
casein,  649,  650 

Schmiedeberg,  O.,  albumin  crystals,  94; 
salmine,  110,  110;  alkali  albuminate, 
126;  onuphin,  171;  nucleic  acids,  179, 
184,  185;  nucleosin,  195;  glucuronic 
acids,  221,  725,  730,  731,  785;  ferra- 
tin, 383;  chondroitin  sulphuric  acid, 
547-549;  urea,  682;  hippuric  acid, 
722,  723;  histozym,  723;  melanin 
substances,  840,  841 

Schmitz,  E.,  775,  780,  786,  818,  826 

Schmitz,  H,  136 

Schmitz,  K.,  517,  519,  724 

Schmutzer,  I.,  708 

Schneider,  A.,  328 

Schneider,  E.,  455,  739 

Schoffer,  A.,  142 

Schonbein,  C.  F.,  766,  871 

Schondorff,  B.,  urea,  333,  572,  664,  679 
690;     thyroidea,    376;     glycogen,    390 
394,  396,  581;    phlorhizin  action,  400 
uric  acid,  701 ;  sugar  in  urine,  803,  808 
protein  metabolism,  908,  909 

Schone,  A.,  208 

Scholz,  H,  728,  729 

Schottelius,  M.,  516 

Schotten,  C.,  fellic  acid,  427;  intestinal 
putrefaction,  720;  fatty  acids  in  urine, 
748,  773;  damaluric  and  damolic 
acid,  758;  behavior  of  aromatic  sub- 
stances in  animal  body,  780 

Schoubenko,  G.,  79 

Schoumow-Simanowski,  E.  O.,  467,  477, 
485 


INDEX  OF  AUTHORS 


975 


Schreiber,  E.,  701 

Schreiner,  Ph.,  G21 

Schrener,  M.,  flesh  nitrogen,  600;  calorific 

value  of  nitrogen,  892;   protein  feeding, 

918 
Schrodt,  M.,  553 
v.  Schroder,  W.,  urea,  12,  333,  079,  682, 

684;  uric  acid,  334,  703,  704 
Schroter,  F.,  182 

v.  Schrotter,  H.,  130,  131,  137,  861,  865 
Schryver,  S.  B.,  43,  427 
Schule,  464 
Schule,  A.,  456 
Schutz,   E  ,  Schutz's  rule,   57;   digestion 

products,    133;     pepsin   determination, 

469;    stomach  movement,  479;    lactic 

acid  in  urine,  749 
Schutz,    J.,    pepsin    determination,    469; 

action,    471;    hydrochloric    acid,    489; 

bile  and  fat  splitting,   502,   511 
Schtitze,  A.,  64 

Schiitzenberger,  P.,  85,  86,  130 
Scbultze,  B.,  563 
Schultze,  E.,  680 
Schultze,  F.  E.,  72 
Schultzen,  O.,  diabetes,  403;    urea,  682, 

683;     lactic   acid   in   urine,    748,  .749; 

sarcosin,    776;     behavior    of    aromatic 

substances  in   the  animal   body,   778- 

780 
Schulz,  Art.,  299 
Schulz,  Fr.  N.,  proteins,  79,  95,  98;   oxy- 

proteins,   83;    histone,    108;    galactos- 

amine,   167,   173,   219;    serumalbumin, 

261,  globin,  288;  starvation  blood,  339; 

premortal  protein  metabolism,  894 
Schulz,  H.,  376,  546,  770 
Schulz,  C,  457 
Schulz,    E.,    products    of    hydrolysis    of 

proteins,  141,  153;   phenylalanine,  152; 

histidine,    160;     arginine,     161;    lysine 

163;   hexone  bases,  164;    vernine,  178- 

180;  hemicelluloses,  231;  phosphatides, 

245;  isocholesterin,  448 
Schulze,  E.,  650 
Schulze,  F.,  22 
Schulze,  H.,  14,  21 
Schumburg,  W.,  474,  889,  926 
Schumm,  O.,  blood,  342,  796;    chyle  fat, 

347;  sugar  formation,  412;    pancreatic 

cvst,  500 
Schunck,  C.  A.,  276,  277,  296,  631 
Schunck,  E.,  740 
Schur,    H.,    uric    acid,    700,    702,    706; 

urinarv  purines,  702,  713 
Schurig,  387 
Schuster,  A.,  531,  938 
Scnuurmanns-Stekhoven,  282 
Schwalbe,  E.,  315,  384,  561 
Schwann,  Th..  414,  528" 
Schwarz,  Carl,  choline,  247;  iodothyrin, 

376;    glycoseuria,  405;    digestion,  483; 

secretin,  498 
Schwarz,  H.,  216 


Schwarz,  Hugo,  116,  117,  118 

Schwarz,  Karl,  572,  575 

Schwarz,  L.,  126,  477,  820 

Schwarz,  (>.,  402,  487,  S25 

Schwareschild,  M.,  502,  508 

Schweisslnger,  O.,  790 

Schwiening,  42,  43 

Schwinge.  \\  .,  33s 

Scofield,  H.,  433 

Scott,  F.  H.,  174,  630,  637 

Scott,  L.,  122 

Sczelkow,  851 

Sebauer,  it.,  555 

Sebelien,  J.,  peptones,  130;  milk,  644, 
651,  652,  655,  656,  658;  casein  diges- 
tion, 651 ;    carbohydrates  in  milk,  655 

Seegen,  J.,  sugar  in  blood,  264,  398,  592 
597;  sugar  formation,  405;  amylolysis, 
456;  respiration,  868;  nitrogen  deficit, 
881 ;  water  and  metabolism,  920 

Seelig,  P.,  517 

Seeman,  J.,  protein  substances,  oxyda- 
tion,  83;  ealactoseamine,  167;  erepsin, 
493;  intestinal  contents,  513;  absorp- 
tion, 527,  528;  creatine  and  creatinine, 
573;  ovomucoid,  635 

Segale,  M.,  72 

Seisser,  Ph.,  702,  705,  709 

Seitz,  W.,  381,  389 

Seliwanoff,  Th.,  217 

Selmi,  47 

Semmer,  G.,  275 

Senator,  H.,  729 

Senter,  G.,  56 

Sera,  Y.,  393,  394 

Sertoli,  E.,  855 

Sestini,  F.  and  L.,  699 

Setschenow,  J.,  851,  853,  855 

Shackell,  L.  F.,  73 

Shepard,  C.  W.,  720 

Shibata,  N.,  384,  561 

Shimamura,  T.,  905,  906 

Shinidzu,  Y.,  238 

Shaw-Mackenzie,  J.  A.,  502 

Siau,  R.  L.,  264,  400 

Sieber,  N.,  protein  sulphur,  79;  blood 
pigments,  280,  292,  293;  ha?matopor- 
phyrin,  295;  glycolysis,  333;  diabetes, 
403;  gastric  juice,  361,  362,  485; 
stomach  enzymes,  475;  intestinal  diges- 
tion, 512;  Umikoff's  reaction,  664; 
urosein,7  33,  740;  urobilinoids,  743; 
nitrobenzaldehyde,  784;  melanins,  841; 
lungs,  869 

Siedentopf,  H.,  19 

Siegert,  F.,  559 

Siegfried,  M.;  peptone  substances  and 
kyrines,  121,  132,  133,  136-138,  146: 
reticulin,  121,  544;  lysine,  163;  car- 
bamino  reaction,  166,  855;  jecorin,  385; 
phosphocarnic  acid,  578,  585,  593,  598; 
orylic  acid,  653;  milk-nucleon  653,663; 
phenol  excretion,  725,  726 

v.  Siewert,  A.,  292 


976 


INDEX  OF  AUTHORS 


Signorelli,  E.,  757 
Sikes,  A.  W.,  663 
Silbermann,  M.,  145,  147 
Simacek,  E.,  407,  583 
Simon,  Fr.,  606 
Simon,  G.,  656,  658 
Simon,  L.,  G.,  510 
Simon,  O.,  45,  364,  870 
de  Sinetv,  L.,  815 
Sittia;,  0\,  355 

Siven,    V.    O.,    uric    acid,    700;     urinary 
purines,  713;   protein  metabolism,  903; 
916,  934 
Sivrc,  A.,  482 
Siwertzow,  D.,  241,  244 
Sjoqvist,  J.,  enzymes,  57;    peptones  137; 
hydrochloric   acid   determination,   488, 
489;       intestinal      concrements,      524; 
urinary     nitrogen,     680,     685;      urea 
determination,  688,  689 
Skita,  A.,  145 
v.  Skramlik,  E.,  675 

Skraup,    Zd.,    protein    nitrogen,    77,    78; 
hydrolysis  of  proteins,    119.    124,    146, 
147;  alkali  albuminate,  126;  oxyamino- 
acids,  165;  carbohydrate,  215 
Skworzow,  W.,  575,  576 
Slanskv,  P.,  585 
Slavw,'379,  380 
Slosse,  A.,  333,  684,  910 
Slowtzoff,  B.,  pentosan,  208;    liver,  389; 
semen,    620;     milk    coagulation,    651; 
metabolism,  923,  926 
v.  Slyke,  D.  D.,  deamidation,  77,  78,  88; 
hydrolysis  of  albumin,   106,    107,    134, 
141,   143,   144;    plastein,   135;    amino- 
acids    in   blood,    266;     casein,  648,  649 
van  Slyke,  L.  L.,  648,  649 
Small.  Fr.,  707 
Smetanka,  F.,  700,  702 
Smirnow,  A.,  784 
Smith,  F.,  847,  848 
Smith,  Herbert,  457,  551 
Smith,  Lorrain,  862,  863 
Smith,  W.  J.,  752 
Socin,  C.  A.,  395 
Socoloff,  N.,  437 
Soldner,  F.,  milk,  647-650,  655-657,  662, 

664-668 
Sorensen,  S.  P.  L.,  isoelectric  point,  20; 
determination  of  the  reaction,  74,  75; 
coagulation   of    proteins   by    heat.    20, 
97;  glycocoll,  140;    phenylalanine,  152; 
proline,   154;    arginine,   161;    ornithin, 
162,   163;    formol  titration,    165;    hip- 
puric  acid,  723;   urinary  nitrogen,  756; 
ammonia,  768 
Soetber,  F.,  763 
Solera,  L.,  455,  456 
Solley,  Fr.,  118,  120 

Sollrnann,  T.,  chyle,  347;  bile,  440;   mus- 
cles, 566,  567,  570;  uterine  fibroma,  627 
Sommer,  A.,  384 
Sommerfeld,  438 


Sommerfeld,  P.,  465 

Sonden,  K.,  respiration    apparatus,   869; 

metabolism,  924,  925,  936 
Sorby,  H.  C.,  636 
Soret,  J.,  281 
Sourder,  C.,  502 
Le  Sourd,  L.,  314 
Soudat,  665 
Southgate,  506 

Soxhlet,    glucose,    216;     galactose,    216; 
maltose,     225;      fat     formation,     563; 
milk,  647,  650,  656,   667.   669;    sugar 
titration,  808,  809 
Spack,  Wl.,  124 
Spanpani,  G.,  669 
Spangaro,  S.,  250 
Spanjer-Herford,  R.,  455 
Speck,  C.,  869,  926,  928 
Spiegler,  A.,  899 
Spiegler,  E.,  790,  841,  842 
Spiro,  K.,  colloids,  precipitation,  25,  102; 
swelling,  31;  diffusion,  32;  gelatin,  119; 
glycocoll,      140;       pyrazinedicarbonic, 
•  acid,  220;    serumglobulins,  259;    blood 
coagulation,     31S,     319,    324;     rennin 
action,  650;    urine  acidity,  675;    oxy- 
butvric  acid  formation,  822 
Spiro," P.,  593 

Spitzer,  W.,  glycolysis,  333,  407;    liver, 
383;     uric    acid    formation,    702,    875 
Spriggs,  E.  J.,  469 
Spring,  14 

Staal,  J.  Ph.,  676,  733 
Stade,  W.,  57,  476 

Stadelmann,  E.,  tryptophan,  155;  icterus, 
301;    adrenal  bodies,   377;    bile,   414- 
416,    438,     441-444,    541;      intestinal 
putrefaction,  519;    nitrogen  excretion, 
685;    ammonia,  768;    pentoseuna,  791; 
diabetes,  blood,  856 
Stadthagen,  M.,  diamines,  47;     adenine, 
191;     xanthocreatinine,    698;     urinary 
sulphur,  752,  cystinuria,  827 
Stadler,  G.,  434 
Staehlin,  R.,  354 
Stanek,  V.,  247 

Stangassinger,  R.,  573,  692,  698 
Stange,  M.,  233 
Starke,  .1.,  102 
Starke,  K.,  262,  263 

Starkenstein,  E.,  398,402,  579,  817,  818 
Starling,  E.  H,  colloids,  16;  enzymes,  5'; 
lymph  formation,  351;    hormones,  375; 
enterokinase,  492,  496;    secretin,    492, 
498;      intestinal     enzymes,     492,     493; 
pancreatic  erepsin,  493,  503;   trypsino- 
gen  and  trvsin,  496,  497 
Stassano,  H.,  496,  497 
Stassow,  B.  [).,  541 
Stauber,  A.,  493 
Stavenhagen,  A.,  41 
Steel,  M.,  768 
Steenbock,  H.,  723 
Steensma,  F.  A.,  158,  488,  732 


INDEX  OF  AUTHORS 


977 


Steiff,  R.,  724 

Steiger,  E.,  161 

Steil,  H.,  599 

Stein,  G.,  445 

Steinitz,     Fr.,    phosphorous    metabolism, 

701;    C:N   quotient,  772,  884;  lactose 

in  urine,  815 
Stenger,    E.,    blood    pigments,    281,   285, 

286,289;  urobilin,  745 
Stepanek,  J.  O.,  628 
Stepp,  W.,  905,  906 
Stern,  E.,  35 
Stern,  Fr.,  750 
Stern,  H.,  441,  442 
Stern,  Heinrich,  621 
Stern,    L.,    uricolysis,    706;     peroxidase, 

873,  874;  oxydation  processes,  874,  875 
Stern,  M.,  248,  630 
Stern,  R.,  440,  724 
Steudel,  H.,  arginine,  162;    hexone  bases, 

164;    mucin,    169;    nucleic  acids,    179, 

181,  183,   184;    pyrimidine  bases,  194, 

195;  glucoseamine,  219 
Stewart,  C.  W.,  471 
Stewart,  G.  X.,  326,  566,  567,  570 
Steyrer,  A.,  571 
Sticker,  G.,  455,  458 
Stiles,  P.  G.,  400,  457 
Stock,  J.,  297,  297 
Stockmann,  R.,  595 
Stoltzner,  H.,  554 
Stoft'regen,  A.,  791 
Stohmann,  F.,  510,  885 
Stokes,  282,  289 
Stoklasa,    J.,    lecithin,    241;     glycolysis, 

407,   408,   509;    lactic  acid  formation, 

583;   fermentation  enzyme  of  milk,  653 
Stokvis,   B.  J.,   bile  pigments.  432,  433, 

743;    benzoic  acid,  723;    urobilin,  745, 

792;  hannatoporphyrin,  797 
Stolnikow,  J.,  793 
Stolte,  K.,  220,  396,  682 
Stoltzner,  W.,  554 
Stolz,  Fr.,  379 
Stomberg,  H.,  256,  322 
Stone,  W.,  208 
Stookev,  L.  B.,  136,  394 
Stoop,  F.,  145,  148 
Starch,  \  .,  646,  671 
Strada,  Fr.,  364 
Stradomskv,  N.,  773 
Strashesko,  N.  D.,  462 
Strassburg,    G.,    gases    of    lymph,     347; 

tension  of  the  gases  of  blood,  861,  864, 

868 
Strassburger,  J.,  523 
Strassuer,  \Y.,  877 
Straub,  W.,  403,  89V),  920 
Strauch,  F.  W\,  124 
Strauss,  Edw.,  122 
.Strauss,    H.,    fructose,    217,    218,    264 

blood,  264;   transudates,  264,  355,  358 

bile,  437;  lactic  acid  fermentation,  485 

amino  acid  storage,  721,  722 


Strecker,  A.,  242,245,  422 
Strickler,  E.,  658 
Strigel,  A.,  870 
Strohmer,  F.,  563 

Strusiewicz,  B.,  912 

Struve,  H.,  797 

Strzyzowski,  C.,  293 

Stubel,  H.,  894 

Stutz,  791 

Subbotin,  V.,  339,  921 

Suckrow,  Fr.,  400 

Sugg,  E.,  653 

Suida,  W.,  445 

Suleima,  Th.,  513 

O'Sullivan,  C.,  54,  55,  57 

Sundberg,  C.,  467,  468 

Sunde,  E.,  655 

Sundvik,  E.,  purin  bases,  188,  190 
glucoseamine,  218;  uric  acid,  699 
conjugated  glucuronic,  acids,  751,  777 
chitin,  839;  psylla  alcohol,  846 

Suter,  F.,  79,  113,  151 

Suto,  K.,  238,  809 

Suwa,  A.,  572,  780 

Suzuki,  \\\,  cystine,  148;  muscles,  572; 
crab  meat,  578;  phytase,  579;  orvza- 
ninc,  905,  906 

Svedberg,  The.,  14,  20 

Svenson,  N.,  919 

Swain,  R.  E.,  159 

Symmers,  D.,  757 

Syniewski,  V.,  227,  229 

v.  Szontagh,  F.,  653,  658,  660,  662 

Szydlowski,  Z.,  474 

Szymonowicz,  L.,  379 

Tachan,  H.,  145 

Takahashi,  D.,  269,  329 

Takaishi,  M.,  579 

Takamine,  J.,  379 

Takada,  K.,  787 

Tallqvist,  T.  W.,  914 

Tammann,  G.,  60,  66 

Tanaka,  T.,  371 

Tangl,  Fr.,  blood  serum,  264,  271;   blood 

analysis,    326;     sugar    in    blood,    398; 

fat,  482;    egg,  development,  637,  638; 

casein,    648,    658;     milk,    668;     C:N 

quotient,  772,  884 
Tanret,  C.,  200 
v.  Tappeimer,  H.,  enzymes,  50;   cellulose, 

510,  515;  bile  acids,  541 
v.  Tarchanoff,  J.,  441,  632 
Tarugi,  B.,  848 
Tarulli,  L.,  675 
Tawara,  932 
Taylor,  A.  E.,  enzymes,  56;     liver    fat, 

384;      fat     formation,     561;      mineral 

starvation,  900 
Tebb,  Chr.,  reticulin,  121;   glycogen,  o!)2; 

amylolysis,  456;   saecliarate,  492;   mal- 
tose, 500;    protogon,  606-609;    choles- 

terin  in  brain,  613 
Tedesko,  Fr.,  676 


178 


INDEX  OF  AUTHORS 


Teeple,  J.,  428,  430,  431 

Teichrnann,  L.,  292 

Tengstrom,  B.  St.,  418 

Terrat,  P.,  758 

v.  Terray,  P.,  bile  and  putrefaction,  518, 

520;    oxalic  acid,   71(5;    lactic  acid  in 

urine,  748,  749 
Terroine,  E.  F.,  105,  500-503 
Terry,  O.  P..  454 
Terunchi,  Y.,  6S2 
Tezner,  E.,  455,  456 
Thannhauser,  S.  J.,  429 
Theissier,  247 
Thesen,  J.,    572,  729 
Thevenot,  247 
Thiele,  O.,  754 
Thiemich,  M.,  384 
Thierf elder,   H.,   barium,   72;    galactose, 

216,   610,   611;    glucuronic   acid,   223; 

cephalin,    248;     digestion    and    micro- 
organism,   516;     protagon,    607,    608; 

cerebron     and     cerebroside,     608-611; 

yolk      phosphatides,    630;     mammary 

glands,    643,    669;     sphingosin,     611; 

cerebronic  acid,  611 
Thies,  Fr.,  379,  380 
Thiroloix,  J.,  406 
Thiry,  L.,  490,  491 
Thorner,  W.,  644 
Thomas,  K.,  595,  611 
Thomas,  Karl,  903 
Thomas,  P.,  492 

Thomas,  W.,  488,  909,  911,  914,  917 
Thompson,  W.  H.,  574,  682 
Thorns,  H.,  687 
Thormahlen,  J.,  799 
Thudichum,    L.    W.,    phosphatides,    239, 

240,    242,    245,    248;     bilirubin,    431; 

brain  phosphatides,  605,  607-609;  cere- 

brosides,     609-611;      sphingosin,     611; 

lutein,  631;  paraxanthine,  7l4;   urinary 

pigments,     740;      alcohol     in     animal 

organism,  921 
Thunberg,  T.,  873,  874,  876 
Tichomirow,  N.  P.,  468 
Tklemann,  F.,  458 
Tiemann,  H.,  652,  658 
Tigerstedt,  K.,  762 
Tigerstedt,     R.,    respiration     apparatus, 

869;    metabolism,  893,  894,  924,  925, 

936;  digestion  work,  930 
Tissot,  J.,  592 
Tobler,  L.,  341,  484,  763 
Toepfer,  G.,  530,  759 
Tollens,  B.,  carbohydrates,  197,  208-211, 

216,  217;    glucuronic  acids,  223,  817; 

urea,    687;     naphthoresorcin-reaction, 

223   817 
Tollens,  C,  724,  725,  750,  817 
Tolmatscheff,  656,  662,  665 
Tomasinelli,  <..,  848 
Tomaszewski,  Zd.,  716 
Tompeon,  E.,  54,  55,  57 
Torup,  S.,     carbon     monoxyhsemoglobin, 


287,     288;      globulins     and     carbonic 

acids,  853,  855 
Totani,  G.,  161,  787 
Tower,  R.  W.,  121 
Towles,  C.,  692,  693 
Toyonaga,  M.,  388 
Traube,    J.,    10,    11;     absorption,    542; 

oxydation,  871 
Traube,  M.,  1,  9 
Traxl,  W.,  78 
Treupel,  G.,  749,  808 
Treves,  Z.,  78 

Trier,  G.,  178-180,  240,  243 
Trifanowski,  D.,  437 
Trillat,  A.,  873 
Tritschler,  F.,  773 
Troisier,  J.,  744 
Troller,  J.,  464 
Trommer,  C.,  214 
Trommsdorff,  R.,  877,  910 
Tnimpy,  D.,  847 
Trunkel,  H.,  119,  120 
Truthe,  W.,  230 
Tschenloff,  R.,  910 
Tschernoruzki,  M.,  307 
Tscherwinsky,  N.,  563 
Tschirjew.  S.,  344 
Tsuboi,  J.,  339,  881 
Tsuschija,  J.,  744,  747,  793 
Tuczek,  F.,  459 
Tullner,  H.,  698 
Turk,  W.,  146 
Turkel,  R.,  585 
Turban,  K.,  912 
Turby,  H.,  491 

Udranszky,  L.,  diamines,  47;  bile  acids, 
419,  800;  urinary  pigments  and  humus 
substances,  740;  carbohydrates  in 
urine,  749,  808;     cystine,  827 

Uffelmann,  J.,  488 

Uhlik,  M.,  280,  282,  283 

Ulrich,  Chr.,  226 

Ultzmann,  R.,  832 

Umber,  F.,  albumoses,  133;  nucleins, 
175;  transudates,  354;  gastric  juice, 
464,  465;  proteins  of  pancreas,  494; 
kevuloseuria,  814 

Umikoff,  N.,  664 

Underhill,  F.  P.,  protozym,  324;  glyco- 
seuria,  400,  402;  saliva,  454;  allantoin, 
717;  lactic  acid  in  urine,  748,  749 

Unna,  P.  G.,  837,  844,  845 

Ulpiani,  C.,  244,  699 

Urano,  F.,  573,  587 

Urbain,  V.,  851 

Ure,  A.,  783 

Usher,  Fr.,  37 

Ussow,  506 

Ustjanzew,  W.,  542 

Vahlen,  E.,  425 
Valenciennes,  A.,  603,  6S6 
Valenti,  A.,  663 


INDEX   OF  AUTHORS 


97  J 


de  Vamossv.  Z.,  370 

Vandegrift,  G.  W  ,  545 

Vandevelde,  A.  J.  J.,  053, 

Vanlair,  C,  129,  521 

Vnsilin.  H..  720,  723 

Vassale,  ( ;.,  374 

Vaubd,  W..  82,  09 

Vauquelin.  L.  N.,  717 

Vav,  Fr.,  3S3,  592 

v.  d.  Velde,  A..  723 

v.  d.  Velden,  R.,  724 

Velichi.  J.,  602 

Vella,  L..  490 

Veraguth,  O.,  910 

Verhaegen,  A.,  405 

Verneuil,  401 

Vernois,  M.,  665 

Vernon,  H.  M.,  erepsin,  57,  492,  493; 
white  of  egg,  05;  pancreatic  enzymes, 
497,  500,  501,  503,  507,  509;  muscle 
rigor,  590 

Verploegh,  H.,  creatin  and  creatinine, 
573,  091-094,  098 

Verzar,  Fr.,  404 

Viault.  P.,  340 

Victorow,  C,  S04 

Vierordt,  K.,  302,  803 

Vigno,  L.,  520 

Vignon,  L.,  122 

Vila,  A.,  blood  corpuscles,  275,  281; 
blood  pigments,  285,  292;  musculamine, 
578 

Ville.  J.,  oxymethyl  furfurol,  215;  blood 
pigments,  281,  285;  bile  acids,  419; 
fat  absorbtion,  539,  540;  urinary 
chlorine  compounds,  758;  Bence-Jones 
protein,  792 

Villiers,  A.,  758 

Vincent,  Sw.,  370-002 

Vines,  S.  H.,  493,  502 

Vinci,  S.,  314,  315 

Virchow,  R.,  171,  301 

Vitali,  A..  790 

Vitali,  D.,  758 

Vitek,  407 

Voegtlin,  C,  507,  092,  094,  703 

Volte,  \Y.,  040,  912 

Vogel,  H.,  372 

Vogel,  J.,  pentoses,  208,  815;  isomaltose, 
225;  lactase,  492;   amylolysis,  450,  500 

Vogelius,  391 

Voges,  O..  080 

Vohl,  H.,  579 

Voit.  C,  glycogen,  390,  393,  395,  534; 
bile  and  putrefaction,  518;  excrements, 
520;  absorption,  525,  537,  538;  fat 
formation,  500,  501,  003;  work  and 
metabolism.  594,  597,  925;  nitrogen 
in  meat,  000,  883;  urea  foimation, 
t>>4;  phosphoric  acid  excretion,  702; 
standard  numbers,  772,  915;  lactose 
detection,  815;  metabolism  experiments, 
879,  881,  920,  920,  929;  starvation 
metabolism,  893,  903;    water  content 


of  body,  SOS;  mineral  metabolism,  899; 
protein  catabolism,  903,  908,  908  910, 
913,  914;  nutritive  value  of  gelatin 
diets,  932-930,  938 

Voit,  E.,  glycogen,  395;  bones,  554,  555; 
fat  formation,  562,  563j  calorific  value 
of  oxygen,  889;  of  nutritive  substances, 
S02;  starvation  metabolism,  894,  898 
897;  nitrogen  excretion,  910;  vege- 
table diet,  915;  protein  minimum,  917; 
surface  rule,  023 

Voit,  Fr.,  galactose  fermentation,  210; 
thyroidea  and  metabolism,  370;  glyco- 
gen formation,  305;  sugar  elimina- 
tion, 305,  534;  feces  formation,  521; 
lactose,  532;  curare  poisoning,  591; 
acetone  bodies,  819 

Voit,  W.,  814 

Voitinovici,  A.,  100,  114,  121 

Volhard,  F.,  409,  470,  500 

Volhard,  J.,  759,  700 

Volkmann,  A.  W.,  899 

Vorlander,  D.,  247 

v.  Vornveld,  J.  A.,  341 

Vossius,  A.,  441 

Voswinckel,  H.,  843 

Vozarik,  A.,  075,  077 

de  Vries,  H.,  2,  5,  0 

Vulpian^A.,  377,  441 

Waage,  P.,  32 

Wachsmann,  M.,  501 

Waehsmuth,  L.,  350 

Wakchli,  G.,  117 

de  Waele,  H.,  053 

Wagner,  B.,  813 

Wagner,  H.,  572,  577 

Wahlgren,  V..  417,  420,  440,  838 

Wait,  Ch.,  595 

Wakemann,  A.  J.,  380,  387,  737,  774 

Walbum,  L.  E.,  50,  793 

Wald vogel,  R.,  820 

Walker,  J.,  28 

Wallace,  G.  B.,  89 

Walter,  Fr.,  082,  851,  856 

Walter,  G.,  173,  030 

v.  Walther,  P.,  535,  537 

Walton,  J.  H.,  35 

Wanach,  R.,  304 

Wang,  E.,  730,  731 

Wanklyn,  J.  A.,  047 

Wauner,  Fr.,  870 

Warburg,  O.,  leucine,  142,  143;  fertiliza- 
tion, 040;  phenylamino  acetic  acid,  780; 
oxvdation  processes,  873,  874 

Warfield,  L.  M.,  455 

Warmbold,  H.,  047 

Warren,  J.,  593 

Wasbuteki,  M.,  519 

Wassiliew,  W.,  499 

Wastenevs,  H.,  73 

Waterman,  N.,  379 

Wavmouth,  Reid,  E..  533 

Weber,  635 


980 


INDEX  OF  AUTHORS 


Weber,  O.  H.,  341,  555,  340 

Weber,  S.,  594,  692, _693 

Wechselmann,  Ad.,  733 

Wechsler,  E.,  83,  117 

Wedenski,  X.,  749 

Wegrzvnowski,  L.,  716 

Wehrle,  E.,  404 

Weidel,  H.,  189 

Weigert,  Fr.,  163 

Weigert,  R.,  265 

Weil,  Arth.,  161,  605,  144 

Weil,  F.  J.,  423 

Weiland,  W.,  786 

Weinland,    E.,    lactase,    53,    492,    532; 

glycogen,   390,   395;    sugar  formation, 

412;    retarding   substances,    466,    487, 

892;  fat  formation,  561; 
Weintraud,  W.,  403,  404,  685,  761 
Weis,  Fr.,  506 
Weisbach,  613 
Weiser,  St.,  264 
Weisberger,  G.,  866 
Weiske    H.,    cellulose    510;     bones,    554, 

555;  asparagin,  nutritive  value,  912 
Weiss,  F.,  78,  83,  109,  111,  162 
Weiss,  H.  R.,  505,  506 
Weiss,  J.,  721 
Weiss,  Sigm.,  395,  592 
Weisz,  Mor.,  741,  752 
Weizmann,  Ch.,  87 
Wellman,  O.,  896 
Wells,  H.  G.,  keratin,  114;    hypophysis, 

380;     liver   nitrogen,    387;     uric    acid, 

701-703,  706 
Wendel,  A.,  143 
v.  Wendt,  G.,  882,  903 
Wenz,  R.,  128 
Wenzel,  F.,  17S-180,  577 
Werenskjold,  F.,  659 
Werigo,  B.,  95,  866 
Werneken,  G.,  651 
Werner,  A.,  552 
Wertheimer,  E.,  440,  498,  499 
Werther,  M.,  459,  590,  591,  593 
v.  Westenrijk,  N.f  309 
Wester,  D.  H.,  839,  840 
Wetzel,'G.,  122,  160,  178 
Weyl,   Th.,    albumin    crystals,    94;     car- 

bonmonoxy  methsemoglobin,  287;  amni- 
otic,   fluid,   642.   643;   creatinine,    695; 

benzoic  acid,  723;  nitrate,  766 
Wheeler,  H.,  L.,  iodogorgonic  acid,  123; 

phenylalanine,  152;    nucleic  acid,  185; 

cytosine,  194 
Whipple,  G.  H.,  253,  442 
White,  B.,  705 
Withnev,  J.  L.,  406 
Wichmann,  A.,  261,  652 
Widdicombe,  J.  H.,  492 
Wichowski,  W.,  urea  determination,  088; 

uric    arid,    and    allantoin,    705,    706, 

716-718;  hippuric  acid,  721 
Wicland,  H.,  423 
Wiener,  H.,  autolysis,  43;  serumglobulins, 


259,  261,  335,  nucleic  acids,  514;  uric 
acid,  699,  700,  702,  704-706;  oxalic 
acid,  716 

Wiener,  K.,  182 

Wihelmv,  33 

Wilke,  K.,  298 

Willcock,  Ed.,  634 

Willdenow,  C.,  164 

v.  Willebrand,  E.  A.,  849 

Willheim,  R.,  103 

Williams,  D.,  510 

Williams,  H.  B.,  827 

Willstatter,  R.,  proline  154;  lecithin,  263; 
glycerinphosphoric  acid,  247;  chloro- 
phyll and  blood  pigments,  277,  296, 
297;  cholesterin,  445,  448;  carotein 
yolk  lutein,  xanthophyll,  631 

Wilson,  R.  A.,  606,  608 

Wimmer,  M.,  914 

Windaus,  A.,  histidin,  159,  methylimi- 
dazol,  201;    cholesterin,  445,  448,  449 

Windrath,  H.,  529 

Winkler,  559 

Winogiadow,  A.,  416 

Winteler,  L.,  416 

Winter,  J.,  660 

Winterberg,  A.,  334,  675,  683 

Winternitz,  Hugo,  blood  pigment  deter- 
mination, 302;  amounts  of  haemoglobin, 
338;  bile,  440;  putrefaction,  517, 
724;  iodized  fat,  560,  668,  669 

Winternitz,  M.  C.,  371,  702 

Winterstein,  E.,  amino-acids,  141,  153; 
arginine,  161;  lysine,  163;  hexone 
bases,  164;  phosphatides,  245;  phytin, 
579;  colostrum,  658;  tunicin,  839; 
chitin,  839 

Wislicenus,  J.,  596 

Wittmaack,  K.,  663 

Woeber,  A.,  126 

Wohler,  Fr.,  hippuric  acid  synthesis,  39, 
783;  urea,  689;  demolition  of  uric  acid, 
705,  allantoin,  716,  717 

Worner,  E.,  607,  611,  711 

Wohl,  A.,  200 

Wohlgemuth,  J.,  autolysis,  43;  enzymes, 
53,  71;  oxyaminosuberic  acid,  147; 
oxvdiamino-sebacic  acid,  165;  pen- 
toses, 203,  208,  210;  diastase  in  blood, 
298;  ferratin,  383;  glycogen,  393,  394; 
cystine  demolition,  442;  gastric  juice, 
476;  pancreatic  juice,  500;  pancreatic 
diastase,  501;  pancreatic  rennin,  509; 
enzyme  of  egg-yolk,  628;  woman's 
milk,  660,  661;  conjugaged  glucuronic 
acids,  751 ;    amino  acids  in  urine,  755 

Wolf,  C.  G.,  glycogen,  397;  urinary 
nitrogen,  680;  creatinine,  693;  cystin- 
uria,  827,  828;  urinary  sulphur,  882 

Wolfenstein,  R.,  737 

v.  Wolff,  E.,  595 

Wolff,  H.,  219,  358,  841 
Wolff,  .1.,  873 

Wolff,  L.  W.,  19 


INDEX   OF  AUTHORS 


981 


Wnlffherg,  8.,  395,  861,  864,  865 

Wolkow,  M.,  735,  736 

Woll,  F.  W.,  645 

Wolter.  O.,  770 

Woltenng,  H.,  340,  383 

Woods,  II.  8..  246 

Wooldridge,  L.  C,  stroma,  of  blood  cor- 
puscles. 274;  tissue  fibrinogen,  306, 
307;  blood  coagulation,  315,  321,  323, 
324 

Worm-Muller,  J.,  blood,  339,  340,  343, 
344;  sugar  te-it,  803;  sugar  determina- 
tion, 811-813 

Worms,  W.,  635 

Woronsow,  W.  N.,  381 

Wright,  A.,  fibrin  ferment  and  coagula- 
tion, 256,  313,  323;  blood  alkalinity, 
309;  diabetes,  400 

Wr6blewski,  A.,  fermentation,  41 ;  pseudo- 
nuclein,  104;  starch,  227;  pepsin,  466, 
470;  enzyme  action,  510;  enzymes 
of  brain,  (J06;  milk,  660,  661 

Wulff,  C.  190,  714 

Wurm,  W.  A.,  843 

Wurster,  C,  768 

Wurtz,  A.,  346,  625 

Yagi,  8.,  448 

Yakuvva,  G.,  912 

Yoshimoto,  443 

Yoshimura,  K.,  579 

Young,  P.  A.,  435 

Young,  R.  A.,  230,  391 

Young,  W.  J.,  co-enzymes,  52,  205;  car- 

bohydrate-phosphoiic    acid    ester,    60, 

205;  dioxyacetone,  205 
Yvon,  752 

Zangerle,  M.,  626 

Zah6r,  H.,  794 

Zaitschek,  A.,  milk,  651,   653,  658-662, 

668 
Zak,  E.,  354,  769,  791 
Zaleski,  J.,  leaf  and  blood  pigments,  277, 

296;     blood    pigments,    291-295,    301; 

ammonia,    334,    336,    528,    683,    768; 

urea,   684;    liver  and  acid  formation, 

685,  703,  704 
Zaleski,  St.,  iron  of  liver,  383,  388;   milk, 

666,  667;  reaction  of  intestinal  contents 

519 
Zaleskv,  N.,  552,  847 
Zander,  E.,  S39 
Zanetti,  C.  U.,  ,seromucoid,   260,   263; 

bile,  417;  ovomucoid,  635 


Zangermcistcr,  \Y.,  302,  642 

Zaribnickv,  It.,  '■>  Is 

Zaudy,  70] 

Zdaiek,  E.,  841 

v.  Zebrowski,  E.,  453 

Zerhui.-en,  II.,  777 

Zeidlite.  P.  V.,  804 

Zegla,  P.,  398 

Zeller,  A.,  75s,  777 

Zemplen,  G.,  839 

v.  Zeynek,  R.„  dermoid-cyst-fat,  239,  627, 
844;  blood  pigments,  283,  285,  289, 
290;  liver,  602;  bile,  438;  sarcomelanin 
841;  chromoproteid,  843 

Zickgraf,  G.,  83,  870 

Ziegler,  E.,  779 

Zieuler,  J.,  32,  708,  716,  717 

Zillessen,  H.,  593 

de  Zilwa,  L.,  500 

Zimmermann,  R.,  372,  725,  726 

Zimnitzki,  S.,  517 

Zink,  J.,  237,  559 

Zinnowsky,  O.,  277 

Zinsser,  A.,  476 

Zisteier,  J.,  917 

Zobel,  S.,  581 

Zoja,  L.,  oxyproteic  acids,  83;  elastin,  122; 
ovalbumin,  633;  urobilin,  744;  uroery- 
thrin,  748;   ha-matoporphyrin,  797,  798 

Zsigmondy,   R.,   colloids,    15,   19,   21,  23 

Zuelzer,  G.,  lecithin,  245;  diabetes, 
405,  406;  skin  breathing,  849 

v.  Zumbusch,  L.,  434,  844 

Zuntz,  N.,  blood,  310,  338,  340,  341; 
glycogen,  391;  sugar  in  blood,  398; 
plilorhizin  diabetes,  400;  digestion, 
506,  542;  protein  assimilation,  525; 
muscle  fat,  586,  596;  muscle  metabol- 
ism, 591,  595,  597;  pig  milk,  659; 
high  altitudes,  341,  848;  skin  breath- 
ing, 849;  gases  of  blood,  851-853,  859, 
866,  carbonic  acid  of  lymph,  856; 
alveolar  air,  S63;  respiration,  868. 
869;  metabolism,  879,  884,  890,  893 1 
922,  923,  926,  927,  929;  calculation 
of  calorific  power,  888;  nutritive  value 
of  albumoses,  912;  alcohol,  921;  diges- 
tion work,  930 

Zuntz,  L.,  309 

Zuntz,  E.,  digestion  products,  132-134, 
139;  gastric  digestion,  482,484;  absorp- 
tion, 484,  526,  532,  540;  trypsinogen 
and  its  activation,  496,  497;  intestinaL 
diaestion,  513;  muscles  ,572 

Zweifel,  P.,  456,  500,  748,  749 


GENERAL   INDEX 


Abderhalden  and  Schmidt's  reaction  for 

proteins,  101 
Abiuret  products,  132,  513,  526 
Absorption,  524-543 

chemical     processes     during, 

524-525 
effect  of  extirpation    of    the 
pancreas       upon, 
531,532 
removing     portions 
of  intestine  upon, 
541,  542 
of  amino  acids    from    intes- 
tine, 526-528 
of  bile  constituents,  540,  541 
carbohydrates,     degrees      of 

rapiditv  of,  532-534 
of  fat,  535-540 
of  nonbiuret  giving  products 

from  intestine,  526-528 
of  mineral  substances,  540 
peptones  from  intestine,  526- 

528 
of    proteose    from    intestine, 

526-528 
of    undigested    protein,    525, 

526 
theory  of,  542,  543 
Acetanilide,  fate  of  in  organism,  779 
Acethaemin,  292 
Acetone  bodies,  776 

in  urine,  818-828 
origin  of,  818,  819 
fate  in  organism,  821 
formation  from  fat,  820-822 
formation  from  protein,  819 
former,  781 
in  urine,  822-824 
quantitative    estimation    of,    in 
urine,  825 
Acetophenone,  fate  of  in  organism,  785 
Acetylation  in  organism,  786 
Acetyldiglucosamine,  839 
Acetyl  equivalent  of  fats,  238 
Acetylglucosamine,  784 
Acetylene  Iwemoglobin,  288 
Acetylpara-amidophenol,  780 
Achilles  tendon,  545 
Acholia,  pigmentary,  439 


Achroodextrin,  229,  458 
Acid  abietinic,  447 

acetic,  in  intestines,  513 
in  stomach,  465 
in  urine,  748,  773 
acetoacetic  in  urine,  821,  824,  825 

fate  in  organism,  821 
acetylaminobenzoic,  7S4 
albuminates,  125-127 

absorption  of,  533 
in  peptic  digestion,  472 
alloxyproteic,  in  urine,  752,  754,  755 
amides,  behavior  in  organism,  775 
aminoacetic.     See  Glycocoll. 
aminocaproic.     See  Leucine,  141 
a-diamino-/3-dithiolactic,  148 
0-7-diaminovaleric,,  163 
a-e-diaminocaproic,  163 
d-a-amino-n-caproic,  144 
aminoethylsulphonic,  150 
a-aminoalutaric,  146,  117 
a-aminoisobutylacetic.     See  Leucine, 
a-amino-valeric,  140.     See  Valine, 
aminobenzoic,    behavior    in    organ- 
ism, 783 
aminoeinnamic,  778,  780 
a-amino-/3-oxypropionic,     145.      See 

Serum. 
a-amino-/3-thiolactic,  148,  150.     See 

Cystine, 
a-aminopropionic,  140.    See  Alanine, 
aminoglucuronic,  548 
aminohippuric,  783 
aminophenylacetic,   behavior   in  or- 
ganism, 781 
aminosuccinic.     See   Aspartic   acid, 

146 
antoxvproteic  in  urine,  752 
arachidic,  232,  627,645,845 
aspartic,  85,  146 

quantitv  in  proteins,   106, 

107,  115,  125 
relation    to    formation    of 

urea,  682,  77*  I 
relation    to    formation    of 
uric  acid,  702 
/3-amino-a-oxypropionic.      See     Iso- 

serine,  14fi 
/3-imidazol-a-aminopropionic,  159 

983 


984 


GENERAL  INDEX 


Acid  benzoic,  conjugation  of,  in  organism, 
782 
in  urine,  723 
glucuronic,  751 

benzoyl-amino  acetic.     See  Hippuric 
acid,  719 

bilinic,  429 

bilianic,  423 

bilirubinic,  430 

bromphenylmercapturic,  780 

butyric,  fermentation,  214,  516 

camphoglucuronic,  222,  751,  786 

capric,  232 

caproic,  232 

caprylic,  232 

carbamic,  267 

conjugation  of,  786 
fate  of  in  organism,  786 
in  blood,  267,  683 
in  urine,  691,  692 

carbamino-acetic,  164,  856 

carboglobulinic,  855 

carbolic.     See  Phenol. 

carminic,  844 

carnaubic,  239,  673 

carnic,  653 

caseanic,  85,  165 

caseinic,  85,  165 

cephalic,  248 

cepholinic,  248 

cerebrinic,  612 

cerebrinic-phosphoric,  612 

cerebronic,  611 

cerotic,  239 

cheno-taurocolic,  422,  427 

chitaminic,  219 

chitaric,  219 

chlorhodinic,  366 

chlorphenylmercapturic,  786 

cholalic,  423-425 

constitution  of,  423 

cholanic,  425 

choleic,  425 

cholesterinic,  423 

cholic,  423,  424 

properties  of,  424 
tests  for,  424 

choloidanic,  423 

choloidic,  427 

cholylic,  423 

chondroitic.       See    Chondroitinsul- 
phuric  acid,  547 

chondroitin-sulphuric,  171,  173,  547 

chrysophanic,  elimination  in  urine, 
787 

cilianic,  423 

cinnamic,     behavior     in    organism, 
719 

citric,  in  milk,  647,  658,  663 

coccinic,  844 

cochenillic,  844 

combined  hydrochloric,  488 

cresol-sulphuric,  724-727 

erotonic,  825 


Acid  cumic,  conjugation   of  in  organism. 
783 
cuminuric,  synthesis  of  in  organism, 

783 
cyanuric,  687,  698 
cysteinic,  149 
damaluric,  758 
damolic,  758 
dehydrobillinic,  429 
dehydrocholeic,  425 
desaminoalbuminic,  127 
desoxycholic,  425,  426 

properties  of,  426 
diacetic.     See  Aceto-acetic 
dialinic,    relation    to    formation    of 

urine,  704 
diamino  acetic,  85 
diaminotrioxydodecanoic,  85,  165 
dimethylaminobenzoglucuronic       in 

urine,  751,  786 
p-dimethylaminobenzoic,  786 
dioxydiaminosuberic,  165 
dioxyphenylacetic  in  urine,  735 
dioxyphenyl-lactic,  738 
dioxystearic,  236,  647 
doeglic,  237 
elaic,  236 
elaidic,  236 
ellagic,  524 
equivalent,  238 
erucic,  232 
ethylidenelactic,  582 
euxanthic,  222,  223 

in  urine,  785 
euxanthonglucuronic,  222 
excretolic,  523 
fellic,  426 
fermentation  lactic,  582 

in  blood,  335 
in  coagulation  of 
milk,  585,  645. 
in  muscle,  604 
in  stomach,  487,, 
488 
formic,  fate  in  organism,  748,  773 
furfuracrylic  in  urine,  784 
furfuracryluric  in  urine,  784 
gadoleic,  237 
galactonic,  217 
gallic,  fate  of  in  organism,  785 

in  urine,  734 
gentisic,  336,  737 

behavior  in  organism,  784 
gluconic,  198 

in  diabetes,  403 
glucosaminic,  201,  219,  221 
in  milk,  643 
glucothionic,  387,  673 

in  milk,  643 
glucuronic,  221-223 

conjugation  with,  785 
elimination,  effect  of  for- 
eign    substances     on, 
750 


GENERAL  INDEX 


985 


Acid  glucuronic,  formation  of  in  organism, 
751 
in  bile,  417,  433 
in  blood,  264 
in  urine,  750 
isolation  of,  222 
preparation  of,  223 
properties  of,  222 
quantitative    estimation 
of,  223 
glutamic,  82,  86,  106,  107,  115,  125, 

147 
glutinic,  119 
glyceric,  145 
glycerophosphoric,  248 

in  urine,  749,  757 
glychollic,  fate  of  in  organism,  713 
glycocholic,  417,  419,  420 

absorption  of,  548 
in  feces,  517 
preparation  of,  421 
properties  of,  420 
glycocholeic,  420-421 

preparation  of,  421 
properties  of,  420 
glycosuric  in  urine,  735 
glycuronic,  221 
glyoxylic,  719 

as  reagent,  100 
guanidineacetic,  fate  of  in  organism. 

See  Glycosamine,  787 
5-guanido-a-aminovaleric.     See  Arg- 

inine,  161 
guano  bile,  421 
guanvlic,  178,  181,  183,  184 

of  liver,  383 
hEematinic,  296,  430 
haematopyrrolidinic,  291 
haemoglobin,  285 
hippuric,  267,  719-723 

formation  of,  139 

in  organism, 
719 
in  urine  of  herbivora,  720, 

721 
occurrence  of,  719  720 
preparation  of,  722-723 
properties  of,  722 
quantitative  estimation  of, 

723 
reactions  for,  722 
synthesis  of  in   organism, 

782 
synthetical  preparation  of, 

719 
theories    of    formation    in 
organism,  721 
homogentisic,  778 

formation  of  in  organ- 
ism, 735-737 
in  urine,  727,  734-740 
mother  substances  of, 

735-736 
preparation  of,  739 


Acid  homogentisic,  properties  of,  738-739 
quantitative      estima- 
tion of,  739 
quantity      eliminated, 

735 
test  tor.  7:;'.» 
homoglytisic,  origin  of,  736 
hydrochloric  action  upon  ptyalin,  455 
secretion  of 

bile,  416 
6ecretion  of 
pancrea- 
tic juice, 
498 
pylorus, 
480,  482 
antifermentive     action, 
485 
hydrochinon  sulphuric,  724 
hydrocinnamic,  behavior  in  animal 

body,  720 
hydrocyanic,    effect    on  blood   pig- 
ments, 285  i 
pepsin    diges- 
tion, 471 
trypsin       di- 
gestion, 506 
hydroparacoumaric,  in  intestinal  pu- 
trefaction^ 15 
in  urine,  734 
hydroquinone  carboxylic,  738 
hydroquinonesulphurir,  691,  724,  728 
hyoglycocholic,  421,  427 
hypogaeic,  239 

imidazolaminoacetic  in  urine,  758 
imidazol  propionic,  82 
indolacetic,  82 

in  urine,  733 
indola:ninoproj;ionic,  82,  155 
indol-carboxylic,  728 
indolTpropionic,  82,  155 
indoxyl-earboxylic,  728 
indoxylglucuronic,  728 
indoxyl-sulphuric,  724 

formation    of    in 

organism,  728 
in  urine,  728-731 
inosinic,  17S,  181-183,  185 
iodogorgonir,  123 
isobilianic,  423 
isocholanic,  425 
isocholic,  427 

isophonopyrrol  carboxylic,  429,  430 
isovaleric,  774 
jecoleic,  237 
kynurenic,  740,  758 

in  urine,  734 
kyroproteic,  83 
lactic,  145 

formation       of       in       active 
muscles,  593 

in  blood,  333 
in  blood,  334 
in  cerebrospinal  fluid,  360 


986 


GENERAL  INDEX 


Acid  lactic,  in  relation  to  diabetes,  408 
origin  of,  583,  584 
quantitative  determination  in 

gastric  contents,  488 
test  for  in  gastric  contents,  488 
transformation  into  uric  acid, 
704 
lactophosphocarnic,  645,  653 
lanoceric,  846 
lanopalmitic,  846 
lauric,  232,  647,  846 
lepidotic,  844 
leucinic,  142 
levulinic,  211,  654 
linolenic,  232 
linolic,  232 
lithobilic,  524 
lithocholic,  427 
lithofellic,  427,  524 
lithuric,  758 
lysalbinic,  127 
lysuric,  164 
maleic,  290 

malic,  fate  in  organism,  668,  676 
mandelic,  780 
mannonic,  212 
margaric,  235 
melanoidic,  841 
menthol-glucuronic,  817 
mesitylenic,  conjugation  of  in  organ- 
ism, 783 
mesitylenuric  synthesis  of  in  organ- 
ism, 783 
metaphosphoric    as    precipitant    of 
proteids,  98,  789 

in  nucleins,  176 
in     pseudonucleins, 
177 
methylethyl-a-aminopropionic,    143. 

See  Isoleucine. 
methylguanidine-acetic.     See    Crea- 
tine, 573 
methylhydantoie,  776 
monoxystearic,  233,  236 
mucic,  217,  394,  654 
muconic,  778 
myristic,  232 
naphtholglucuronic,  787 
neurostearic,  611 
nitrobenzoic,  784 
nitrohippuric,  783 

nitrophenolpropiolic,  fate   in  organ- 
ism, 728, 731 
test     for     dex- 
trose, 216,808 
norisosaooharic,  219,  630 
nucleic,  622 

of  milk,  643 
yeast,  178,  185 
nucleotinic,  179 
nuoleotinphosphoric,  179 
oleic,  232 

properties  of,  236 
tests  for,  237 


Acid  ornithuric,  163 

synthesis  of  in  organism, 
783 
orotic,  654 
orylic,  653 

oxalic  in  urine,  716,  717 
oxaluric,  699,  715 
oxaminic,  83 
oxonic,  699 

oxyaminosuberic,  85,  148 
oxyaminosuccinic,  85,  147 
oxy-a-pyrolidinecarboxylic.   See  oxy- 

proline. 
oxybenzoic,  fate  in  organism,  783 
/3-oxybutyric,  821,  825-827 

detection  of  in  urine, 

826 
fate  in  organism,  821  \ 
formation  from  amino 

acids,  821 
properties  of,  825,  826 
quantitative      estima- 
tion of,  in  urine  826 
oxydiaminosebacic,  85,  165 
oxydiaminosuberic,  85,  164 
oxyhydroparacoumaric,  in  urine,  734 
oxyisovaleric,  774 
oxymandelic,  739 

oxymethylpyrazine-carboxylic,  221 
p-oxyphenylacetic,  515 

in  urine,  734,  784 
p-oxyphenyl-a-aminopropionic .     See 

Tyrosine,  152 
p-oxyphenylpropionic,  515,  734 

in  urine,  784 
oxyphenylpyroracemic,  737 
oxyproteic  in  urine,  752,  754 
oxyprotosulphonic  acid,  83 
oxyquinoline  carboxylic.     See  Kynu- 

renic  acid, 
palmitic,  232,  235 

properties  of,  236 
parabanic,  699 
paralactic,  582 

origin  of,  583,  584 
in  urine,  749 
paranucleic,  652 
paraoxyphenyl-acetic   in  urine,  734, 

735 
paraoxyphenylpropionic,  82 

in       urine, 
734, 735 
pepsin-hydrochloric,  473 
peroxyproteic;  83 
phenaceturic  in  urine,  723 
phenol  glucuronic,  222,  725,  749,  751 
phenol-sulphuric,  724,  725-727 
phenylacetic,  82 

conjugation   of   in   or- 
ganism, 783 
phenylaminoacetic,  780 
phenyl-a-aminonropionic.     See  Phe- 
nylalanine, 151 
phenylbutyric,  781 


GENERAL  INDEX 


987 


Acid  phenylcaproic,  781 

phenylketopropionic,  781 
phenyllactic,  736,  778,  780 
phenyl  oxypropionic,  781 
phenylpropiomc,  82 
phenyl  pyroracemic,  737 
phenylvaleric,  781 

in  perspiration,  848 
in  urine,  724 
phonopyrrolcarboxylic,  298 
phosphocarnic,  267,  572,  577-579 

in  active  muscles,  594 

in  urine,  757 

source    of    muscular 

energy,  598 
as    nuclein    amounts 
in  blood  of  differ- 
ent animals,  326 
in  urine,  quantitative 
estimation  of,  763- 
764 
phthalic,  fate  in  organism,  779 
physetoleic,  239 
plasm  inic,  186 
polypeptide  phosphoric,  652 
protalbinic,  127 
protic,  572 
protocatechuic,    fate    in    organism, 

726 
pseudonucleic.     See  Paranucleic  acid 
psyllic,  845 
pulmotartaric,  870 
pyinic,  366 

pyridine-carboxylic,    fate   of   in    or- 
ganism, 784 
pyridinuric,  784 
pyrocatechin-sulphuric,  724 

in  urine,  727 
pyrocholoidanic,  424 
a-pyrolidine    carboxylic.     See    Pro- 
line, 154 
pyromucic  in  urine,  784 
pyromucinornithuric  in  urine,  784 
pyromucuric,  784 
pyroracemic,  150 

effect   of   yeast   upon, 
206 
pyrrolidone-carboxylic.     See    a-Pro- 

line,  113 
pyruvic,  effect  of  yeast  upon,  206 
quinic,  719 

racemic,  fate  in  organism,  773 
renosulphuric,  673 
rhodizonic,  580 
saccharic,  198,  222 

behavior  in  diabetics,  403 
relation    to  glycogen   for- 
mation, 394 
salicylic,  conjugation  of  in  organism- 

783 
salmo-nucleic,  178 
sarcolactic,  582 

in  brain,  606 

in  lymphatic  glands,  366 


Acid  sarcolactic,  in    muscular    work    and 
rigor,  591-595 
in  the  bones,  556 
passage  into  urine,  748 
earcomelanic,  841 
scymnol,  417 
scymnol-sulphuric,  417 
sebacic,  236 
silicic,  in  blood,  268 
in  bones,  555 
in  connective  tissue,  549 
in  feathers  and  hair,  839 
in  hen's  eggs,  632,  637,  639 
in  urine,  769 
skatol  acetic,  82,  156 
skatolaminoacetic,  156 
akatol-carboxylic  in  urine,  733 
skatoxylglucuronic,  222,  732,  750 
skatoxyl-sulphuric,  724,  732,  733 
origin  of,  732 
stearic,  232 

properties  of,  235 
succinic,  83 

in  fermentation  of  milk,  645 
in  intestines,  513 
in  perspiration,  848 
in  spleen,  370 
in  thyroid,  373 
in  transudates,  355,  359, 361 
in  urine,  749,  773 
sulphosalicylic  as  a  reagent,  790 
sulphuric,  action  on  peptic  digestion, 
469.     See  also  Ethereal  sulphates 
and  Mineral  substances, 
tannic,  fate  of  in  organism,  785 
tartaric,  relation    to    glycogen    for- 
mation, 394 
in  organism,  773 
in  perspiration,  848 
tartronic,  704 
taurocarbamic,  776 
taurocholic,  417,  421-422 

preparation  of,  421,  422 
properties  of,  422 
taurocholeic,  422,  423 
tetraoxyaminocaproic,  548 
therapinic,  237 
thiolactic,  150 
thiophenuric  in  urine,  784 
thymic,  179 

thymonucleic,  groups,  181 
thiolactic,  85 
toluic,  conjugation  of  in  organism, 

783 
toluric,  synthesis  of  in  organism,  783 
triehlorethylglucuronic.      See    Uro- 

chloralic  acid, 
triticonucleic,  178,  185 
2-,  4-,  S-trimethvlpvrrol-S-propionic, 

429 
trioxy  benzoic.     See  Gallic. 
turpentine  glucuronic,  785,  817 
tyrosinesulphuric,  153 
uraminobenzoic,  783 


988 


GENERAL  INDEX 


Acid  uram in-salicylic,  783 
urea  glucuronic,  750 
uric,  187,  267 

amounts  formed  in  organism, 

707 
as  a  pigment,  844 
effect  of  food  on  elimination  of, 

700 
endogenous  origin,  702 
exogenous  origin,  702 
fate  of  in  organism,  705,  706 
formation  of  from  purines,  701, 
702 
in  birds,  703,  704 
of  in  the  organism, 

701-707 
from  lactic  acid,  704 
in  blood,  334 
in  muscles,  572 
in  urinary  sediment,  830 
in  urine,  698-712 
occurrence  of,  699,  700 
preparation  from  urine,  710 
preparation  of,  698 
properties  of,  698-699,  707,  708 
quantitative  estimation     of   in 
urine,  710 
relation  to     urea 
elimination,  701 
quantity  in  various  urine,  699, 

700 
synthetic  formation  of,  in  or- 
ganism, 705 
tests  for,  709 

various  factors  effecting  elim- 
ination, 700-702 
Urocanic,  758 
urochloralic,  777 
uroferric  in  urine,  752,  755 
uroleucic  in  urine,  736,  739 
uronitrotoluolic  in  urine,  786 
uroproteic  in  urine,  754 
uroxanic,  699 
ursalicylic  in  urine,  783 
ursocholeic,  427 
valeric,  80,  142 
vanillinic,  780 
whey,  645 

xanthobilirubinic,  429 
xanthopyrrolcarboxylic,  299 
yeast-nucleic,  179,  185 
Acids,  amino,  92 

aromatic,  fate  of  in  organ- 
ism, 780-783 
investigations  on, 
of  demolition  in 
organism,  780- 
783 
as  acetone  formers,  819 
as  carbon  dioxide  binders, 

866,  856 
conjugation  with,  786 
deamidation,  682 
fate  of,  in  organism,  774-776 


Acids,  amino,  formation  in  tryptic  diges- 
tion, 507  ; 
of,  in  liver,  530 
how  absorbed,  528 
in    globin    of    blood    pig- 
ments, 289 
in  homogentissic  acid  for- 
mation, 736-738 
in  liver  tissue,  383 
in  lymphatic  glands,  366 
in  muscles,  572 
in    transudates    and    exu- 
dates, 355 
in  serum,  267 
in  urine,  756,  827 
investigation  on  the  demo- 
lition   of,    in    organism, 
774-776 
of  pseudomucin,  626 
Sorensen's,  formol  titration 

for,  139-167 
synthesis  of,    in   organism, 

786 
sugar    formation    from,    in 

liver,  412 
transformation      of,      into 
urea,    in    the    organism, 
683,684 
aromatic,  fate  of,  in  organism,  784 

-oxy,  in  urine,  733,  734 
bile,  267 

detection  of,  418,  419,  427,  428 
biliary,   properties   of   alkali  salts, 

418 
caseonphosphoric,  652 
cholic,  preparations  of,  426 
conjugated    glucuronic    in    urine, 

817-818 
desaminoproteic,  83 
diamino,  84,  159-163 
ethereal  sulphuric,  723-733 

amounts     in 

urine,  724 
in  urine,  765 
quantitative  es- 
timation    of, 
726 
synthesis         in 
liver,  381 
excitants  for  bile  secretion,  416 
fatty,  232-239,  265 

amounts  in  blood  of  differ- 
ent animals,  328 
in  brain,  605 
in  blood,  334 
in  perspiration,  848 
in  pus,  365,  366 
in  urine,  748,  773,  829 
investigation  on  the  demol- 
ition of,  in  organism,  773, 
774 
series,  fate  of,  in  organism, 
773,  774 
glucuronic,  conjugated,  222 


GENERAL   INDEX 


989 


Acids,  glycocholie,  preparation  of,  421 

in   large    intestine,  from  putrefac- 
tion, 515 
in  spleen,  370 
in  thymus,  368 
in  thyroid  gland,  373 
in  transudates  and  exudates,  355 
kyroproteic,  83    ' 
lactic,  582-586 

detection  of,  585 
in  bones,  556 
in  urine,  703,  704,  748 
mercaptic,  elimination  of,  786 

properties  of,  585 
melanoidic,  841 

mineral,  alkali-removing  action  of, 

and  action  on  the  elimination  of 

ammonia,  675,  676,  762,  768,  856 

893 

monoamino.     See  Acids,  Amino. 

neutralization  of,  in  the  organism, 

675,  676 
nucleic,  175,  177-186 
complex,  181 
effect  of  gastric  juice  on, 

473 
enzymotic,  cleavage  of,  508 
plant,  185-186 
simple,  181 
organic,  behavior  in  animal  body, 

767,  772,  773,  776,  777 
oxyamino,  79,  80,  84,  148,  219 
oxyfatty,  in  animal  fat,  232,  238 
oxy,  in  urine,  detection  of,  735 
oxypropionic,  582 
oxyproteic    in    urine,    quantitative 

estimation  of,  755,  751} 
phthalic,  fate  of,  in  the  organism, 

77S 
proteic  in  blood,  267 
in  serum,  267 
in  urine,  752,  754 
thymonueleic,  178 
thymua-onucleic,  173 
uramino,  786 
ureido  glucuronic,  750 
volatile  fatty,  in  urine,  748 
Acidosis,  820,  821 
A. Tire,  212 
Acrolein,  234,  237 
a-aerose,  212 
/3-acrose,  212 
Actinioehrom,  844 
Activators.  60 

Adamkiewicz-Hopkin's  reaction  for  tryp- 
tophane, 157 
Adamkiewicz'a  reaction  for  protein,  100 
Adaptation  of  the  glands,  454,  462,  463, 

494-496 
Addison's  disease,  377,  379 
Adelomorphic  cells,  489 
Adenase,  48,  188 

Adenine,  178,  181,  185,  187,  188,  192,  193, 
712 


Adenine-hexose  compound,  180 
Adenosine,   180 

Adialyzable  bodies  in  urine,  757 
Adipoeere,  560 
Admissibility,  theories  of,  9 
Adrenalin,  378-380 

bodies,  377,  378 

constitution  of,  378 

function  of,  379,  380 

properties  of,  379 

tests  for,  379 
"  Adsorpates,"  12 
Adsorption,  49,  62,  63,  69 

in  relation    to    permeability, 
11,  12 
J^agrophiUe,  524 
/Erotonometric  method,  864 
Age,  effect  upon  metabolism,  922,  924 
Agglutenins,  47,  69 
Agmatine,  162 

Air  bladder,  of  fishes,  gases  of,  867 
Alanine,  85,  106,  107,  109,  111,  113,  125, 

140,  145 
Alanylalanine,  88 
Alanvlalanineglycin,  86 
Alanylglycine,  87,  508 
Alanylleucine,  86,  508 
Albamine,  220 
Albumin,  49 

detection  of,  in  urine,  791 
Albuminates,  104,  125-127 
acid,  92,  126 

alkali,  91,  92,  125,  126,  533 
Albuminoids,  92,  112 
Albuminose,  623 
Albumins,  91,  92,  93,  102,  103,  106 

quantitative  estimation  of,  in 
urine,  71)3 

properties  of,  102,  103 
Albumoids,  preparation  of,  549 
Albuminuria,  771,  787 
Albuminoids,  92 

Albuminous  bodies,  in  general,  94-97 
Albumoid.  properties  of,  618 
Albumose,  alkali,  127 
Albumoses,  130 
Alcapton  in  urine,  727 
Alcaptonuria,  735-740 
Alcohol,  aminoethvl,  240 
cetyl,  239 
melissyl,  239 
myricyl,  239 
Alcoholase,  47,  48 
Alcoholases,  875 
Alcoholic  fermentation,  41 
Aldehydases,  875 
Aldehydes,  fate  of,  in  organism,  775,  780, 

781 
Aldoses,  197 
Alexines,  266 

Alimentary  acetonuria,  820 
glycosuria,  533 
Alizarin.'elimination  in  urine.  556,  787 
Alkali  earths.     See  Mineral  substances. 


990 


GENERAL  INDEX 


Alkaloids,  fate  of,  in  organism,  787 
Alkyl  sulphides,  846 
Allantoin,  699,  717-719 

elimination   of   during  poison- 
ing, 717,  718 
formation  of,  in  organism,  717 
occurrence  of,  717 
preparation  of,  718 
properties  of,  718 
tests  for,  718 
Alloisoleucine,  145 
Alloxan,  86 
Alloxuric  bases,  186,  712 

preparation  of,  714.     See 
Purines. 
Alveolar  air,  853 
Ambergris,  524 
Amboceptors,  69 
Ambrain,  524 
Amicrons,  19 
Amidases,  702 
Amidoguanine,  718 
Amidomj'elin,  609 
Amidulin,  227 

Amino  acids.     See  Acids,  Amino. 
Amino  aldehyde,  219 

butyrobetaine  in  urine,  787 
cerebrinic  acid,  glucoside,  612 
oxypurine.     See  Guanine, 
oxypyrimidine.     See  Cytosine. 
sugars,  219-221 
Aminophenol,  779 
Aminopurine,  191 
Aminosuccinic-acid  amide.     See  Aspara- 

gine 
Ammonia,  amounts  in  urine,  766 

detection  of,  in  urine,  768 
elimination  in  relation  to  acid 

formation,  766,  767 
importance  of  to  organism,  767 
in  blood,  334 
in  urine,  766-768 
in  venous  blood,  336 
quantitative  estimation  of,  in 

urine,  768 
transformation  of,  in  the  liver, 
767 
Ammonium     magnesium    phosphate,     in 
intestinal  calculi,  232 
in  urinary  calculi,  834 
salts,  relation     to   "glycogen 
formation,  394 
relation    to    urea  for- 
mation, 683,  767 
relation    to    uric    acid 
formation,  702 
urate,  in  urinary  calculi,  834 
in    urinary   sediment, 
829 
Amniotic  fluid,  642 
Amphicreatine,  578 
Arnygdalase,  48 
Amygdalin,  cleavage  of,  59 
Amylase,  47,  48 


Amylodextrin,  227,  229 

Amyloid,  172,  231 

preparation  of,  174 

Amylopectin,  227 

Amylopsin,  501 

Amylose,  227 

soluble,  227 

Amylum.     See  Starch,  226 

Anaphylaxis,  70 

Aniline,  fate  of  in  organism,  779 

Animal  oxidation,  42 

gum  in  urine,  749 

Anions,  5 

Anisotropous  muscle  substance,  565 

Antedonin,  844 

Antialbumate,  472 

Antialbumid,  472 

Antibodies,  66,  68 

Antidonin,  844 

Antienzyme,  63 

Antigens,  66,  68,  69 

Antiketoplastic  action,  820 

Antimony,  action  on  N-elimination,  679 

passage  into    milk,    671.     See. 
Mineral  substances. 

Antipepsin.     See  Enzymes,  464 

Antipeptone,  130 

Antipyrine,  fate  of,  in  organism,  785 

Antirennin,  69,  474 

Antithrombin,  322 

Antitoxins,  47,  66 

Antitrypsins,  503 

Anuria  in  cholera,  848 

Aorta  elastin,  116 

Apatite  in  bone  ash,  553 

Amphopeptone,  129 

Aporrhegmas,  167 

Aqueous  humor,  361 

Arabinoses,  198,  210.     Structural  formu- 
lae for,  198 

d-arabinosinine,  201 

Arabite,  198 

Arachnoidal  fluid,  355 

Arbacin,  108 

Arbutin,  394,  727 

Arginase,  48,  91,  161 

Arginine,  85,  106,  107,  108,  109,  111,  113, 
115,  117,  119,  125,  161,  162 
in  various  proteins,  165 
cleavage  into  creatine,  575 

Arginine-histone  peptone,  139 

Argon  in  blood,  850.     See  also  Gases 

Arnold    and    Lipliawsky's    reaction    for 
acetoacetic  acid  in  urine,  824 

Arnold's  reaction  for  proteins,  100 
urine  reaction,  696 

Aromatic  combinations,  fate  in  organism,. 
776-784 

Arsenic  poisoning,  439-441 

on  nitrogen  elimination,  679 

Arterin,  276 

Ascitic  fluid,  357 

Asparagine,  146 

Asparagus,  effect  on  odor  of  urine,  764 


GENERAL  INDEX 


991 


Assimilation  limit,  533 

Atheromatous  cysts,  142 

Atmidalbumin,  130 

Atmidalbumose,  130 

Atmidkeratin,  113 

Atmidkeratose,  113 

Atropine,  effect  on  uric  acid  elimination, 

700 
Auto  digestion,  43 
Auto-oxidation, 
Autolysis,  43 

as  a  protective  agent,  46 
effect  of  arsenic  upon,  44 
C02  on,  44 
in  organic  colloids,  on 

44 
oxygen  upon,  44 
radium  on,  44 
reaction  upon,  43 
importance  of  enzymes  on,  45 
various  processes  in,  44 
Autolytic  processes  in  life,  45 
Autotoxines,  influence  on  putrefaction,  529 

Bacteria,  influence  on  putrefaction,  520 

Bacterial  action  in  intestinal  canal,  518 

Bacteriolysins,  69 

Bacterium  urea?,  829 

Balsam  of  copaiba,  fate  of,  in  organism, 

787 
Bang's  method  of  estimating  sugar,  809 
Barium,  22 

Basal  requirement,  S97,  922 
Basedow's  disease,  375 
Baumann's  test  for  dextrose,  216 
Beer  vinegar  bacteria,  enzymes  of,  10 
Beeswax,  239 

Bela  v.  Bitto's  reaction  for  acetone,  823 
Bence-Jones'  protein,  792 
Benzaldehyde,  36 

Benzene  homologues,  fate  of,  in  organism, 
779 
ring,  theory  for  splitting  of,  in 
organism,  77S 
Benzidine  blood  test,  798 
Benzoylation  of  carbohvdrates,  215,  216, 

749,  808 
Benzoylchloride  test  for  dextrose,  215 
Benzoyl  cystine,  150 
Betaine,  246,  572 
Bezoar  stone,  oriental,  426,  532 
Bifurcated  air,  853 
Bile-acids,  419-428 

detection  of,  418,  419 

in    animal    fluids, 
427,  428 
formation  of,  442 
in  blood,  267,  333 
in  urine,  799-800 
orisrin  of,  440 

relation  to  bile  pigments,  444 
Bile,  413,  442 

absorption  of,  from  the  liver,  444 
amounts  secreted,  414-445 


Bile,  chemical  formation  of,  440-444 

coloring    matters,    detection    of,    in 
blood,  435 

composition  of,  in  disca.se,  43!) 
concretions,  cholesterin  stones,  445 

pigment  atones,  445 
constituents,  absorption  of,  540,  541 
constituents  of,  417,  435 
effect  of  in  absorption  of  fats,  536- 

540 
effect  on  putrefaction,  518,  519 
gases  of,  857 
Hufner's,  419 
human,  437 

composition  of,  438 
pigments,  in,  438 
properties  of,  437 
importance  in  absorption  of  fat,  511 
mucus,  415,  437 
passage  into  blood,  444 
urine,  444 
phosphorized  constituents  of,  439 
properties  of,  416,  417 
putrefaction  of,  in  intestine,  516 
quantitative  composition  of,  436,  437 
pigments,  427 

formation  of,  440-442 
in  feces,  522 
in  urine,  800,  801 
origin  of,  440,  441 
relation  to  bile  acids,  444 
relation     to     blood     pig- 
ments, 430,  442,  443 
relation    to    urinary    pig- 
ments, 743.  744 
test  for,  432,  433 
salts,  417-419 

properties  of,  418 
tests  for,  418,  419 
secretion,  cholagogues,  415 

effect  of  therapeutic  agenta 
on,  415 
Biliary  fistula,  413,  421 
Bilicyanin,  435 
Bilifulvin,  427 
Bilifuscin,  434 
Bilihumin,  435 
Biliphaein,  427 
Biliprasin,  434 
Bilipurpurin,  435 
Bilirubin,  427,  431,  516 

derivatives  of,  428-430 
detection  of,  in  blood,  435 
Ehrlich's  test  for,  429 
Hedenin's  test  for,  433 
hemi-,  429 

Huppert's  test  for,  429 
hydro-,  428 
of  bloo;l  serum.  26S 
preparation  of,  433 
properties  of,  430-432 
quantitative  estimation  of,  433 
Biliverdin,  433,  434 

in  excrements,  530 


992 


GENERAL  INDEX 


Biliverdin  in  feces,  530 
properties,  434 
preparation  of,  434 
Biological  equivalence,  917 

protein  reactions,  266,  533 
Bismuth,  passage  into  milk,  670 
Bitter  substances,  effect  upon  secretion, 

461,  482 
Biuret,  86 

base,  86 

reaction  for  proteins,  101 
Blister-fluid,  362 
Blood,  308-344 

acid  lactic,  in,  334 

alkalies  in,  310,  311 

ammonia  in,  334 

analyses    of    blood    from    various 

animals,  328 
arterial,  335,  336 

quantity    of    carbon    di- 
oxide in,  851 
quantity  of  oxygen  in,  851 
at  different  periods  of  life,, 
"  buffy  coat,"  312 
casts  in  urine,  796 
clot,  251 

coagulation,    accelerating    sub- 
stances, in  cell,  315, 
316 
calcium  salts  in,  316, 

317 
lime  salts  in,  316 
methods  of  retarding, 

312 
prevention  of,  251 
retarding     substances 

in,  312-326 
theories  for,  317,  321- 
323 
changes  in  viscosity,  311 
coloring   matters,    in   urine.     See 
also      Blood 
p  i  g  m  e  n  ts, 
795-799 
relation  to  bile 
pigments, 
442,  443 
corpuscles,  250,  272-305-308^ 
constituents  of,  274 
determination    of    vol- 
ume   in   blood,    326, 
327 
effect  of  water  on,  6 
aalte  on,  6 
experiments  with,  6 
ha;maKKlutination,  275 
haemoglobin,  274,  276 
haemolysis  of,  273 
in  lymph,  346 
isolation  of,  274 
mineral  bodies  in,  304 
number  of,  272 
size  of,  272 
non-nucleated,  250 


Blood-corpuscles,  nucleated,  250 

protection  of  salts  upon, 

6 
quantitative     constitu- 
tion of,  304 
quantitative     determi- 
nation of,    326,  327, 
328 
red,   osmotic    phenom- 
ena with,  8 
stromata,  274 
stroma-fibrin,  275 
stroma,  272,  273,  274 
white,  250,  305-308 

number  of,  305 
constituents    of, 
306-308 
defibrinated,  252 
degree  of  dissociation  in,  271 
determination  of  reaction,  271,  272 
form  elements  of,  272-276 
detection  of,  in  urine,  796 
determination  of  H  ions  in,  310 
during  pregnancy,  337 
effect    of    alkalinity    on    carbon- 
dioxide  content,  856 
electrical  conductivity,cleavages  in, 
enzymes  in,  53,  332 
fat  in,  334 
fatty  acids  in,  334 
from  muscular  veins,  337 
from  veins  of  glands,  337 
gas  exchange  in,  858-870 
gases  of,  857 
gas  tension  in,  859-868 
hepatic  vein,  from  the,  336 
human,  analysis  of,  328 
importance  of  haemoglobin  in  oxy- 
gen-carbon dioxide  exchange  in,. 
853 
influence  of  food  on,  338 
injection  of,  343,  344 
in  urine,  795-799 
laky,  312 
leucsemic,  properties  of,  342 

constituents  of,  342 
leucocytes,  increase  in  number  of, 

342 
manner     of     binding     of     carbon 

dioxide  in,  853,  854 
menstrual,  337 
mineral  substances  in,  335 
non-coagubility  of  circulating,  the- 
ories for,  319,  320 
of    various    animals,    analysis    of,, 

328 
of  the  two  sexes,  337 
of  woman,  analysis  of,  328 
oxidation  in,  858 
pigment  arterin,  276 
pigments,  276-305 

acid  haematinic,  296 
acid  hemoglobin,  286 
carbohsemoglobin,  288 


GENERAL  INDEX 


993 


Blood  pigment -<,  carbon     dioxide -haemo- 
globin, 288 

carbon   monoxide-haemo- 
globin, 286,  287 
carbon-monoxide  methae- 

moglobin.  287 
ehlorocruorm,  303 
cryptopyrrol,  297,  299 
cyanluemoglobin,  2N5 
cyanmethsemoglobin.  285 
decomposition,    products 

of,  288,  289 
detection  of,  286,  287 
formation  of,  286 
globin,  289 

properties  of,  289 
haematin,  277,  290-292 
formula  for,291 
preparation  of, 

291 
properties     of, 

291 
spectroscopic 
action  of ,292 
haematin,   reduced,   289 
haematinogen,  300 
haMiiatocrystallin,  278 
haematoglobulin,  278 
haematoidin,  301 

relation    to 
bile      pig- 
ments, 301 
haematoporphyrin,  277 
haematoporphyrin,    spec- 
troscopic   examination 
of,  300 
haematoporphyrin,    prep- 
aration of,  300 
haematoporpbvrin,     pro- 
duction of,  294-301 
haematoporphyrin,      for- 
mula for,  294 
haematoporphyrin,     rela- 
tion to  haematin,  294 
haematoporphyrin,     rela- 
tion to  bile  pigments, 
295 
haematoporphyrin,       be- 
havior in  animal  body, 
295 
haematoporphryin,     rela- 
tion to  plant  pigments, 
296 
haemin,  292-294 

formula  for,  292 
properties  of,  293 
preparation      of, 
293 
ha?machromogen,2S9,  290 
haemerythrin,  303 
haemin  crystals,  292 

prepara- 
tion, of, 

293 


Blood  pigments,  hacmochrom,  276 
haemocvanin,  303 
haemoglobin,  274,  276 
haemoglobin,       composi- 
tion of,  277 
hemoglobin,     molecular 

weight  of,  278 
haemoglobin,  gas  combin- 
ing ability,  280 
haemoglobin,        prepara- 
tion   from    oxyhaemo- 
globin,  283 
haemoglobin,       quantita- 
tive determination  of, 
301 
haemoglobin  Hoppe-Sey- 
ler's  colonmetric  meth- 
od, 301 
haemoglobin,        reduced, 

282 
haemoglobin,         spectro- 
scopic        quantitative 
method  for,  302 
haemopyrrol,        297, 

299 
hsemorrhodin,  288 
haemochromogen,  276 
haemoverdin,  288 
in  urine,  795-799 
isohaemopyrrol,  297 
kathaemoglobin,  288 
mesoporphyrin,  294, 

300 
methaemoglobin,        283- 

285 
methaemoglobin,   proper- 
ty of,  284,  285 
methaemoglobin,     prepa- 

aration  of,  285 
nitric  oxide-haemoglobin, 

288 
neutral  haematin,  288 
phlebin,  276 
phonoporphyrin,  295 
phvllohaemin,  296 
phyllopyrrol,  298,  299 
phylloporphyrin,  296 
porphyrinogen,  295 
oxyhaematin,  290 
oxyhaemocyanin,  303 
oxyhaemoglobin,       prop- 
erties of.  280,  281 
parahaemoglobin,  281 
parahaemoglobin,      prop- 
erties of,  281 
photomethsmoglobin, 

285 
purple  cruorin,  282 
quantitative     estimation 

of.  301-304 
sulphhaemoglobin.  287 
sulnhur  methaemoglobin, 

287 
tetronerytl'.rin,  303 


994 


GENERAL  INDEX 


Blood  plasma,  250,  252-264 

analysis  of,  269 
plates,  250,  308 

in  coagulation,  314 
poikillocytosis,  342 
portal  vein,  from  the,  336 
quantitative  composition  of,  326- 
335 
estimation  of  urea  in, 
•691 
quantity  of,  343 

bleeding  of,  343 
in  organs,  344 
reaction  of,  76,  309-311 
red  corpuscles,  decrease  in  number 
of,  341 
effect  of  hemorrhage 

on,  341 
effect  of  transfusion, 

340 
effect    of   transuda- 
tion from,  340 
effect  of  pressure  on, 

340 
increase  of,  340 
increase  of,  theories 
for,  341 
refraction  coefficient  of  serum,  311 
rennin  acting  enzyme  in,  474 
serum,  acids  in,  267 

action  of,  on  starch,  226 
analyses  of,  269 
constituents  of,  264-269 
properties  of,  264 
rest  in  nitrogen,  267 
pigments  of,  268 
quantitative      analysis      of 

mineral  bodies,  270,  271 
specific  gravity,  determina- 
tion of,  309 
spleenic  vein,  from  the,  336,  337 
"  sucre  virtual,"  331 
"  sucre  immediat,"  331 
urea  in,  333,  334 
uric  acid  in,  334 
vascular    regions,    composition    of, 

335-344 
venous,  335,  336 

quantity   of   carbon   diox- 
ide in,  851 
quantity  of  oxygen  in,  851 
Blueberry,  pigments  of,  in  urine,  787 
Blue  milk,  671 
Blue  stentorin,  844 
Boar  sperms,  622 

Boas'  test,  for  lactic  acid,  in  gastric  juice, 
487 
for  HCL,  486,  487 
Body,  relation  of  weight  and  age  of,   to 
absolute   consumption   of  mate- 
rial, 913 
weight  decrease  during  starvation, 
885 
Boiling-point,  elevation  of  by  colloids,  17 


Bombicesterin,  449 

Bondi-Schwarz's  test  for  acetoacetic  acid, 

824 
Bone,  551-558 

ash,  analysis  of,  553 
at  different  ages,  555 
catabolism  in  starvation,  896 
components  of,  551 
earth,  analysis  of,  552 
effect  of  food  upon,  556 
marrow,  554 
rachitic,  557 
rachitis  of,  555 
softening  of,  555 
Bonellin,  844 

Bony  structure,  matrix  of,  551 
Borneol,  fate  of,  in  organism,  785 
Bottcher's  spermine  crystals,  621 
Bottger-Almen's  test,  215,  802 
"  Bowman's  disks,"  566 
Boyle-Marriot's  law,  28 
Bradoxidizable  substances,  4 
Brain,  constituents  of,  604-606 
epileptic,  analysis  of.  613 
gray   and    white   substance,    com- 
pared, 605 
human,  analysis  of,  612-613 
paralytics,  analysis  of,  613 
phosphatides  of,  606-609 
quantitative  composition  of,  612 
British  gum,  229 
Bromhaemin,  294 
Bromides,  in  saliva,  459 

relation   to  formation   of  gas- 
tric juice,  477 
Bromine,  22,  123 

Bromoform,  behavior  in  animal  body,  775 
Bromthymine,  195 
Bromtoluene,  behavior  in  animal  body, 

775 
Brownian  molecular  motion,  20 
Briicke's    quantitative   method   for   pep- 
sin, 469 
Buccal  mucus,  453 
"  Buffy  coat,"  312 
Bufidin,  846 
Bufonin,  846 
Bufotalin,  846 
Bufotenin,  846 
Bufotin,  846 
Bunge    and    Schmeideberg,    quantitative 

method  for  hippuric  acid,  723 
Burbot,  sperms  of,  181 
Bursae  mucosa;,  361 
Butalanine,  495 
Butter,  647 

Butterflies,  pigments  of,  844 
Butylmercaptan,  844 
Butyrinase  in  blood,  266 
Butvrine,  mono  enzymotic  synthesis  of,  60 
Byssus,  92,  122,  123 

Cadaver  alkaloids,  82 
Cadaverine,  47,  82,  164 


GENERAL  INDEX 


995 


Cadaverine  in  urine,  827 
Caecum,  514 
Caffeine,  187 

Calcium  carbonate  in  urinary  sediment, 
831 
importance  to   enzymotic   proc- 
esses, 255,  256,  307,  312,  313, 
498,  649.  650 
in  urine,  768(769 
manner  of  excretion,  769 
oxalate  in  urinary  sediment,  830 
phosphate  in  urinary  sediment, 

831 
salts  in  blood  coagulation,  308, 

309 
sulphate   in   urinary   sediments, 
831.     See   also    Mineral   sub- 
stances. 
Calculi-ammonium  urate,  833 
-calcium  carbonate,  834 

oxalate,  833 
-cystin,  834 
-fibrin.  834 
Heller's   scheme   for  investigating, 

833 
hemp  seed,  833 
intestinal,  531 
-mulberry,  833 
pancreatic,  509 
-phosphate,  833 
salivary,  459 
-uric  acid,  833 
urinary,  828-829,  832-836 
urinary,  scheme  for  chemical  anal- 
ysis of,  836 
urinarv,  chemical  investigation  of, 

834-836 
-urostealith,  834 
-xanthine,  834 
Caliphora  larvae,  fat  formation,  561 
Calomel,  effect  upon  excrement,  529 
Calorific  coefficients,  888 
value  of  fat,  888 
value  of  proteins.  888 

milk  protein,  888 
starch,  887 
urine  quotient,  884 
Cammidge's  reaction  for  sugar,  815 
Camphors,  fate  of,  in  organism,  000 
Cane-sugar,  49,  224.     See  Sucrose. 
Canirine,  578 
d-Caprine,  144 

Capronica's  test  for  guanine,  191 
Capsule  of  crystalline  lens,  170,  617 
Caramel,  214,  225 

Carbamino  reaction,  Siegfried's,  166 
Carbazole,  fate  of.  in  organism,  779 
Carbohaemoglobin,  287 
Carbohydrate  (phosphatized),  244'. 
Carbohydrates,  absorption  of,  532-534 

as  a  source  of  muscular 

energy,  598 
benzoyl  esters  of,  202 
classification  of,  197 


Carbohydrates,  cyanhydrin  synthesis  for, 
200 
effect  of  gastric  juice  on, 

473 
ether-iike     combinations, 

202 
fate  of,  in  organism,  885 
fermentation  of,  203-207 
hydrazones,  202-203 
in  urine.  749-752 
in  venous  blood,  336 
osazones,  202-203 
phosphoric  acid  esters  of, 

204-205 
relation  to  histidine  and 

purines,  202 
See  Various  sugars. 
Carbolic  urines,  728 

Carbon  dioxide,  acids,  amino  as    binders 
of,  855,  856 
effect    of    alkalinity    on 

content  in  blood,  856 
formation,  calculation  of, 

889 
haemoglobin,  287,  853 

as  a   binder 
of,  853 
influencing    oxygen    ab- 
sorption, 859 
manner    of    binding    in 

blood,  853-854 
mechanism    of    elimina- 
tion, 852-853 
physical    explanation    of 

the  giving  up  of,  866 
proteins  as  binders  of,  855 
quantity-  in  arterial  blood, 
851 

venous  blood 
851 
tension,  865 
tension  in  tissue,  868 
Carbon,  elimination  in  organism,  883-884 
monoxide  blood,  test  for,  286 

haemochromogen,  288 
haemoglobin,  279,  285, 

288,  300 
methaemoglobin,  286 
poisoning,     285,     402, 
582,  679 
Carboxylase,  206 
Carnaubon,  240,  673 
Carniferrine,  578 
Carnine,  572,  577,  712 
Carnitine,  572,  577 
Carnomuscarine,  578 
Carnosine,  572,  576-577 
Carotin,  631 
Cartilage,  546-550 

components  of,  540 
constituents  of,  549-550 
gelatin,  549 

hyalin,  action  of  trvpsin  upon, 
508 


996 


GENERAL  INDEX 


Caseid,  649 

Casein,  49,  84,  91,  106,  647-652 
cleavage  products,  of,  106 
coagulation  of,  649 

a  two-faced  proc- 
ess, 650 
theory,  of,  650,  651 
composition  of,  647 
properties  of,  647,  648 
hexone  bases  in,  165 
human,  preparation  of,  652 
of  woman's  milk,  661 
peptic  digestion  of,  651,  652 
preparation  of,  652 
solutions  of,  properties  of,  649 
Caseinates,  648,  649 
Caseinokyrin,  136 
Caseoses,  130 
Castoreum,  846 
Castor  lipase,  234 
Castorin,  846 

Catabolism  of  protein,  907,  908,  909,  911 
Catalases,  43,  872 

definition  of,  33 
equilibrium  constant  of,  33 
in  heterogeneous  systems,  36 
mass  action  on,  32 
measurement  of,  32 
of  benzaldehyde,  36 
of  diaceton  alcohol,  35 
of  diazoacetic  ether,  34 
of  esters,  32 

of  hydrogen  peroxide,  35 
reaction  velocity  of,  32 
velocity  coefficient,  34 
function  of,  7 
Catalysts,  35 
Catalytic  processes,     compared     (organic 

and  inorganic),  37 
Cataphoresis,  20,  50 
Cataract  of  lens,  619 
Cations,  5 
Cell-globulin,  274 

-membrane,  animal  effect    of    gastric 
juice  on,  473 
of  plant,  effect  of  gastric 
juice  on,  473 
Cells,  adelomorphic,  461 

boundary  layer  of,  21 
<cover,  46l 
delomorphic,  461 
lymphoid,  300 
mineral  constituents  of,  22 
pepsin,  461 
permeability  of,  34 
rennin,  461 
Cellobiose,  231 
Cellose,  231 
Cellulose,  231 

fermentation  of,   in  the  intes- 
tine, 511 
Celluloses,  226 
Cement,  of  teeth,  557 
Cephalin,  248-249,  605 


Cephalopoda,  flesh  of,  572,  604 
Cerebrin,  607,  609-610 

in  pus-corpuscles,  364,  365 
Cerebron,  605,  609,  611-612 

cleavage  products  of,  611 
preparation  of,  611 
properties  of,  611 
Cerebrosides,  605,  607,  609 
Cerebrospinal  fluid,  360-361 
Cerolein,  239 
Cerumen  of  skin,  845 
Cetin,  238,  239 
Cetyl  alcohol,  239,  627,  845 
Chalaza,  634 
Charcoal  bone,  ability  to  absorb  trypsin, 

703 
Charcot's  crystals,  871 
Charcot-Leyden  crystals,  621 
Chemical  processes,  plants  and  animals, 

37 
"  Chemical  tonus,"  591 
Chief  cells.     See  Adelomorphic  cells. 
Chitin,  122,  839,  839-840 
Chitosamine,  219,  626 
Chitosan,  168,  840 
Chitose,  221 
Chloral  hydrate,  fate  of,  in  organism,  777 

secretin,  415,  500 
Chloramine,  40 
Chlorbenzene,  behavior  in  animal  body, 

786 
Chlorides.     See  Mineral  substances, 
of  urine,  758-761 
in  urine,  quantitative  estima- 
tion of,  759 
quantity  of,  759 
Chlorine,  in  teeth,  558 
in  blood,  333 
Chlorochrome,  384 
Chlorocruorin,  299 

Chloroform,  behavior  in  animal  body,  760, 
775 
influence  upon  elimination  of 

chlorine,  760 
influence  upon  muscles,  591 
influence  upon  protein,  96 
Chlorometer,  762 
Chlorophan,  617 
Chlorophyl,  38,  844 
Chlorosis,  340 
Chlortoluene,  behavior  in  animal  body, 

783 
Cholagogues,  415 
Cholecyanin,  430 
Choleprasein,  434 
Cholepyrrhin,  427 

Cholera  bacilli,  behavior  toward  gastric 
juice,  485 
blood,  334 
perspiration,  848 
Cholestanol,  a  and  /3,  446 
Cholestenon,  443 
Cholesteriline,  445 
Cholesterin,  10,  265,  613 


GENERAL   INDEX 


997 


Cholesterin,  amounts  in  blood  of  different 
animate,  328 

constitution  of,  445 

derivatives  of,   146 

ester,  264,  360,  444,  445,  613. 

844 
importance  of.   149 
in  ascitic  fluid,  359 
in  chyle,  347 
in  lymphatic  glands,  366 
in  lymph,  348 
in  pus  corpuscles,  364 
in  pus  serum,  363,  365 
in  spleen,  370 
occurrence  of,  446 
of  sebum  of  skin,  845 
preparation  of,  450 
properties  of,  446 
tests  for,  447-448 
Cholesterone,  445 
Choline,  240,  246-248,  607 

in  cerebrospinal  fluid,  360 
in  thyroid,  373 
occurrence  of,  247 
preparation  of,  247 
properties  of,  247 
Cholohaematin,  435 
Chondrigen,  546 
Chondrin,  121 
Chondrin-balls,  549 

Chondroalbuminoid,  elementary  composi- 
tion of,  551 
Chondroproteins,  168  ,172-174 
Chondromucoid,  172,  546-547 

elementary    composition 

of,  551 
preparation  of,  548 
Chondroitin,  547 
Chondrosin,  171,  547 
Chorda  saliva,  448 
Choroid  coat,  619 
Chromaffine  tissue,  378 
Chromhidrosis,  849 
Chromogens.     See  Urinary  pigments. 
Chromoproteins,  92,  93,  167 
Chyle,  quantitative  composition  of,  346- 

347 
Chyluria,  827 
Chylous  ascites,  358 
Chyme,  478 
Chymosin,  474 
Ciamician  and  Magnanini's  reaction  for 

indol,  158 
Circulating  proteins,  908-910 
Cleavage,  hydrolytic,  16 

processes,  15 
Clupeine,  110 
Coagulation,  intravascular,  324 

method  for  quantitative  pro- 
tein in  urine,  793 
Coagulins  of  blood,  320,  321 
Coagulose,  59 
Coagu  loses,  135 
Coapeptides,  136 


Coaproteoscs,  130 
Cobra-poison,  310,  320 

Cochineal,  s  J  \ 
Cocosite,  581 
Codfish,  eggs,  630 

sperms,  107 
Co-enzymes,  60 

Coffee,  action  on  metabolism,  912 
Coilin,  92 
Collagen,  92,  549,  551 

analysis  of,  118 
in  lymphatic  glands,  366 
preparation  of,  118,  121 
properties  of,  119 
Colloid,  171,  624,  625 

cysts,  624 

effect  of  charge  upon,  23 

effect  of  various  ions  upon,  22 

envelope,  45 

from  uterine  fibroma,  627 

substances,   non-permeability  of, 
9 

suspension,  electrolytic  precipita- 
tion, 21 
"  Colloidal  nitrogen,"  795 

substances,  preparation  of.  14  ' 
Colloids,  49 

adsorption,  27-30 

boiling-point,  elevation  of,  17 

character  of,  14 

classification  of,  15 

diffusion  of,  18 

disperse  phase,  27 

dispersion  means,  27 

effects  of  the  different  ions,  25 

electrical  transportation  of  sus- 
pended particles,  20 

electrolyte  precipitation  of,  24 

emulsion,  15 

examples  of,  14 

filterability  of,  17 

freezing-point,  depression  of,  17 

hydrophile,  15 

in  relation  to  surface  tension  and 
adsorption,  24 

irreversible,  21 

migration  of  to  poles,  21 

molecular  movement  of,  20 

optical  properties  of,  19 

precipitation  of,  21 

precipitation    phenomena,    the- 
ories of,  25 

protective,  23 

relationship  to  crystalloids,  14 

relative  size  of,  18 

reversible,  21 

suspension,  15 

suspension,  precipitation  of,  70 

Tyndal's  phenomenon,  19 

ultra  microscope,  use  of  in,  19 
Colon,  effect  of  extirpation  of,  549 
Coloring  matter.     See  Pigments. 
Colostrum,  658 

composition  of,  658 


998 


GENERAL  INDEX 


Colostrum  of  woman's  milk,  665 
Combustion,  physiological  heat  of,  879 
Complements,  69 
Conalbumin,  633 
Conchiolin,  122,  123 

Concrements,   intestinal,    523,    524.     See 
also  Calculi, 
prostatic,  623 
Concretions  of  lungs,  871 
Conglutin,  878,  879 

Conjugated  glucuronic    acids,    fate   of   in 
s  organism,  777 

sulphuric    acids,    fate    of    in 
organism,  777 
Connective  tissue,  analysis  of,  545 

components  of,  544 
fibrils  of,  545 
Copper,  occurrence  in  blood,  268,  333 
in  bile,  416,  433,  440 
in  liver,  387 
in  pigments,  299 
Cornea,  550 

Corneal  mucoid,  elementary  composition 
of,  551 
tissue,  619 
Cornein,  122,  123-124 
Cornicrystallin,  123 
Corpora  lutea,  623 
Corpse-wax,  560 
Corpus  callosum,  613,  614 
Corpuscles,  blood.     See  Blood  corpuscles, 
colloid,  624 
colostrum,  645 
Gluge's,  624 
Corpuscula  amvlacea,  612 
Cover  cells,  456,  476,  478 
Crab  extract,  576 
Crangitine,  578 
Crangonine,  578 
Creatine,  267 

detection  of,  576 
formation  of,  in  active  muscles, 
594 
in  organism,  787 
in  ascitic  fluids,  359 
in  urine,  692 

mother  substance  of,  574 
origin  of,  574 
preparation  of,  573,  576 
production  from  arginine,  575 
properties,  575-576 
relation  of  to  creatinine,  692-693 
relation   to  catabolism   of  pro- 
tein, 574 
Creatinine,  572,  573r576 

elimination,  effect   of   disease 
upon,  694 
effect  of  muscular 
activity    upon, 
694 
effect   of   starva- 
tion upon,  694 
Folin's    folorimetric    method 
for,  697 


Creatinine,  formation  of,  in  active  mus- 
cles, 594 
mother  substances  of,  693 
preparation  of,  697 
properties  of,  695,  696 
quantitative  estimation  of,  697 
quantity  of,  in  urine,  692 
relation  of,  to  creatine,  692- 

693 
-zinc  chloride,  695 
Crenilabrine,  110 
Crenilabrus  pavo,  844 
Croners-Conheim's  test  for  lactic  acid  in 

gastric  juice,  488 
Crude  fibre,  digestion  of,  549 

silk,  121,  122 
Cruor,  252 
Cruorin,  purple,  282 
Crusocreatinine,  578 
Crustaceorubin,  844 
Crusta  infiammatoria,  306 

phlogistica,  306 
Cryptopyrrol,  430 
Crystalbumin,  618 
Crystalfibrin,  618 
Crystallin,  alpha  and  beta,  properties  of,, 

618 
Crystalline  lens,  617-619 
Crystalloids,  13 
Crystals,  Charcot-Leyden,  621 
Cuorin,  249 

Curare,  348,  398,  588,  920 
Cyanhaemoglobin,  284 
Cyanhydrins,  formation  of,  200 
Cyanmethaemoglobin,  284 
Cyanocrystallin,  637,  844 
Cyanurin,  741 
Cyclopterine,  110 
Cymene,  fate  of,  in  organism,  779 
Cyprinine,  110 

hexone  bases  in,  165 
Cysteine,  80,  85,  148.  150,  619 
Cystic  fluid,  protein  bodies  in,  626 
Cystine,  80,  85,  100,  107,  113,  114,  115,. 
116,  125,  148-150 
in  urine,  827-828 
preparation  of,  828 
protein-.  148 
stone-,  148 
Cystinuria,  827,  828 

Cysts,  characteristic  constituents  of.  624 
colloid,  624 
dermoid,  627 
intraligamentary,  627 
myxoid,  624 
papillary,  627 
parovarial,  627 
proliferous,  624 
serous,  624 
tubo-ovarial,  627 
Cytidine,  180,  185 
Cytin,  368 

Cvtoglobin,  307,  315,  366 
Cytosine,  178,  181,  185,  193-194 


GENERAL   INDEX 


999 


Cytosine,  detection  of,  194 
Cytotoxin,  69 
Cytozym,  319 

Deamidation,  411.  530,  582,  702,  774 
Dehydrochloride  nsemin,  293 
Dehydrocholon.  418,  423 
Deniges's  test  for  tyrosine,  154 
Dentin,  553 
Dermocerin,  844 
Derrnolein,  844 
Desamidoprotein,  78 
Descernet's  membrane,  171,  551,  018 
Desoxyha-matoporphyrin,  291 
Deutercaseoses,  129 
Deuteroelastose,  117 
Deuteromyosinose,  130 
Deuteroproteose,  129 
Deuterospongenose,  122 
Deuterovitellose,  130 
Development,  work  of,  042 
Dextrin,  229 

hydrolytic  cleavage  products  of, 
229 
Dextrins,  220 

Dextrose,  212.     See  Glucose. 
Diabetes,  duodenal,  400 
mellitus,  403 

acetone  bodies  in,  818 
pancreas,  artificial,  405 

relat  ion    to    adrenals 
and  thyroids,  400 
phlorhizin,  400 

sugar  eliminated   in,  origin  of, 
409-411 
Diabetic  sugar,  212 
Diaceton  alcohol,  35 
Diamine,  24,  131,  103,  757,  928 
Dialysis,  14 
Diarginylalanine,  111 
Diarginylproline,  111 
Diarginylserine,  111 
Diarginylvaline,  111 
Diastase.     See  Enzymes,  48,  04. 

action  of,  on  starch  paste,  226 
Diazca^etic  ether,  34 

Diazohenzenesulphonic-acid  test  for  dex- 
trose, 210 
Diazo-reaction,  Ehrlich's,  755 

for  histidine,  101 
Dibenzoylornithine,  161 
Diet,  average  daily  adult,  923 

for  people  in  different  vocations,  924, 
927 
Diffusion,  1 

of  colloids,  18 
streams,  5 
Digestion,  effect    of   extirpation    of  pan- 
creas upon,  531-532 
gastric,  effect  of  fats  on,  481 
in  the  stomach,  478-489 

time  of,  481 
movement  of  food  in  stomach 
during,  479-480 


Digestion,  movements  of  stomach  during, 

1 7'.  i 
peptic,  132 

Digestion-work,  930-931 
Diglucosamine,  220 
Diglycyl-gfycine,  85 
Dihydrocholesterin,  443,  446 
1  K-isobutyldiacytpiperazine,  142 
Di-iodotyrosin,  123 
Di-leucyl-cystine,  85 
Di-leucy  1-glycyl-glycine,  85 
Dimethylaminobenzaldehyde,  fate  of,  in 

organism,  786 
Dimethylfulvene,  7 
Dimethylguanidine  in  urine,  758 
Dimethylindol,  729 
Dimethylketone,  822 
Dimethyltoluidine,  fate  of,  in  organism, 

786 
Dimethylxanthine  (1,  7),  713 
Dioxyacetone,  205 
Dioxybenzene,  778 
o-Dioxybenzene.  727 
yj-Dioxybenzene,  728 
Dioxymethylene  creatinine,  574 
Dioxynapththaline,  778 
Dioxypurine,  188 
Dioxypyrimidine.  220 
Dipalmitylolein,  233 
Dipentosamine,  220 
Dipeptides,  85-89 
Diphosphates,  239 
Disaccharides,  223-226 
Disperse  phase,  47 
Dispersion  means,  47 
Dissociation,  degree  of,  5 
Di-stearyl  lecithin.  148,  241,  242 
Di-stearylolein,  233 
Di-stearyl-palmatin,  233 
Di-tetraoxybutylpyrazine,  220 
Dithiopiperidine,  88 
Donne's  pus  test,  798 
Dotterplattchen,  93,  628,  638 
Dulcite,  198 
Dye    stuffs,     behavior    of    living    cells 

toward,  10 
Dyslvsins,  427 
Dysoxidizable  substances,  4 
Dyspeptone,  472 
Dysproteoses,  129 

Ear,  fluids  of  the  inner,  619 

Earthy      phosphates.      See    Phosphates. 

earthy 
Echinochrom,  299 
Echinoooccus  cysts,  fluid  of,  362 
Eck'e  fistula  operation,  398,  537,  542,  680 
Edestan,  108,  408 
Edestin,  84 

cleavage  products  of,  107 

hexone  bases  in,  165 
Eel-meat,  601 

scrum.  250,  327 
Egg  shell,  030-641 


1000 


GENERAL  INDEX 


Egg,  white  of  the,  632-636 

yolk  of  hen's,  628 
Eggs,  chemical  energy  in,  639 
development  of,  637 
fertilization  of,  639-640 
incubation,  637-638 

change    in    solid    con- 
tent during,  638 
exchange  of  gases,  637- 
638 
Loeb's  experiments  on  fertilization, 
639 
Ehrlich's  diazo  reaction,  753,  847 
glucosamine  test,  221 
side  chain  theory,  67,  71 
test  for  bilirubin,  429 
urine  test,  826 
Eicosyl  alcohol,  844 
Elaidin,  236 

Elastic  substance,  action    of   trypsin   on, 
508 
tissue,  analysis  of,  545 
Elastin,  92 

analysis  of,  116 
effect  of  gastric  juice  on,  473 
hexone  bases  in,  165 
in  lymphatic  glands,  366 
peptone,  117 
preparation  of,  117 
properties  of,  117 
Elastoses.  130 

Electrolytes,  amphoteric,  90,  93 
Elephant,  bones  of,  553 
Emulsin,  48,  58,  59,  62,  64 
Emulsoids,  properties  of,  15 
Enamel  of  teeth,  557,  891 
Encephalin,  607,  609,  610 
Endocrinic  glands,  374,  375 
Endoenzymes,  52 
Endolymph,  619 

Energy  content  of  various  food  stuffs,  886 
development,  calculation  of,  889 
exchange,  891 

calculation  of,  889 
metabolism    calculation   of,   886, 
887 
Enterokinase,  496 
Enzymes,  37-70 

action  on  glucosides,  62 
action,  specificity  of.  61 
action,  retardation  of,  62 
activators,  52 
adenase,  703 
alcoholases,  875 
aldehydases,  875 
amylopsin,  501 
anti,  63-64,  266 
ant i pepsin,  467 
arginase,  682 
butyrinases,  266 
catalases,  266,  872 
classification  of,  47 
co-,52 
deamidizing,  703 


Enzymes,  deviation,  64 
diastases,  266 

effect  of  bile  upon,  511-512 
endo-,  52 
esterases,  266 
extra  cellular,  52 
fat  splitting,  501-503 
fermentation,  653 
formation  of,  52 
gastric  lipase,  476-478 
general  properties,  48 
glutinase,  505 
glycolytic,  332 
glyoxylase,  584 
guanase,  703 
heat  production  of,  54 
histozyn,  723 
hydrogenases,  876 
in  amniotic  fluid,  642 
in  ascitic  fluids,  359 
in  blood,  52 
in  blood  serum,  266 
in  bile,  435 
in  brain,  608 
in  fatty  tissue,  558 
in  gastric  juice,  466 
in  intestinal  juice,  491-492 
in  leucocytes,  307 
in  liver,  386 
in  lungs,  870 
in  lymph,  346 
in  mammary  glands,  643 
in  milk,  653 
in  muscle,  572 

in  pancreatic  gland,  495,  500 
in  pancreatic  juice,  496 
in  placenta.  641 
in  prostate,  621 
in  pus  cells,  365 
in  pyloric  secretion,  478 
in  saliva,  455 
in  spleen,  370,  371 
in  thymus,  369 
in  thyroid  gland,  373 
intracellular,  52 
in  urine,  757 
in  yolk,  628 
lipases,  260 
.    maltase,  266,  458 
modes  of  action  of,  54 
myosin  ferment,  570 
nuclease,  504 
nucleases,  703 
oxidases,  266 
oxidones,  875 
oxygenase,  872 
pancreas  press  juice,  action  of, 

62 
pancreatic  rennin,  509 
pepsin,  466-476 
peroxidases,  872 
phenol-oxidases,  875 
phytase,  579 
polypeptide-splitting,  266 


GENERAL   INDEX 


1001 


Enzymes,  proteolytic,  82,  266,  703 
pseudopepsin,  166 
ptyalin,  156  400 

purine-oxidases,  875 
quantitative  determination  of, 

58 
reactivation,  52,  64 
reductases,  s7l>,  877 
rennin,  266,  474 
retardation  by  charcoal,  62 
retarding  substances,  65 
reversibility  of  enzyme    action, 

58 
salivary  diastase,  456 
Schutz's  rule  for,  58 
secretion  of,  52 
steapsin,  501-503 
syntheses,  38,  39 
synthesis  of  hippuric  acid,  39 
trypsin,  503-509 
tyrosinases,  875 
urease,  829 
uricase,  706 
uricolase,  706 
urocolytic,  706 
xanthin  oxidase,  703 
Enzymotic  processes,  40 

fermentation  processes,  41 
hydrolytic  cleavages,  40,  58 
synthetic  processes,  58 
reactions,  55 

laws  of,  55,  56,  57 
Epidermis,  112,  834,  835,  845 
Epidermoidal  structures,  837-838 
Epiguanine,  187 
Epiguanine  in  urine,  712,  714 
Epinephrin,  378 
Episarkine,  187 

in  urine,  712,  714 
Epitoxiod,  71 
Equilibrium  constant,  33 

nitrogenous,  906 
Erepsin.     See  Enzymes,  48,  593-494 

action  of,  493 
Ereptases,  503 
Erythrite,  relation  to  glycogen  formation, 

393 
Erythrocytes,  275 
Erythrodextrin,  229 
Erythropsin.     See  Visual  purple,  615 
Esbach's   quantitative   method    for   pro- 
tein in  urine,  794 
Ethal,  239 

Ether,  action  on  blood,  273 
Ethyl  alcohol,  action  on  metabolism,  912 
behavior  in  animal  body, 
775,  912 
benzene,  behavior  in  animal  bodv, 

778 
butyrate,  enzymotic  synthesis  of,  60 
mercaptan,     behavior     in     animal 

bodv,  776 
secretin,  823 
sulphide,  from  protein,  79 


Ethyl  sulphide,  behavior  in  animal  bodv, 
776 
sulphuric  acid,  behavior  in  animal 
body,  776 
Ethylene  glycol,  glycogen  formation,  394 
Euglobulin,  259 

Euxanthon,  fate  of,  in  organism,  785 
Excelsin,  108,  160 
Exchange  of  force,  871,  877,  879 
Excrements,  521,  549,  873,  874 

in  biliary  fistula-,  519 
Excreta,  regular  and  constant.  ,S79 
analysis  of,  880 

nitrogenous  constituents  of,  880 
Excretin,  523 
Exostosis,  556 

Expectorations  of  lungs,  870-871 
Extirpation  of  large  intestine,  effect  of,  548 
Extra  cellular  enzymes,  52 
Extractive  bodies  of  brain,  606 

of  kidneys,  673 

of     mammary     glands, 

643 
of  milk  plasma,  647 
non-nitrogenous,  579 
of  pancreatic  gland,  495 
substances  of  liver,  386 

of  muscles,  572-588 
Extractives  nitrogenous,  of  muscle,  572 
of  bone  marrow,  554 
of  bodies  of  cystic  fluids,  627 
of  testes,  620 
of  yolk,  628 
Exudates,  gases  of.  857 
Eye,  fluids  of,  615-619 

lens  of,  insoluble  protein  of,  618 
soluble  protein  of,  618 
pigments  of,  615-617 
tissues  of,  615-619 

Fat-cells,  membrane  of,  558 

action  of  trypsin 
on,  508 
-globule,  646 
Fats,  38,  232-249 

absorption  of,  535-540 

effect    of   bile    upon, 
536-540 
acetone  formers,  820,  822 
amounts  in  blood  of  different  animals, 

328 
catabolism  in  starvation,  894 
chemical  methods  for  investigating, 

238 
deposition  of,  920 
destruction  of,  during  work,  596 
detection  of,  237 
effect  of  extirpation  of  pancreas  on 

absorption  of,  539.  540 
effect  of  gastric  juice  on,  473 
effect  on  glycogen  content  of  liver, 

394 
emulsion  of,  511 
fate  of,  in  organism,  885 


1002 


GENERAL  INDEX 


Fats, "format ion  from  carbohydrates,  563 
from    glycogen    in    liver, 

397 
from  protein,  560-562 
of,  in  organism,  559 
hydrolysis  of,  234 
human,  559 
in  blood,  334 

serum,  265 
in  chyle,  346,  347 
in  kidney,  673 
in  liver,  384 
in  lymph,  348 
in  lymphatic  glands,  366 
in  muscle,  586 
in  pus  corpuscles,  364 
in  pus-serum,  363 
in  spleen,  370 
in  synovial  fluid,  362 
in  thymus,  368 
in  urine,  827 
in  woman's  milk,  660 
in  yolk,  630 

manner  of  absorption  of,  535-536 
metabolism  of,  in  starvation,  894 

with   an  exclusive    pro- 
tein diet,  907-908 
muscular  energy,  source  of,  598 
of  different  animals,  559 
pancreatic  splitting  of,  502 
properties  of,  233 
saponification  of,  234 
storing  up  of,  563 
syntheses  of,  60 
Fatty  degeneration,  385,  560 

series,  fate  of,  in  organism,  773-774 
tissue,  558-564 

analysis  of,  558 
constituents  of,  558 
Feathers,  mineral  substances  of,  838 

pigments  of,  843-844 
Feces,  appearance  of,  521,  522 
constituents  of,  521,  879 
pigments  in,  522 
reaction  of,  522 
Feeding   experiments   to   show   value    of 

different  foodstuffs,  904-906 
Fermentation,  8,  9,  203,  207 

lactic  acid,  in  stomach,  485 
processes,  41 

test  in  urine,  803,  804,  809, 
810,  812 
for  sugar,  803 
Ferments,  41 
Ferratin,  of  liver,  383 
Ferrine,  384 
Fertilization  membrane,  bringing  about, 

640 
Fever  elimination  of  ammonia.  768 
Fibrin,  100,  251,  254-261 
action  of,  257 
cleavage  products  of,  106 
coagulation,  256,  257 
elementary  composition  of,  263 


Fibrin,  ferment,  256 

globulin,  258,  259 
Henle's,  620 

in  blood  coagulation,  317 
in  blood  during  pregnancy,  337 
in  venous  blood,  336 
manner  of  formation,  322 
peptic  digestion  of,  472 
plastic  substance,  258 
preparation  of,  254-255 
production,  manner  of,  315 
properties  of,  255 
quantitative  estimation  of,  255 
Fibrinogen,  91,  252-261 

amounts  in  blood,  in  poison- 
ing, 253 
detection  of,  254 
elementary  composition,  263 
formation,  seat  of,  252 
in  coagulation  of  blood,  317 
in  venous  blood,  336 
occurrence  of,  252 
preparation  of,  254 
properties  of,  253 
purification  of,  254 
quantitative  estimation,  254 
relation  to  fibrin,  257,  258 
transformation  into  fibrin,  256 
Fibrinolysis,  255,  322 
Fibroin,  92,  122,  124 

analysis  of,  125 
properties  of,  124 
Filterability  of  colloids,  17 

preparation  of  the  filter,  18 
Fischer-Weidel's  reaction,  189 
Fleischl's  hsemometer,  299 
Florence's  sperm  reaction,  621 
Fluorine  content  in  teeth,  558 

content  in  organs  and    tissues, 
553 
Folin  and  Dennis'  test  for  tyrosine,  154 
Folin's  method  for  urea,  689 
Folin-Schaffer's  quantitative  method    for 

uric  acid,  711-712 
Food,  amount  of,   for   an   average   daily 
diet,  933,  934 
chemical   energy   introduced   with, 

932 
definition  of,  878 
influence  on  blood,  338 
necessity  under  various  conditions, 

932-939 
needs  of,  in  work  and  rest,  937-939 
requirements    for    men    in   various 

vocations,  933 
stuff,  determination  of  heat  value, 
891 
energy  content  of,  886 
organic,  uses  for  in  organism, 

890 
physiological    availability  of, 
891 
Foods,  energy  of,  885 

essential  to  life,  878 


GENERAL  INDEX 


1003 


Foods,  importance  of  various,  878 
Formaldehyde,  38 

formation  of,  from  carbon- 
ic acid,  2 
relation   to   glycogen   for- 
mation, 397 
transformation  into  sugar, 
1 
Formol  titrable  nitrogen  in  urine,  755,  756 
titration,    Sorensen's,    for    amino 
acids,  166 
Freezing-point  depression,     for    mamma- 
lians, 12 
in  the  frog,  12 
in  the  inverte- 
brates, 12 
in     the     lower 

fishes,  13 
in    the    higher 

fishes,  13 
in  the  eel,  13 
in  urine,  13 
of  colloids,    17 
molecular  lowering  of,  4 
Fructosazine,  221 
Fructose,  217-218 

in  blood  serum,  265 
in  urine,  814-815 
structural  formula1  for,  197,  199 
tests  for,  217-218 
Fruit-sugar,  217.     See  Levulose. 
Fuld  and   Levison's  method    for  testing 

pepsin,  470 
Furbringer's  test  for  proteid,  790 
Furfurol,  208-209 

fate  of,  in  organism,  784 
Fuscin,  617 

Gadushistone,  108 
Galactosamine,  220 
Galactose,,  654,  816 

structural  formula  for,  199 
tests  for,  217 
Galactose,  202 

Gall  bladder,  secretion  of,  416,  437 
Gall-stones,  444 
Gallois'  inosite  test,  578 
Ganassini's  reaction  for  uric  acid,  707 
Gas  exchange,  a  measure  of  metabolism, 
927-928 
between    blood    and    pul- 
monary air,  858-870 
between  blood  and  tissues, 

858-870 
in  muscle  activity,  592 
in  starvation,  895 
methods  for  the  quantita- 
tive   determination     of, 
868-870 
through  skin,  849 
rise  of,  931 
tension  in  blood,  859-868 

methods  of  determining,  864- 
866 


Gases,  in  bile,  857 

in  birds'  eggs,  636 
in  blood,  857,  850-856 
in  exudates,  867 
in  blood  serum,  26'.) 
in  gastric  digestion,  485-486 
in  lymph,  346,  856-858, 
in  milk,  657,  658 
in  muscles,  588 
in  saliva,  857 
in  stomach,  485-486 
in  transudates,  355 
in  urine,  770,  857 
in  woman's  milk,  68  I 
produced  in  putrefaction,  515-516 
Gastric  contents,  indicators  for  determin- 
ing acids  in.  487 
nature  of  acids  in,  487 
quantitative  determina- 
tion of  lactic  acid  in, 
488 
test  for  lactic  acid  in, 
488 
digestion,  absorption  of  cleavage 
products  in  stomach, 
484 
degree  of,  483,  484 
effect  of  fats  on,  481 
gases  in,  485,  486 
time  of  passage  through 
the   stomach   of   dif- 
ferent foods,  482,  483 
juice,  461-466 

action  of  foreign  substances 

on  ser-retion  of,  462 
action   of  saliva   on   secre- 
tion of,  463 
composition  of,  465 
constituents  in,  466 
degree  of  acidity  in,  487 
obtainment,    free   of  saliva 

461-482 
origin  of  hydrochloric  acid 

in,  477 
secretion  of,  461,  462-463 
in  man,  464 
lipase,  476-478 
Geissler's  albumin-test  papers,  790 
Gel,  15 
Gels,  30-32 

Gelatin,  31,  78,  80,  118,  119-121,  152 
analysis  of,  118 
as  a  foodstuff,  912 
forming  substances  of  bones,  pep- 
tic digestion  of,  472 
forming  substances  of  cartilage, 

peptic  digestion  of,  472 
forming  substance  of  connecting 
tissue,  action  of  trypsin  on,  508 
forming  substances  of  connective 
tissue,  peptic  digestion  of,  472 
from  peptic  digestion,  473 
hexone  bases  in,  165 
in  egg  development,  639 


1004 


GENERAL  INDEX 


Gelatin,  in  protein  catabolism,  911 
oxidation  of,  83 
pancreatic  digestion  of,  508 
peptic  digestion  of,  473 
peptones,  120,  121 
preparation  of,  118 
properties  of,  119 
protein-sparer,  911,  912 
Gelatose,  proto-,  120 

deutero-,  120 
Gelatoses,  120,  473 
Generation,  organs  of,  620-642 
Generative  organs,  female,  623-642 
secretions,  male,  620-623 
Gerhardt's  test  for  acetoacetic  acid,  824 
Glands,  albuminous,  541 

Brunner's,  secretion  of,  489 
fundus,  460,  461 
Lieberkuhn's,  secretion  of,  490 
mammary,  643 

constituents  of,  643 
mixed,  451 
mucous,  451,  460 

membrane,  in  the  intes- 
tine, 489-493 
membrane    of    stomach, 
460 
pancreatic,  494-495 
pyloric,  460,  461 
salivary,  451-460 

analysis  of,  451 
Gliadin,  cleavage  products  of,  107 
Globan,  104,  276 
Globulins,  78,  91,  92,  93,  106 

detection  of,  in  urine,  791 
properties  of,  104 
quantitative  estimation  of,  in 
urine,  791,  793 
Globuloses,  130 
Glucocyanhydrin,  200 
Glucomaines  in  urine,  757,  758 
Glucoproteins,  167,  168-174 
Gluconose,  206 
Glucopeptose,  200 
Glucoproteins,  phosphorized,  105 
Glucoproteose,  133 
Glucosamine,  84,  219 

from  blood  globulin,  260 
from  seralbumin,  262 
preparation  of,  220 
tests  for,  219 
d-glucosamine,  201 
Glucosan,  213 
Glucose,  59,  212-217 
in  blood,  330 
in  urine,  749,  802-814 
from  blood  globulin,  260 
osimine  formation,  201 
preparation  of,  216 
properties  of,  213 
structural  formula  for,  197,    198, 

199 
tests  for,  213-216 
Glucosidee,  202 


Glucosides,  action  of  enzymes  on,  62 
Glucoside-splitting  enzymes,  16,  202,  491 
Glucosoxime,  200 

Glucuronates,  conjugated,    properties    of, 
751, 752 
conjugated  in  urine,750-752 
Glucurone,  222 
Gluge's  corpuscles,  623 
Gluteins,  119 
Glutelins,  92 
Gluten  casein,  hexone  bases  in,  165 

proteins,  hexone  bases  in,  165 
Glutin,  92 
Glutinase,  505 
Glutokyrin,  138 
Glycerides,  tri-,  232 
Glycerine,  265 

relation  to  glycogen  formation, 
394,  397,  412 
Glycine.     See  Glycocoll,  139 
Glycocoll,  85,  106,  107,  109,  113,  115,  119, 
124,  125,  139 
amounts  in  proteins,  106,  107, 

113,  115,  125 
conjugation  with,  782-785 
formation  of,  in  organism,  721 
importance    of,    in    uric    acid 
formation,  720,  721 
Glycocyamine,  693 

Glycogen,  229,  390^14,  581-582,  637 
amount  in  liver,  390,  391 
amount  in  muscles,  390,  391 
consumption  of,  in  muscles,  592 
content  increased  by,  393,  394, 

396,  397 
fat  formation  from,  397 
formation,  a  cell  function,  398 
formation  from  sugar,  395 
in  lymph,  346 
in  lymphatic  glands,  366 
in  placenta,  641 
origin  of,  393 
origin  in  muscles,  397 
preparation  of,  392 
properties  of,  391,  392 
pseudoglycogen-formers,      395, 

396 
quantitative  estimation  of,  393 
synthesis  of,  58 
synthesis  of,  in  liver,  381 
true  glycogen  formers,  395,  396 
transformation  into  sugar,  398, 
399 
Glycolaldehyde,  38 
Glycolysis,  265,  332,  407-409,  570 
Glycolytic  enzyme,  265 
Glycoproteins,  92,  167,  168-174 

in  blood,  264 
Glycosuria  adrenalin,  402,  403 

relation  of  pancreas 
and  adrenals,  406 
alimentary,  401 
diabetic,  relation  of  pancreas 
to,  405 


GENERAL   INDEX 


1005 


Glycosuria  piqure,  402 
*alt,  401 

BUgar-puncture,  402 
Glycylalanine,  86 
Glycylasparaginyl  leucine,  86 
Glycyiglycin,  86 
Glycyl  leucine,  86 

proline  anhydride,  86 

tyrosine,  86 

valanine  anhydride,  86 

Glyoxal-methyl,  202 

Glyoxyldiureide.     See  Allantoin,  717 

Gmelin's  test,  for  bile  pigments,  429 

in  urine,  800 
Goitre,  376 
Gorgonin,  123 
Graafian  follicles,  623,  624 
Grape  sugar,  212.     See  Glucose. 
Guaiac  test  for  blood,  281,  795 
Guanadine,  162 
Guanase,  48,  188,  187,  3S2,  368,  702.     See 

Enzymes. 
Guanidobutvlamine,  162 
Guanine,  178, 181,  183,  185,  187,  188,  190, 
191,  712 
Capronica's  test  for,  191 
epi-,  187 
-hexoside,  180 
Weidel's  reaction  for,  191 
Guano,  188,  699 
Guanosine,  ISO 
Guanovulit,  637 
Gulose,  211 
Gums,  229,  230 
plant,  226 
vegetable,  230 
Gunning's  modified  Lieben's  test,  822 
Gunzburg's  test  for  HC1  in  gastric  juice, 

487 
Gvnesin  in  urine,  758 


Hacmaphilia,  329 
Hu'inase,  50 
Ha;mataerometer,  861 
Hiematin,  289-291 

neutral,  215 
reduced,  288 
Haematinogen,  296 
Haematinometer,  297 
Haematocrit,  326 
Haematocrystallin,  278 
Haematogen,  629,  637 
Hsematoidin,  871 
Haematoblasts,  308 
Hajmatoporphyrin,  294-296,  844 

relation    to    bilirubin, 

295,  428,  440 
relation  to  chlorophyl, 

276,  295 
relation    to    urobilin, 

295,  440,  742 
in  urine,  740,  797-798 
Haematoscope,  303 


Haematuria,  795,  796 
Hsemerythrin,  299 
Bsmin,  292  294,  297,  800 

crystals,  292,  293,  800 

Haemochrom,  275,  27'.) 
Hsemochromogen,  276,  288,  289 
Haemocyanin,  92 
Haemoglobin,  92,  108 

amounts  in  blood,  328 
carbon  dioxide  binder,  853 
exudation  of,  <i 
in  blood  during  pregnancy, 

337 
in     (X)2  —  ()2     exchange     in 

blood,  853 
in  venous  blood,  326 
transformation  into  bile  pig- 
ments, 442 
Haemoglobinuria,  796 
Haemoglutination,  275 
Haemolysins,  69 
Haemolysis,  273 
Haemometer,  303 

Ha>mopyrrol,  276,' 291,  295,  428,  843 
Haemorrhodin,  287 
Haemoverdin,  287 
Hair,  837 

-balls,  524 

human,  sulphur  content  of,  838 
lanugo,  642 
Hammarsten's  test  for  bile  pigments,  430, 

800 
Haptogen-membrane,  646 
Haptophore,  67 

Hanriot  and  Richet's  method  for  deter- 
mining respiratory  exchange,  869 
Haser's  coefficient,  771 
Heat  regulation,  chemical,  929 
physical,  929 
Hedenius'  bilirubin  test,  433 
Helicoproteid,  174 
Heller's  blood  test,  797 

scheme  for  investigating     calculi, 
837 
Heller-Teichmann's  test,  797 

in  urine,  788 
for  proteid,  98 
Hemicelluloses,  231 
Hemicollin,  120 
Hemielastin,  117 

in  blood,  264 
Hemipeptone,  130 
Hemolysins,  70 
Hemp  seed  calculi,  835 
Henle's  fibrin,  620 
Henoeque's  haematoscope,  299 
Henriques  and  Gammeltoft's  method  for 

urea,  690 
Heparphosphatide,  386 
Hepatopancreas,  494 
Heptapeptides,  85,  87 
Heptose  in  urine,  817 
Heptoses,  197 
Heterocaseoses,  129 


1006 


GENERAL  INDEX 


Heterocyclic     compounds,     fate     of,     in 

organism,  778-787 
Heterolysis,  18 
Heterosponginose,  122 
Heterosyntonose  hexone  bases  in,  165 
Heteroxanthine,  187 

in  urine,  712,  713 
Hexapeptides,  85 
Hexobioses,  224 
Hexone  bases,  161 

in  various  proteins,  165 
Hexoses,  197,  211-218 

syntheses  of,  212 
Hippokoprosterin,  449 
Hippomelanin,  841 
Hirudin,  251 

Histidine,  85,  106,  107,  109,  110,  111,  115, 
117,  119,  125,  159-161 
diazo  reaction  for,  161 
in  various  proteins,  165 
Weidel's  reaction  for,  160 
Histidyl-histidine,  85 
Histone  in  urine,  795 
Histone-peptone,  109 
Histones,  78,  91,  92,  93 
Gadus-,  108 
hexone  bases  in,  165 
Lota-,  108 

properties  of,  108-109 
Histozym,  48 

Hoffmann's  test  for  tyrosine,  153 
Holozym,  319 

Homocerebrin,  607,  609,  610 
Homocyclic  compounds,  fate  of,  in  organ- 
ism, 778-787 
Hopkin's   quantitative    method    for   uric 

acid,  711 
Hoppe-Seyler's  reaction  for  xanthine,  190 
CO  blood  test,  286 
colorimetric  method,  297 
test  for  bile  acid,  179 
Hordein,  107,  156 
Hormone,  375 
Hormones,  407 
Horn  structures,  837 

mineral  content  of,  838 
sulphur  content  of,  838 
See  also  Keratin. 
Hufner's  bile,  419 
Humor,  aqueous,  352,  359,  617 
Huppert-Messinger  method  of  estimating 

acetone,  825 
Huppert-Schiiltz's     method     of     testing 

pepsin,  468 
Huppert's    test    for   bile    pigments,    429, 

430,  799 
Hyalin,  840 
Hyalines,  171 
Hyaline  substance,  364 
Hyalogens,  171 
Hyalomucoid,  617 
Hydantoins,  786 
Hydremia,  340.  352 
Hydramnion,  642 


Hydrazine  poisoning,  718 

Hydrazones,  202-203 

Hydrobilirubin,  743 

Hydrocele  and  spermatocele  fluids,  359, 

360 
Hydrochinon  in  urine,  727 
Hydrogel,  14 
Hydrogen,  colorimetric  method,  75 

determination  of,  75 

determination  of,  in  fluids  con- 
taining CO2,  76 

electromotive  method,  75 

ion  content,  75 

peroxide,  35 
Hydrogenases,  876 
Hydrolytic  cleavage  processes,  40 
Hydroquinone,  727 
Hydrosol,  14 

Hydroxylamine  poisoning,  718 
Hyperacidity,  486 
Hyperglycemia,  401,  402 
Hyperthyreoidismus,  378 
Hypertonic  solution,  6 
Hypnotics  and  glycogen  formers,  394 
Hypophysis,  380 
Hypotonic  solution,  6 
Hypoxanthine,  178,   187,    188,    191,    192, 
572,  712 
detection  of,  191 


Ichthidin,  630,  636 

Ichthin,  636 

Ichthulin,  92,  174,  630,  636 

Ichthylepidin,  122 

Icterne,  413,  428,  431,  440,  441 

urine,  799 
Ignotine,  576 
Ileum,  extirpation  of,  549 
Imbibition,  30 
Imidazol  derivatives  in  urine,  757,  826 

structural  formula  of,  186 
Immune  bodies,  66,  69 
Immunity,  63-70 

active,  70 
passive,  70 

theory  of,  Arrhenius',  67,  68 
theory  of  Erlich,  67,  68 
Immunization,  66 
Indican,  517,  728 

elimination  of,  728 

effect  of  putrefaction 
on,  729 
excretion  of,  729,  730 
quantitative  method  for,  731 
tests  for,  730,  731 
Indigo-blue  of  urine,  741 
Indigo-red  formation,  source  of,  731 
Indigotin  in  urine,  728,  731 
Indirubin  in  urine,  731 
Indol,  46,  82,  117,  157-159,  267,  515,  522, 
843 
fate  of,  in  organism,  786 
formation  of,  in  organism,  728,  729 


GENERAL  INDEX 


1007 


Indol,  methyl-,  157 
tests  for,  158 
Indoxyl,  724 
Infraproteins,  93 
Inosine,  ISO,  577 
Inosite,  579-581 

in  adrenal  bodies,  377 
preparation  of,  580 
properties  of,  580 
Inositogen,  577 

"Integral  factor,"   in   uric  acid  elimina- 
tion, 705 
Internal  friction,  18 

effect  of  acids  and  alka- 
lies upon, 19 
effect  of  NaCl  upon,  19 
of  hydrophile  colloids,  19 
of  suspension  colloids,  18 
Intestinal  fermentation,  512,  513 

juice,  constituents  of,  491,  492 

properties  of,  491 
putrefaction,  512,  513 
Intestine,  chemical  processes  in,  510-524 
decomposition  by  microbes  in, 

513 
large,  putrefaction  in,  515 
small,  digestion  and  absorption 
of  various  foods  in,  513-514 
walls  of,  chemical  processes  in, 
527-529 
Intracellular  enzymes,  52 
Intraligamentary  cysts,  627 
Inulin,  228 

Inversion  of  sugars,  224 
Invert  sugar,  224 
Invertase.     See  Enzymes,  48 
Invertin,  225.     See  Enzymes. 
Iodine  combinations,  passage    into    milk, 
667 
passage     into     per- 
spiration, 849 
passage  into  saliva, 
457 
in  blood,  268,  333,  337 
in  glands,  378,  379 
in  perspiration,  848 
in  proteins,  81,  125 
Iodoform,  fate  in  organism,  795 
Iodospongin,  124 
Iodothyrin,  373,  376 

of  thymus,  368 
Iodothyrioglobulin,  373,  376 
Ions,  5,  70-76 

action  of,  on  enzymes,  70 
Iron,  absorption,  338-339 
and  blood,  339,  639 
elimination,  433,  440,  770 
importance  of.  in  metabolism,  903 
in  spleen,  371 
in  urine,  769 
results  in  lack  of,  895 
Islands  of  Langerhans,  407 
Iso-casein,  649 
Isocholesterin,  449 


Isodynamic  law  in  metabolism,  890-892 
Isoelectric  point,  21,  26 
Isolactose  svnthesis  of,  58 
[soleucine,  86,  106.  107,  143-145 
I  s<  .maltose,  223,  224,  225-226 

in  urine,  749 

synthesis  of,  58 

splitting  of,  58 
Isoserine,  146 
Isosmotic  solutions,  3 
Isotonic  solution,  5 
lsotropous  muscle  substance,  564 


Jacoby's  quantitative  method  for  pepsin,. 

470 
Jaffe's  creatinine  reaction,  695 
Jaffe-Obermayer's  indican  test,  729 
Janthinin,  844 
"Jaune  indien,"  222 
Jecorin,  605 

in  liver,  385 

in  spleen,  370 
Jejunum,  effect  of  extirpation  of.  549 
Jerusalem's  test  for  lactic  acid  in  gastric 
juice,  488 


Kaolin,  16 
Karyogen,  623 
Kephir,  518,  654,  659 

effect  on  putrefaction,  518 
-lactase,  57.  58 
Kerasin,  607,  609,  610-611,  613 
Keratin,  A,  B,  C  of  horn  and  hair,  prop- 
erties of,  837 
action  of  gastric  juice  on,  473 
Keratins,  92,  112-116 

composition  of,  113 
Ketone,  behavior  in  animal  body,  776,  777 
Ketoplastic  action,  819 
Ketoses,  197 
Kidneys,  constituents  of,  672-674 

formation  of  hippuric  acid   in,. 

722 
quantitative  analysis  of,  673 
relation  to  formation  of  urea,  686 
Kinase,  in  thrombin  formation,  323 
occurrence  in  intestine,  492 
Kinases,  51 
Kjeldahl's    method    for    total    nitrogen, 

688 
Klupeovin,  698 

Knapp's    method    of    estimating    sugar, 
812 
reaction  for  dextrose,  215 
Knop-Hupner's  method  for  urea,  696 
Koilin,  115 
Koprosterin,  44s 

Kossel's  reaction  for  hypoxanthine,  192 
Kossler  and  Penny's  method  for  phenol- 
sulphuric  acids,  726 
Kramm's  creatinine  reaction,  696 
Kumyss,  654,  659 


1008 


GENERAL  INDEX 


Kyrin,  137 

caseino-  137 

gluto-,  137 

proto-,  137 
Kyrines,  167 

carbamino  reaction  with,  167 

Lactacidase,  207 
Lactalbumin,  91,  652-654 

cleavage  products  of,  106 
composition  of,  652 
properties  of,  653 
Lactase,  48,  64 
Lactimide,  156 
Lactocrit,  657 
Lactoglobulin,  652 
Lactones,  198 
Lactoprotein,  653 
Lactose,  223,  224,  647,  654-655 

in   milk,    conversion   into   lactic 

acid,  644 
in  urine,  815 
preparation  of,  655 
properties  of,  654 
tests  for,  655 

theory  of  formation  in  mammary 
gland,  643 
Lactosuria,  811 
Laiose  in  urine,  815 
Langerhans,  islands  of,  494 
Lanocerin,  844 
Lanolin,  446 
Lanugo  hair,  642 
Large  intestine,  effect  of  extirpation  of, 

548 
Latebra,  624 
Lead  in  blood,  333 

in  the  liver,  389 
passage  into  milk,  670 
Lecithin,  105,  586 

amounts   in   blood    of   different 

animals,  328 
from  blood  serum,  261 
in  chyle,  347 
in  lymph,  348 
in  pus  corpuscles,  364,  365 
in  pus-serum,  363 
quantitative    determination    of, 
246 
Lecithins,  10,  242-249 

hydrolytic  products  of,  342 
in  metabolism,  importance  of, 

902 
preparation  of,  246 
properties  of,  244 
structure  of,  242 
Lecithoproteins,  92 
Legal's  reaction  for  indol,  158 
Legumin,  cleavage  products  of,  107 
Lense,  of  oxen,  analysis  of,  619 
Leo's  sugar,  810 
Lepidoporphyrin,  844 
Lethal,  238,  239 
Leucaemia,  blood,  187,  341,  698 


Leucaemia,  spleen,  375 

urine,  710,  764 
Leucine,  85,  106,  107,  109,  111,  113,  115, 
123,  124,  125,  141-143,  267 
conversion  into  isoamyl  alcohol, 

206 
in  brain,  607 
in  lymphatic  glands,  366 
in  spleen,  370 
in  thyroid  gland,  373 
in  urine,  755,  827 
Leucinimide,  143 
Leucocytes,  305 

constituents  affecting  coagu- 
lation, 315 
in  venous  blood,  336 
osmotic  phenomena  with,  8 
See    also    white    blood  cor- 
puscles 
Leucomaines,  25 
Leuconuclein,  367 

in  blood,  316 
Leucopolun,  609 
Leucyl  peptides,  85-88 
Levulins,  228 
Levulose,  217 

in  urine,  814-815 
Lichenin,  228 

Lieberkiihns  solid  alkali  albuminate,  126 
Lieben's  iodoform  test  for  acetone,  822 
Liebermann-Burchart's,    for    cholesterin, 

447 
Liebermann's  reaction  for  protein,  100 
Liebig's  method  for  urea,  688 
Lienases,  371 

Lifschiitz's  reaction  for  cholesterin,  445 
Ligamentum  nuchas,  115,  116,  546 
Lignin,  231 

Lime  salts,  manner  of  excretion,  769 
Lipanin,  absorption  of,  545 
Lipase,  47,  48 

pancreatic,  57 
Lipases,  plants,  64.     See  also  Enzymes. 
Lipochromes,  268 
Lipopeptides,  88 
Lipoids,  241 

as  a  special  limiting  layer,  10 
in  brain,  605 

in  relation  to  osmosis  in  cells,  10 
sulphurized,  605 
Lipuria,  827 
Lithium,  22,  333 
Liver,  381-450 

acetone  formation  in,  819,  822 

amino  acids,  synthesis  of,  382 

autolysis  of,  387 

assimilation  in,  381 

bile  pigments,  formation  of,  381 

blood    pigments,     destruction     of, 

388 
-cells,  reaction  of,  382 
conjugation  of  glucuronic  acid  in, 

382 
constituents  of,  382 


GENERAL   INDEX 


1(09 


Liver,  creatin-creatine   metabolism,   694, 
695 
deamidation  in,  382 
destroying  of  uric  acid  in,  707 
ethereal  sulphuric  acids,  formation 

of,  382 
formation  of  amino  acids  in,  530 
formation  of  bile  in,  382 
glycogen  formation,  381,  390 
glycogen  content  in,  extirpation  of 

the  adrenals,  402 
glycolysis  in,  theories  for,  408 
iron  in,  387,  388 
"  organ  plasma  "  of,  382 
pigments  of,  384 
processes  going  on  in,  381,  382 
protein  synthesis  in,  529,  530 
storehouse  for  protein,  389 
synthetical  formation  of  uric  acid 

in,  705 
urea  formation,  382 

of.  from  ammonia, 
767 
uric  acid  formation  in,  702,  703 
Livetin,  629 
Lotah  istone,  108 
Lungs,  concretions  of,  871 
constituents  of,  870 
expectorations  of,  870,  871 
gas,  exchange  in,  867 
Lutein,  631 

Luteins,  in  blood  serum,  268 
Lymph,  345-380 

amount  secreted,  349 
composition  of,  345-348 
coagulation  of,  346 
formation  of,  350-352 
gases  of,  856-858 
origin  of,  345 
osmotic  pressure  in,  348 
secretion  of,     circumstances     in- 
fluencing, 349,  350 
Lymphagogues,  350 
Lymphatic  glands,  366 

quantitative    composi- 
tion of,  366 
Lymphocytes,  300 
Lymphoid  cells,  305 
Lysatine,  160 
Lysatinine.  160 

Lysine,  85,  106,  107,  109,  111,  113,  115, 
117,   119,   123,    124,    125,    163, 
164,  267 
in  various  proteins,  165 
peptone,  138 
Lysines,  47 

Lysylglycylpeptide,  89 
Lysyl  lysine,  85 

Magnesium  in  urine,  768,  769 

triphosphate  in  urinarv  sedi- 
ment, 831 
Maintenance  value,  897 
Maltase,  48,  458.     See  also  Enzymes. 


Malt  dextrin,  229 
Malt  diastase,  225 

action  of,  upon  starch,  226 
Maltose,  223,  224,  225 
in  urine,  815 
synthesis  of,  58 
Malt  sugar,  225 
Mammary  gland,  theory  of  formation  of 

lactose  in,  643 
Mandelic  acid  ester,  racemic,  62 

nitrile  glucoside,  59 
Manganese,  268,  333,  442 
Mannite,  198 
Mannose,  198,  216 
Manonose,  206 
Marcitine,  47 
Margarin,  235 

Maschke's  reaction  for  creatinine,  695 
Mass  action,  32,  74 
Mastic,  22 
Meconium,  523 

Medicinal  coloring  matters  in  urine,  801     ' 
Medulla,  607 

Medullary  fibers,  composition  of,  614 
Melanin,  617,  842 

in  urine,  799 
Melanins,  of  skin,  841 

mother  substances  of,  843 
Melanogen  in  urine,  799 
Melanoidin  nitrogen,  78 
Melanoidins,  841 
Melano-protein,  842 
Melanotic  cancers,  802,  842,  843 
Membrane,  semi-permeable,  26 
Membranin,  171,  550 

properties  of,  617 
Menthol,  fate  of,  in  organism,  785 
Mercuric  salts  poisoning,  23 
Mesitylene,  fate  of,  in  organism,  779 
Mesoinosite,  582 
Mesoporphyrin,  295 
Mesoxalyl  urea,  698 
Metabolism,  878-939 

affected  by  sleep,  928 
as  affected  by  external  tem- 
perature, 929 
as    affected    by    high    alti- 
tude, 930 
as  affected  by  ingestion  of 

food,  930 
as  affected  by  light,  928 
as  affected  by  mental  activ- 
ity, 928 
basal  requirement,  897,  898 
calculation  of,  889 
conditions  affecting,  922-931 
effect  of  age  upon,  922-925 
effect  of  alcohol  on,  921,  922 
effect  of  coffee  and  tea  upon, 

922 
effect  of  rest  and  work  upon, 

926-«)2-< 
effect  of  salts  on,  921 
'  effect  of  sex  upon,  925,  926 


1010 


GENERAL  INDEX 


Metabolism,  effect    of    weight    of    body 
upon,  922-925 
effect  of  water  on,  920,  921 
importance   of   lecithins   in, 

902 
importance     of     phosphates 

in,  902 
in  active  and  inactive  mus- 
cles, 591-598 
inanition  condition,  897-898 
in  starvation,  892-906 
lack  of  mineral  substances  in 
food,  899,  900 
water  in  food,  898 
maintenance  value,  897-898 
measured  by  gas  exchange, 

927,  923 
with    absence    of    carbohy- 
drates in  food,  903,  904 
with  absence  of  fats  in  food, 

903-904 
with     absence     of     proteins 

in  food,  903 
with  a  mixed  diet,  913-920 
of    fat    with    an    exclusive 

protein  diet,  907,  908 
with  foods  rich  in  protein, 

906-913 
with    insufficient    supply    of 

chlorides,  900,  901 
with  lack  of  bases  in  foods, 

901 
with  lack  of  earths  in  food, 

901,  902 
with  lack  of  iron  in  foods, 

903 
with  lack  of  phosphates  in 

food,  901,  902 
with  various  foods,  906-922 
Metacasein,  651 
Metalbumin,  624,  625 
Metanitrobenzaldehyde,    fate   of,   in    the 

body,  784 
Metaproteins,  93 
Metazym,  319 
Methaemoglobin   of   thyroid   gland,   374. 

See  also  Blood  pigments. 
Methal,  239 
Mf-those,  212 

Methylation  in  organism,  787 
Mfthylenitan,  212 

Mfthylethylmaleic  acid  anhydride,  290 
Methylglycocoll.     See  Sarcosin. 
Methylglyoxal  in  relation  to  lactic  acid 

fermentation,  584 
Methylguanidine,  578,  698 

in  urine,  758 
Methyl  guanine  (7),  714 
Methylindol,  157      See  Skatol. 
Methylindolm,  729 

Methylimidazol  formation  of,  201-202 
Methyl  pentoses,  199,  208 
Methylphenylhydrazine,  test  for  levulose, 
218 


Methyl  pyridine,  behavior  in  animal  body,. 
778,  781 
chloride  in  urine,    756, 
758 
Methylpyridylammonium  hydroxide,  787 
Methylthiophene,  784 
Methyluracil,  194 
Methyluramine,  574,  694 
Methyl-urea,  691 

in  urine,  712,  713 
Methylxanthine,  187 
Mett's  quantitative  method  for  pepsin, 

469 
Micro-organisms  in  intestines,   511,  514, 

517,  520 
Microrespirometer,  874 
Microtonometer,  861 
Milk,  643-671 

albumin,    quantitative    determina- 
tion of,  656 
ash,    quantitative    composition   of, 

666 
casein,   quantitative  determination 

of,  656 
coagulation,    a   two-faced   process, 

650 
coagulation  of,  645 
cows,  644,  645 
cows,  quantitative  composition  of, 

657 
effect  on  putrefaction,  518 
fat,  quantitative  determination  of, 
657 
constituents  of,  647 
theory  for  origin  of,  669 
foodstuffs  in.  666 
gases  of,  857 
globules,  645-647 
goat's,  659 
human,  660-671 
human,    compared   to   cow's   milk,. 

660,  661,  663 
influence  of  food  on  composition  of, 

667,  668 
mare's,  659 
lactose  quantitative  determination,. 

of,  657 
mineral    bodies,    quantitative    de- 
termination of,  656,  657,  658 
of  carnivora,  659 
of  various  animals,  composition  of, 

660 
passage  of  foreign  substances  into, 

670 
plasma,  647 

preparation,  composition  of,  659 
proteins,    quantitative    determina- 
tion of,  656 
quantitative  analysis  of,  656,  657 
secretion,  chemistry  of,  668-671 
solids,    quantitative   determination! 

of,  656 
souring  of,  645 
sugar,  226.     See  Lactose. 


GENERAL  INDEX 


1011 


Milk,  sugar,  theory  for  origin  of,  670 

quantitative     determination 
of,  657 
theory  for  origin  of  casein,  tills 

permanent  emulsion,  646 
uterine,  641 

various  fermentations  of,  til.") 
woman's,  quantitative  composition, 
652,  653 
quantity  of  mineral  sub- 
stances in,  654 
Millon's  reaction  for  proteins,  99 

in      hyurocele      an  I 
spermatocele  Quids, 
.  359 
Mineral  bodies  in  milk,  656 
Mineral  substances,  absorption  of,  540 

amounts  in  blood   of 
different     animals, 
328 
distribution  of,   72 
effect  on  metabolism, 

921 
importance  to  life  of 

cells,  72 
in  bile,  436 
in  blood,  335 
in  blood  serum,  26S, 

269 
in  bone  structure,  552 
in  brain,  615 
in  cartilage,  550 
in  cells,  72 
in  cerebrospinal  fluid, 

361 
in  chyle,  347 
in  connective  tissue, 

546 
in  cystic  fluid,  627 
in  egg  shell,  636 
in  feathers,  838 
in  gastric  juice,  466 
in  hair,  838 
in  intestinal  juice,  491 
in  kidneys,  673 
in  liver,  387-389 
in  lungs,  870 
in  lymph,  346,  348 
in  muscles,  586-588, 

599 
in  nails,  838 
in   pancreatic   gland, 

495 
in    pancreatic    juice, 

500 
in    pericardial    fluid, 

356 
in  pus  corpuscles,  365 
in  pus-serum,  363 
in  retina,  615 
in  semen,  621 
in    smooth    muscles, 

602 
in  spermatozoa,  622 


Mineral  substances,  in    starvation,     895, 
896 
in  synovial  fluid,  362 
in  urine,  758-770 
in  white  of  egg,  635 
in  woman's  milk,  654 
in  volk,  632 
lack  of,  in  food,  899, 

900 
of  spleen,  361 
toxicity  of,  73 
Mingen  in  urine,  756,  75s 
Mohr's  quantitative  method  for  chlorides, 

759 
Molisch's  test  for  sugar,  216 
Monaminophosphatides,  241 
Monomethylxanthine,  712 
Monosaccharides,  197-218 

cyanhydrin    formation, 

200 
oxime  formation,   200 
transformation  of,  201 
Moore's  test  for  sugar,  214,  224 
Morner-Sjoqvist  and  Folin's  method  for 

urea,  6S9 
Morphine,  elimination  in  urine,  749,  787 

in  milk,  670 
Moss-starch,  228 
Mucin,  619 

action  of  trypsin  on,  508 
"  dissolved,"  794 
effect  of  gastric  juice  on,  473 
in  urine,  757,  794 
pseudo,  171 
substances,  168-172 
Mucilages,  226 

vegetable,  230 
Mucinoids,  171 
Mucins,  analysis  of,  169 

amino  acids  in.  170 
cleavage  products  of,  170 
true,  168,  169 
Mucoid,  osseo-,  172 
Mucoids,  169,  171 

effect  of  gastric  juice  on,  473 
Mucous  of  urine,  674 
Mulberry  calculi,  833 
Murexide  test,  707 
Muscarine,  246 
Muscle-pigments,  571 
Muscle-plasma,  566 

preparation   and   proper- 
ties of  566,  567 
proteins  of,  569   570 
Muscle-serum,  566 

smooth,  601-603 

constituents  of,  602 
extractives  in,  602 
"  snow,"  566 
"-stroma,"  569 
svntonin,  569 
Muscles,'565-603 

acid  rigor  of.  590 

active,  acid  reaction  of,  593 


1012 


GENERAL  INDEX 


Muscles,  active,  characteristics  of,  596 

amount  of  fat  in,  600 

an   isotropous  substance  of,  555, 
556 

chemical  rigor  of,  590 

dead,  proteins  of,  567 

elementary  analysis  of,  601 

extractive  bodies  of,  572-588 

"flesh  quotient,''  601 

haemoglobin,  571 

heat  rigor  of,  590 

imbibition  rigor  of,  590 

isotropous  substance  of,  555,  556 

metabolism  in,  591-598 

of  different  animals,   ash  anal- 
yses of,  599 

of  different  animals,  analyses  of, 
599 

proteins  of,  566-572 

quantitative  composition  of,  598- 
601 

reaction  of,  565 

rigor  mortis  of,  588-591 

striated,  565-601 

water  rigor  of,  590 
Musculamine,  578 
Muscular  action,  source  of,  597 
Musculin,  568,  570 
Myelin,  608 
"  Myeline  forms,"  605 
Myoalbumin,  569 
Myocynine,  578 
Myogen,  570,  571 
Myogen-fibrin,  570,  571 

soluble,  570 

preparation  of,  570,  571 
Myoglobulin,  568,  571 
Myohaematin,  571 
Myoproteid,  571 
Myosin,  84,  567,  568,  570 

as  related  to  fibrinogen,  254 

coagulation  of,  570 

ferment,  570 

fibrin,  568,  570 

in  blood  corpuscles,  306 

preparation  of,  568 
Myosinogen,  569,  570,  571 
Myosinoses,  130 
Myri»in,  239 
Mytilite,  581 
Mytolin,  569 
Myxcedema,  374 
Myxoid  cysts,  624 

Nails,  837 

mineral  substances  of,  838 
Naphthalene,   fate  of,    in  organism,  779, 

785 
Napthoresorcin,   reaction   for   conjugated 
glucuroric  acid,  218,  223, 

818 
reaction  for  levulose,  218 
Naphthylisocyanate  compounds  of  amino- 
acids.     See  various  Amino-acids. 


Narcotics,  relation  to  glycogen  formation, 

394 
Neosine,  578 
Neossin,  171 
Neottin,  631 
Neozym,  320 

Nephrorosein  in  urine,  734 
Nerve  fibres,  analysis  of,  614 
Nerves,  604-615 
Neubauer's    and    Rolide's    reaction    for 

proteins,  100 
Neuberg's  test  for  levulose,  218 
Neuberg-Rauchwerger's  reaction  for  cho- 

lesterin,  444 
Neuridine,  606,  612,  628 
Neurine,  246 
Neurochitin,  614,  615 
Neuroglia,  601 

Neuroglobulin,  a  and  /3,  604,  605 
Neurokeratin,  605,  614,  in  various  nerves, 

amounts  in,  614 
Nicotine,  effect  upon  gases  of  stomach, 

485 
Ninhydrin  reaction,  101 
Nitrates  in  urine,  766 
Nitriles,  fate  in  organism,  775 
Nitrobenzaldehydes,  fate  of,  in  organism, 

783,  784 
Nitrobenzene,  fate  of,  in  organism,  782 
Nitrogen  colloidal,  795 

elimination  in   starvation,   893, 
894 
through  various 

channels,  880,  881 
gaseous    elimination    of,  881. 
See  also  Gases  in  various  tis- 
sues and  fluids. 
Nitrogenous  equilibrium,  918,  919,  934 
Nitrotoluol,    behavior  in    animal     body, 

786 
Novaine,  577 

in  urine,  758 
Nuclease,  504,  508 

Nucleases,  48,  182.     See  also  Enzymes. 
Nucleinases,  182 
Nuclein  bases,  186 

in  lymphatic  glands,  366 
plates,  308 
in  pus-cells,  365 
true,  176 
Nucleins,  176,  177 

action  of  trypsin  on,  508 
effect  of  gastric  juice  on,  473 
pseudo-,  177 
Nucleoalbumins,  91,  92,  93,  177 

detection  of  in  urine,  795 
properties  of,  104 
in  urine,  794 
Nucleohistone,  307 

in  blood,  316 
in  urine,  795 
of  thymus,  367,  368 
Nucleon,  578.  620,  666 
Nucleo  proteins,  92,  167,  174-177,  252 


GENERAL  INDEX 


1013 


Nucleo  proteins,  action  of  trypsin  on,  508 
cleavage  products  of ,  177— 

195 
of  blood,  258 
of  milk,  653 
of  the  spleen,  369 
peptic  digestion  of,  472 
Nucleosidases,  182 
Nucleosides,  180,  211 
Nucleosin,  196 
Nucleotides,  179 

mono-,  179 
poly-,  179 
Nucleotin,  179,  182 
Nucleotoprotamines,  174,  623 
Nutritive  need  of  man,  916 
requirement,  908 
Nylander  Almen's  test,  215 

sugar  test,  802 

Obermayer's  indican  test,  729 

Obermuller's  cholesterin  reaction,  445 

Oblitine,  578 

Ochronose,  550 

Octodecapetide,  85,  87 

Oedematous  fluid,  and  its  constituents,  363 

Oesophageal  fistula,  459 

Oil-turpentine,  behavior  in  animal  body, 

749,  784 
Olein,  236 

mono  and  tri  enzymotic  synthesis 
of,  60 
Oligemia,  340 
Oligocythemia,  340 
Oliguria,  771 
Olive  oil,  absorption,  544 

effect  upon  bile  secretion,  415 
Onuphin,  171 
Oocyan,  636 
Oorodein,  636 
Opalisin,  652 

Opium,  passage  of,  into  milk,  670 
Organic  phosphorous  compounds  in  urine, 
757 
in  urine,  762 
sulphur  compounds  in  urine,  752, 
765 
Orcin-hydrochloric  acid  test,  209,  223 
Orcin,  test  for  pentoses  in  urine,  817 
Oriental  bezoar-stone,  524 
Ornithine,  85,  162,  163 
Orthonitrobenzaldehyde,    fate  of    in   the 

body,  784 
Orthonitrophenylpropiolic    acid,   test    for 

dextrose,  etc.      See  Acid   nitrophenyl- 

propiolic. 
Orthonitrotoluene,  fate  of,  in  organism,  785 
Osamines,  204 
Osazones,  202,  203,  215 
Osimines,  201 
Osmometer,  16 
Osmosis,  5 
Osmotic  pressure,  1-13 

determination  of,  16 


Osmotic  pressure,  effective,  12 

effect   of   various   sub- 
stances upon,  17 
electrolytes,    abnormal, 

due  to  ionization, 
in  higher  animals,  13 
in  lower  sea  animals,  13 
in  Lymph,  13 
in  milk  and  bile,  13 
non-electrolytes,  4 
of  animal  fluids,  12 
of  colloids,  16-18 
of  the  blood,  in  selachii, 

13 
in  saliva,  13 
in  urine,  13 
Osones,  203 
Ossein,  551 

Osseoalbuminoid,     elementary     composi- 
tion of,  551 
Osseomucoid,  172,  551 

elementary  composition  of. 
551 
Osteomalacia,  556,  557 
Osteosclerosis,  556 
Otoliths,  619 
Ovalbumin,  633-635 

cleavage  products  of,  106 
preparation  of,  634 
Ovaries,  623 

cysts  in,  624 
Ovin,  631 

Ovoglobulin,  84,  633 
Ovokeratin,  114 
Ovomucin,  634 

Ovomucoid,  171.  633,  635,  636 
properties,  635 
preparation,  635 
Ovovitellin,  84,  91 

composition  of,  628,  629 
preparation  of,  630 
Ovum,  623,  628 
Oxamide,  83 
Oxidases,  188 

artificial,    873.     See    also    En- 
zymes 
Oxidation  in  animal  tissues,  theories  of,  42 

in  the  organism,  850-877 
Oxidones,  874,  875 
Oxydase,  47,  48 

Oxygen  absorption,  calculation  of,  889 
activation,  4-8 
tension  in  blood,  859-861 
tension,  regulation  of  in  organism, 
868 
Oxygenases,  872 
Oxyhaematin,  289-291 
Oxyhaemocyanin,  299 
Oxyhemoglobin,  276,  278-282 

absorption  bands  of  281, 

282 
action  of  trypsin  on,  508 
effect  of  gastric  juice  on, 
473 


1014 


GENERAL  INDEX 


Oxyhemoglobin,  law    of    dissociation    of, 
859 
preparation  of,  282 
See    also    Blood    pig- 
ments. 

Oxyphenylethvlamine,  82 

Oxyproline,  85,A106,  107,  125,  155 

Oxyprotein,  83 

( )xypurine,  190 

Oxypyrimidine,  195 

Oxyquinolines,  fate  of,  in  organism,  785 

Ozone,  occurrence  in  the  organism,  4 

Palmitin,  235 
Pancreas,  494-510 

importance  of,  to  absorption  of 
carbohydrates,  535 
.Pancreatic  diastase,  501 

gland,  constituents  of,  495 
juice,  494-501 

action  on  fats,  502 
action  of,  on  starch,  226 
amount  secreted,  500 
constituents  of,  500 
excitants  for  the  secre- 
tion of,  498 
properties  of,  500 
lipase,  57.     See  also  En- 
zymes, 
secretion  of,  496 
rennin.     509.     See     also     En- 
zymes, 
secretion,  human,  500,  501 
Papain,  51 
Paracasein,  650 
Parachymosin,  474,  567 
Paracresol,  515 

formation  in  putrefaction,  515, 
723,  724 
Paraglobulin,  258 
Parahsemoglobin,  280 
Parahistone,  109 
Paralbumin,  624,  625 
Paraminophenol,  779 
Paramucin,  626 
Paramyosinogen,  568,  570,  571 
Paranuclein.     See  Pseudonuclein 
Paraovarial  cysts,  627 
Paraoxypropiophenone,  785 
Parapeptone,  472 
Parathyroids,  377 

Paraxanthine  in  urine,  712,  713,  714         1 
Parenchymatous  organs,  action  of  trypsin 

on,  508 
Parenteral    canal,    introduction    of    pro- 
tein by  way  of,  533-511 
Parotid,  448 

saliva,  450 
Peaglobulin,  84 
Pectin  bodies,  229,  231 
Pemphigus  chronicus,  359 
Pennac-frin,  S  10 
Pennatulin,  123 
Pentacrinin,  844 


Pentamethylenediamine,  47.     See  Cada- 

verin 
Pentapeptides,  85 
Pentosamines,  220 
Pentosans,  207 

Pentoses,  197,  198,  199,  207-211 
in  urine,  816 

quantitative  estimation,  209 
test,  orcin-hydrochloric,  209 
tests  for,  209 
Pentosides,  180 
Pentosuria,  816 

Penzoldt's  test  for  acetone,  823 
Pepsin,  57,  64,  466-476 

action  on  proteins,  468-473 
characteristic  property  of,  468 
digestion  of  proteins,  products  of, 

472 
digestion,   effect   of   foreign   sub- 
stances upon  rapidity  of,  470 
digestion,  rapidity  of,  471 
digestion  of    various  bodies,  472, 

473 
mother  substances  of,  477 
nature  of,  467 
occurrence  of,  466 
preparation  of,  468 
properties  of,  467,  468 
quantitative  methods  for,  469,  470 
relation  to  rennin,  475 
test  for,  469 

testing;  for,  in  gastric  juice,  487 
Pepsinogen,  477 

Peptases,  503.     See  also  Enzymes. 
Peptic-glutin  peptone,  135 
peptones,  135 

zymolysis,  effect  of  bile  upon,  511 
Peptidases,  48 
Peptides,  68,  85-89,  93,  126,  127,  156 

behavior    toward    enzymes,    8, 
266,  492,  510,  511.     See     Poly- 
peptides, 
in  urine,  755 

relation  to  alcaptonuria,  735 
relation  to  urea  formation,  777 
Peptinuria,  791 
Peptochondrin,  549 
Peptoid,  507 
Peptone-plasma,  251 
Peptones,  92,  127-132 
anti-,  132 
as  foodstuff,  912 
carbamino  reaction  with,  167 
from  peptic  digestion  472 
in  urine,  791 
properties  of,  131 
quantitative  estimation,  138 
separation  from  proteoses,  136, 
138 
Percaglobulin,  637 
Pericardial  fluid,  356,  357 

analysis  of,  356 
Perilymph,  619 
Peritoneal  fluid,  357 


GENERAL   INDEX 


1015 


rermeability,  causes  for, 

of  blood  corpuscles,  7 

( hrerton'a  solubility  theory, 

10 
shortcomings  of,  10,  11 
Traube's   solution   tenacity 
theory,  1 1 
Peroxidases,  872.     See  also  Enzymes. 
Peroxydase,  50 
Perspiration,  847-849 

circumstances      influencing, 

847 
constituents  of,  848 
properties  of,  847 
Pettenkofer's  method     for     determining 
respiratory  change,  869 
Pit  test  for  bile-acid,  798 
test  lor  bile,  418 
Pfaundler's  method  of  precipitating  urine, 

681 
Phaseomannite,  579 
Phenol,  515 

as  precipitant  of  proteids,  98 
in  urine,  515,  723,  726,  785,  787 
Phenol-oxidases,  875 
Phenols,  117,  723 

fate  of,  in  organism,  775,  784 
Phenylalanine,  85,  109,  114,  151,  152 

amounts  in  proteins,  106, 
107,  113,  115,  125 
Phenylethylamine,  82 
Phenylieocyanate  compounds     of     amino 
acids.      See    various 
Acids,  amino, 
peptone,  138 
Phenylmethvlketone,  785 
Philothion,  876 
Phlebm,  276 

Phosphates,  importance  of,  in  metabolism, 
902 
in  urine,  761-764 
in   urine,    quantity   of,    762 
See  also  Mineral  substances. 
Phosphatides,  239-249,  265,  586 

in  adrenal  bodies,  377 
in  bile,  435,  439 
in  kidney,  673 
in  liver,  385 
in  yolk,  630 
Phosphaturia,  763 
Phosphoglycoproteins,  174,  177 
Phosphoproteins,  91,  92,  93 

properties  of,  104 
Phosphorus  compounds,  action     on     bile, 
437,  441 
action  on  blood, 

252,  254 
action     on     urea 
elimination, 
679,  685 
organic,  in  urine, 

756 
metabolism,  875, 
876,  894 


Phosphorus-containing   urinary    constitu- 
ents, 756,  875 
elimination  in  organism,  883 
elimination   in   relation  to  ni- 
trogen elimination,  762,  763 
elimination  in  relation  to  pur- 
ine metabolism,  7*13 
Phosphatides,  properties  of,  241 
Photometha;moglobin,  2.S4 
Phrenin,  612 
Phrenosin,  609,  610,  611 
Phyllocyanin,  295 
Phylloervthrin,  435 
Phylloporphyrin,  276,  295 
Phymatorhusin,  841 

in  urine,  799 
Physical  chemistry  in  biology,  1 
Physiological  oxidation  processes,  871-877 

salt  solution,  7 
Phytase,  579 
Phytin,  579 
Phytosterines,  446 

Picolin,  behavior  of,  in  animal  body,  784 
Pigments,  fate  of,  in  organism,  787 
medicinal  in  urine,  801 
of  bile,  427,  433,  435,  436 
of  birds'  eggs,  636 
of  blood,  275,  298 
of  blood  serum,  268 
of  butterflies,  844 
of  corpora  lutea,  297,  623 
of  eye,  614,  616 
of  feathers.  843,  844 
of  human  skin,  840-843 
of  liver,  384 
of  lobster,  638,  844 
of  lower  animals,  299,  844 
of  lungs,  870 
of  muscle,  571 
of  placenta,  641 
of  pus,  366 
of  skin,  840-844 
of  stones,  441 
of  urine,  733.  734,  740,  748,  798, 

799 
of  yolk,  631 
Pilocarpine,  effect    upon    elimination    of 
C02  in  stomach,  485 
effect    upon    elimination    of 

uric  acid,  700 
effect   upon   secretion   of  in- 
testinal juice,  490 
effect  upon  secretion  of  sali- 
va, 457 
Piperdine-glvcosuria,  403 
Piquie,  402 

Pina's  test  for  tvrosine,  153 
Placenta,  641 
Plant  cells,  5 

osmotic  experiments  with,  5 
gums,  226 
Plasmolysbg   power   of   differently   con- 
structed salts,  6 
Plasmolysis,  5 


1016 


GENERAL  INDEX 


Plaamolvsis,  substances  bringing  about,  5 
Plasmolytic     experiments     with     animal 

cells,  7 
Plasmoschisis,  314 
Plasmozym,  319 
Plastein,  59 
Plasteines,  135 
1  lastin,  178 

Plattner's  crystallized  bile,  396 
Pleural  fluid,  357 
Pnein,  874 
Pneumonic  infil .rations,  solutions  of,  18, 

368,  86j 
Poikilocytosis,  Oj.2 
Polycythtc..  ia,  3*0 
Polynucleotides,    179 
Polypeptide-like  1  odies  in  urine,  756 
Polypeptides,  bd-ui,  92.     See  Peptides, 
action  of  try]  sin  on,  509 
cleavage  of,  by  enzymes,  62 
E.      Fischer's      synthetical 

preparation  of,  88 
methylated  b8 
syntheses  ol ,  86 
synthetically  produced,  87 
sulphur  containing,  88 
synthetic,     comparison     of 
properties  with  natural  pro- 
teins, 90.     See  also  Pep- 
tides. 
Polyperythrin,  844 
Polysaccharides,  colloid,  226-231 
Polyuria,  771 

Potassium  in  urine,  766.     See  also  Min- 
eral substances. 
Praglobin,  307 

Precipitation,  Traube's  membrane,  1 
Precipitins,  66 
Preglobulin,  315,  368 
Preputial,  secretion  of  skin,  846 
Proenzymes,  51 
Prolamine,  78 
Prolamins,  106 
Proline,  85,  109,  111,  154.  155 

amounts   in    protsins,    106,    107, 
113, 115,  125 
Propepsin,  477 
Propeptone,  126 
Propylalanine,  85 
Propyl  benzene,  behavior  in  animal  body, 

778 
Propylene    glycol,    relation    to    glycogen 

formation,  394 
Prosecretin,  463,  499 
Prostate,  secretion  of,  621 
Prostatic  concrements,  172,  611,  623 
Prosthetic  group,  174 
Protagon,  605,  606-608.  609 

cleavage  prod  rets  of,  607 
elementary  composition  of,  606 
nature  of,  606,  607 
preparation  of,  608 
properties  of,  608 
Protamine  nucleate,  623 


Protamines,  91,  92,  93,  108 

hexone  bases  in,  165 
preparation  of,  112 
properties  of,  109-112 
Proteans,  93 
Protease,  47,  48 

alpha  retardation  of,  63 
Proteases,  48 

Protective  colloids,  37,  44,  131 
Protein,  absorption,  effect  of  cellulose  on, 
531 
catabolism,  907,  908,  909,  911 

importance  of  sulphur 

in,  882,  883 
in  active  muscle,  594 
in  starvation,  894 
destruction  of,  during  work,  595 
ochrome,  155 
requirement,  933-936 

lower  limit,  915J 
sparers,  .913-915 

substances,  diarginylalanine,  111 
diarginylproline,  111 
diarginylserine,  111 
diarginylvaline,  111 
Proteins,  38,  77-195 

absorption  of,  525-531 
acid,  125 

action  of  neutral  salts  on,  98 
action  of  nitrous  acid  upon,  79 
action  of  pepsin  on,  468-473 
action  of  trypsin  on,  505,  507 
adsorption  compounds,  95 
adsorption  of,  97 
albuminates,  104 
albuminoids,  112 
albuminous,  102-138 
albumins,  106 
albumose,  alkali,  127 
alcohol  soluble,  92 
alkali,  126 

amino  acids  in,  linking  of,  90 
amounts  in  blood   of  different 

animals,  328 
analysis,  118 
atmidkeratin,  113 
atmidkeratose,  113 
Bence-Jones,  792 
byssus,  122,  123 
carbon  dioxide  binders  in  blood 

855 
casein,  106 
chitin,  122 
chondrin,  121 
chondro-,  168,  172-174 
chromo-,  167 
circulating,  908-910 
classification  of,  91-93 
cleavage  products,  93,  106 
clupeine,  110 
coagulated,  93 
coagulation  of,  96 
collagen,  118,  119 
color,  reaction  for,  99 


GENERAL  INDEX 


1017 


Proteins,  composition  of,  77,  94 
compound.  Ill',  ld7-177 
concliiolm,  122,  12:1 
conjugated,  (.*2,  93 
cornein,  122,  123,  124 
cornicrystalline,  123 
crystalline,  94 
cyclopterine,  110 
cyprinine,  1 10 
derived,  93 
deutero,  120 
deuteroelastose,  117 
effect   on   glycogen   content   of 

liver,  394,  397 
elastin,  116-118 
fate  of,  in  organism,  886 
fattening,  919 
fibrin,  106 
fibroin,  122,  124  . 
foodstuff  purposes  of,  917,  918 
forms  of  nitrogen  in,  77,  78 
gelatin,  118,  119-121 
gelatin-peptones,  120 
gelatose,  120 
gelatoses,  120 
globan,  104 
globulins,  103-104 
gluco-,  167,  168-174 
glucoproteins,  phosphorized,  105 
gluteins,  119 
glyco-,  92,  167,  168-174 
gorgonin,  123 
helico-,  174 
hemicollin,  120 
hemielastin,  117 
hordein,  cleavage  of,  107 
hydrolysis  of,  81 
ichthylepidin,  122 
in  amniotic  fluid,  642 
in  aqueous  humor,  361 
in  ascitic  fluids,  359 
in  bone,  551 
in  bone  marrow,  554 
in  brain,  604 
in  cartilage,  546 
in  cerebrospinal  fluid,  360 
in  chyle,  347 
in  connective  tissue,  544 
in  crystalline  lens,  617,  618 
in  cystic  fluids,  625-627 
in  dead  muscles,  567 
in  fat  globules,  647 
in  horn  substance,  837 
in  hydrocele  and  spermatocele 

fluids,  359 
in  kidneys,  672 
in  liver,  382,  383 
in  lungs,  870 
in  lymph,  346,  348 
in  lymphatic  glands,  366 
in  mammary  glands,  643 
in  milk,  647-654 
in  milk  plasma,  647 
in  muscles,  566-572 


Proteins,  in  muscle-plasma,  569,  570 

in  pancreatic  gland,    l{'."> 
in  pericardial  Quid,  356 

in  placenta,  til  1 

in  prostate  secretion,  621 

in  pus  corpuscles,  364,  365 

in  pus-serum,  363 

in  retina,  615 

in  salivary  glands,  451 

in  sebum  of  .skin,  844 

in  semen,  620 

in  smooth  muscle,  602 

in  spermatozoa,  623 

in  sputum,  S71 

in  synovial  fluid,  362 

in  testes,  620 

in  thymus,  366,  367 

in  transudates  and  exudates, 
353,  354 

in  urine,  787-795 

in  urine,  detection  of,  788 

in  urine,  quantitative  estima- 
tion of,  in  urine,  793 

in  veins,  336 

in  venous  blood,  336 

in  white  of  egg,  633-636 

in  yolk,  628-630 

iodo.  123 

keratins,  112-116 

koilin,  115 

lecithalbumins,  105 

lecitho,  92 

Lieberkuhn's  solid  alkali  albu- 
minate, 126 

metabolism  calculation  of,  889 

metabolism  of,  in  starvation, 
893, 894 

metabolism  with  food  rich  in 
proteins,  906,  913 

method  of  synthesis,  86 

membranins,  617 

modified,  96 

molecular  weight,  98 

mucins,  true,  analysis  of,  168- 
172 

mucinoi  Is,  171 

mucoids,  171 

native,  96 

Neubauer  and  Rohde's  test, 
100 

nuclei  of  aliphatic  series,  85 

nuclei  of  carbocyclic  series,  85 

nuclei  of  heterocvclic  series,  85 

nucleo,  92,  105,  167,  174-177 

nucleo  albumins,  104,  105 

ovokeratin,  114 

ovovitellin,  105,  106 

parenterally  introduced,  absorp- 
tion of,  525 

pennatulin,  123 

phospho,  92,  104 

phosphoglvcoproteins,  174,  177 

phosphorized  importance  of,  in 
metabolism.  902 


1018 


GENERAL  INDEX 


Proteins,  precipitation  of,  95,  96,  98,  99 
preparation,  118,  121 
prolamine,  106 
properties  of,   94-96,    107,   108, 

119 
protamine,  constitution  of,  111 
protamines,  109-112 
proto-,  120 
protoelastin,  117 
protones,  110 
pseudomucins.  171 
pseudonucleins,  104 
putrefaction  of, 
putrefactive  products  of,  82 
quantitative  estimation,  101 
reaction  of,  91 
reactions,  precipitation,  98 
reticulin,  121,  122 
salmine,  110 
salting  out  of,  95 
scombrine,  110 
semiglutin,  120 
seralbumin,    cleavage    products 

of.  106 
[serglobulins,  cleavage  of,  106 
sericin,  122,  124 
simple,  91,  92,  93,  94 

cleavage     products     of, 
125-195 
skeletins,  122 

constitution,  122 
source  of  muscular  energy,  595- 

597 
Bpongin,  122,  123 
sturine,  110 
sulphur  content, 
syntheses    from    amino    acids, 

529,  530 
synthesis  of,  in  organism,  530 
syntheticallv  formed,  59 
tests  for,  99,  100,  101 
tissue,  908-910 
xanthoprotein  reaction,  99 
zein,  cleavage  products  of,  107 
Proteose-like  substances  in  blood  serum, 

264 
Proteoses,  92,  127-132 

absorption,  534,  538 

as  foodstuff ,  912 

carbamino  reaction  with,  167 

detection  of,  in  urine,  792 

deutero,  130 

dys,  130 

from  peptic  digestion,  472 

gluco-,  134 

hetero   130,  134 

in  blood,  262,  263,  534,  535 

in    intravascular    coagulation, 

in  lungs,  870 

in  stomach,  483.  484 
in  urine,  791 
primary,  130 
properties  of,  131 


Proteoses,  proto-,  130,  134 

quantitative  estimation,    138 

secondary,  130 

separation  from  peptones,  136, 

138 
syn-,  134 
Prothrombin,  255,  256,  257,  312,  314,  315, 
317 
in  circulating  plasma,  318 
Protocaseoses,  129 
Protoelastose,  117 
Protogelatose,  119 
Protogen,  126 
Protokyrins,  138 
Protones,  110 
Protoplasm,  5 

Protoproteose,  131,  133,  135 
Protosyntonose  hexone  bases  in,  165 
Proto-toxoids,  71 
Pseudocerebrin,  611 
Pseudoglobulin,  259 
Pseudohsemoglobin,  283 
Pseudomucin,  171 

beta,  625,  626 
detection  of,  626 
hydrolytic    cleavage   prod- 
ucts of,  626 
Pseudonuclein,  629;  651 
Pseudonucleins,  104,  177 
Pseudopepsin,  464,  489 
Pseudoxanthine,  578 
Psittacofulvin,  843 
Psylla-alcohol,  845 
Ptomaines,  46,  82 

in  urine,  757,  758 
Ptyalin,  48,  456-460 

action  of,  456,  457 
best  reaction  for,  457 
condition  of,  in  the  intestine,  510 
effects  of  foreign  substances  upon, 

457,  458 
preparation  of,  456 
Purine  bases,  186-193,  267,  715 
detection  of,  193 
in  active  muscles,  594 
in  adrenal  bodies,  377 
in  spleen,  370 
preparation  of,  193 
quantitative  estimation  of, 

715 
transformation     into     uric 
acid,  702,  703 
bodies,  in  pus-corpuscles,  364 

composition  of,  187 
oxidases,  875.     See  Enzymes, 
skeleton,  186 

structural  formula  of,  186 
tri-oxy-,  2,  6,  8,  187 
Purines,  in  lymphatic  glands,  366 
in  thymus,  36S 
in  thyroid  gland,  373 
Pus,  363 

cells,  analysis  of,  365 
corpuscles,  364-366 


GENERAL  INDEX 


1019 


Pus,  constituents  of,  364 
in  urine,  7W 
serum,  363 

analyses  of,  363,  364 
analyses  of  ash  of,  364 
Putrefaction,  43,  Hi 

factors  influencing,  516-520 
in  intestines,  importance  of, 

517 

preventive    substances  for, 

518 

Putrefactive  products,  absorption  of,  515 

conjugation  of,  515 

elimination  of,    in 

urine,  515 
in   large  intestine, 
515 
Putrescine,  47,  82,  162 

in  urine,  827 
Putrine,  47 
Pyin,  363,  366 
Pyloric  secretion,  478 
Pyocyaneus  protease,  57 
Pyocyanin,  366 
Pyogenin,  364 
Pyosin,  364 
Pyoxanthose,  366 

Pyrazine,  formation  from  glycocoll,  220 
Pyridine,  fate  of  in  organism,  787 

structural  formula  of,  186 
Pyrimidine,  193 

bases,  177,  178,  193-195 
Pyrin,  357 

Pyrocatechin  in  urine,  727 
Pyrrol  derivatives,    154,    158,   276,  290, 

740 
Pyrrolidone-carboxylic  acid,  113 


Quercinite,  581 

Quercite,  394 

Quinidine,  as  catalyst,  60 

Quinine,  as  catalyst,  60 

effect  upon  spleen,  375 

Cal 
■Quotient,  T  ^r.  ,  888 

Cal      ftfi 

LA'888 

—  892 

C  to  N,  771 

flesh,  602 

nitrogen  to  homogentistic  acid, 

735 
respiratorv  quotient,  412,  563, 

593,  879,  887,  918 
urea  to  nitrogen,  777,  877 
calorific  urine,  892 

^,887 

C 


N 


in  feces,  £84 


Quotient,  ^,  in  urine,  771 

N 

=-,  in  urine,  883 

N 

■5-,  in  urine,  882 

ft 

calculation  of,  889 
in  starvation,  895 
respiratory,  887,  927,  928 


Rachitis,  556,  557 

Reaction,  of  a  solution,  determination  of 
74 
velocity,  32 
Reactivation  enzymes,  52 
Receptors,  67 

Reducing   substances   in  urine,   749-752 
Reductase,  47,  48 
Reductases,  876,  877 
Reductions  in  animal  body,  876 
Reductonovaine  in  urine,  758 
Regnault-Reiset's  method  for  determining 

respiratory  exchange,  869 
Reichert-Meissl's  equivalent  for  fats,  238 
Rennet,  649 
Rennin,  48,  49,  57,  474-476 

action  of  on  charcoal,  62 

anti,  64,  69 

occurrence  of,  474 

pancreatic,  509 

preparation  of.  475 

properties  of,  475 

relation  to  pepsin,  475 

retardation  of,  63 

retarding  substances  for,  474 

testing  for  in  gastric  juice,  487 

See  also  Enzymes, 
zymogen,  474 
Resacetophenone,  785 
Resins,  fate  of  in  organism,  787 
Respiration,  850-877 

apparatus,  experiments  with, 

889,  890 
external,  858 

gas  tension  in  blood,  859-868 
importance  of  haemoglobin 
in  oxygen-carbon  dioxide 
exchange  in  blood,  853 
internal,  858,  867 
mechanism  of  carbon  dioxide 

elimination,  852, 853 
processes  taking  part  in  the 
gas  exchange,  858 
Respiratory  exchange,  methods  for  deter- 
mining. 86S-S70 
quotient  in  active  muscle,  596 
in  diabetes  412-413 
Rest  carbon,  268 
Rest  nitrogen,  in  serum,  267 

in  stomach,  483,  484 
and    work,    effect   on   metabolism, 
926-928 


1020 


GENERAL  INDEX 


Reticulin,  92,  121,  544 

composition  of,  121 
in  lymphatic  glands,  336 
properties  of,  121 
preparation  of  J  122 
Retina,  constituents  of,  615 

pigments  of,  615-617 
Reynold's  test  for  acetone,  823 
Rhamnose,  208 

relation  to  glycogen  formation, 
394 
Rhodophan,  617 
Rhodopsin,  615 

Ribose,  structural  formula  for,  199,  211 
tf-Ribose,  178 
Ricin,  470,  505 

in  testing  pepsin,  470 
Rigor  mortis,  601 

of  muscles,  588-591 
Ringer-Locke's  solution,  75 
Ritthausen's  method  of  determining  pro- 
teins in  milk,  656 
Roch's  test  for  proteid,  995 
Rosenbach's  bile  pigment  test,  799 

urine  test,  827 
Rotation,  specific,  199 
Rovida's  hyaline  substance,  274,  302,  367 
Rubner's     reaction     for     dextrose,     215 
sugar  test  in  urine,  809 

Saccharase,  48,  49,  57.     See  also  Enzymes, 
action  of  charcoal  on,  63 
retardation  of,  63 
Saccharides,  mono,  197-218 
di,  197,  223-226 
poly,  crystalline,  197 
poly,  colloid,  197,  226 
tri,  197-226 
Saccharose,  223,   224,  225.     See  Sucrose. 
Sachsse's  reaction  for  dextrose,  295 
Sahidin,  609 
Sahli's  hsemometer,  299 
Saliva,  the,  451-460 

action  of,  in  stomach,  480 

on  starch,  226 
chorda,  452 
constituents  of,  453 
diastatic  power  of,  71 
gases  of,  857 

human,  quantitative   composition 
of,  458 
quantity  secreted,  459 
mixed  buccal,  constituents  of,  455 
paralytic,  452 
parotid,  453,  454 
physiological    importance  of,  459, 

460 
properties  of,  453,  454,  455 
sublingual,  453 
submaxillary,  452 
sympathetic,  452 
Salivary  concrements,  460 
Salkowski's  creatinine  reaction,  696 

reaction  for  chloesterin,  444 


Salmine,  110 

Salt  action  on  enzymes,  70-76 
glycosuria,  402 
plasma,  251 
Samandarin,  846 
Sandmeyer's  method  of  inducing  pancreas 

diabetes,  405 
Santonin,  elimination  of  in  urine,  787,  800 
Sapokrinin,  499 
Saponin,  273,  446,  447 
Sarcolemma,  relation  to  permeability,  9 
Sarcomelanin,  841 
Sarcosine,  fate  in  organism,  786 
Sarkine.     See  Hypoxanthine,  190 
Sarkosine,  776 
Schalfejeff's,  haemin,  292 
Scherer's  inosite  test,  578 
Schiff  s  reaction,  686 

reaction  for  cholesterin,  445 
Schreiner's  bases,  621 
Schutz-Borrissow's  rule  of  ferment  action,. 

54,  471,  476,  503,  506 
Schutz's  rule,  58 
Schweitzer's  reagent,  231 
Scleroproteins,  93  ^ 
Sclerotic,  619 
Scombrine,  110 
Scombron,  108 
Scyllite,  370,  581 
Scymnol,  418 
Sebelien's  method  of  determining  proteins 

in  milk,  656 
Sebum  of  skin,  844 
Secretin,  463,  492,  498,  499 
Secretion  enzymes,  52 

of  prostate,  621 
Sedimentum  lateritium,  708 
Segregation,  52 
Seliwanoff's    reaction    for    levulose,   21Sr 

815 
Semen,  620,  621 

Semicarbazide,  poisoning  therewith,  718 
Semiglutin,  119,  120 
Seminose,  216,  217 
Semi-permeable,  membrane,  1 
Senna,  elimination  of  coloring  matter  of, 

in  urine,  787,  802,  827 
Sensibilizators,  69 
Sepia,  381,  841,  843 
Sepsine.  47 

Seralbumin,  84,  91,  252,  261-264 
crystalline,  262 
composition  of,  262 
elementary    composition    of,. 

263 
preparation  of.  262,  263 
properties  of,  262,  263.     See- 
also  Proteins, 
quantitative    estimation    of, 
263 
Serglobulin,  84,  91,  252,  258-261 
detection  of,  261 
elementary    composition    of, 
263 


GENERAL  INDEX 


1021 


Serglobulin,  para-,  258 

preparation,  259,  201 

properties,  260 
quantitative  estimation,  261. 
See  also  Proteins. 
Sericin,  92,  122,  124 
Serine,  106,  107,  111,  113,  115,  125,  145 

is<>-,  146 
Serolin,  265 
Seromucoid,  261,  264 
Serosamucin,  354 

Serum,    casein,     258.      See    Serum    glo- 
bulin. 
normal.     See  Blood  serum. 
Sham  feeding,  450,  464,  821 
Shark,  bile,  41(1,  433,  681 
urea,  334,  433,  681 
Shell,  membrane  of  hen's  egg,  636-641 
Side-chain  theory,  71 
Siegfried  and  Zimmermann's  method  for 

phenol-sulphuric  acids,  726 
Siegfried's  earbamino  reaction,  166 
Silk  gelatin,  123,  124 
Sinistrin.  animal,  174 
Skatol,  46,  82,  117,  157-159,  515 
522,  723,  732,  843 
fate  of  in  organism,  786 
Skatosine,  159 
Skatoxyl,  724 

tests  for,  732 
Skeletins,  122 

composition  of,  122 
Skin,  837-849 

exchange  of  gas  through,  849 
horn  formations  of,  840 
human,  pigments  of,  840-843 
melanins  of,  841 
perspiration  of,  847-849 
pigments  of  840-844 
sebum  of,  844 
secretion,  preputial,  846 
secretions  of,  837-849 

of  various  animals,  846 
Smegma  praputii,  844 
Snake  poison,  effect  upon  blood,  255,  310, 

325 
Soaps,  265 

in  chyle,  347 
Sodium    alcoholate    as    a    saponification 
agent,  234,  237 
compounds,  division  among  form 

elements  and  fluids,  22,  23 
in  urine,  766.    See  Mineral  sub- 
stances. 
Solanin,  273 
Solution  tenacity,  10 

in  relation  to  Osmosis  Relation- 
ship to  surface  tension,  1 1 
Sorbin,  218 
Sorbinose,  218 
Sorbite,  198 
Sorensen's   formol   titration    for   amino- 

acids,  166 
Specific  dynamic  action,  918 


Speck's  method    for  determining  respira- 
tory exchange,  860 
Spermaceti,  238 

oil,  238,  239 
Spermatin,  623 
Spermatocele  fluids,  359,  360 
Spermatozoa,  020,  622,  623 

properties  of,  622 

constituents  of,  622 

Florence's  test  for,  621 
osmotic  phenomena  with,  8 
Spermine,  621,  022 

crystals,  Bottcher's,  621 
Sphygmogenin,  378 
Sphyngomyelin,  608,  609 
Sphyngosin,  011 
Spiegler's  test  for  proteids,  789 
Spinal  marrow,  014 
.Spirograph  in,  171 
Spleen,  369-373 

analyses  of,  372 
constituents  of,  370 
pathological  processes  in,  372 
physiological  functions  of,  372 
uric  acid  formation  in,  702,  703 
Spongin,  92,  122,  123 

iodo-,  123 
Sponginoses,  122 
Spongosterin,  449 
Sputum,  871 

form  constituents  of,  871 
Stachyose,  226 
Starch,  220-228 

artificial,  227 
cellulose,  227 
granulose,  hydrolysis,  228 
gum,  229 
soluble,  227 
Starvation,  bone,  catabolism  in,  896 
catabolism  of  fat  in,  894 
gas  exchange  in,  895 
loss    in    weight    of    different 

organs  in,  S90 
metabolism,  892-906 
metabolism  of  fats  in,  894 
mineral    substances    in,    895, 

896 
nitrogen  content  of  urine  in, 

897 
nitrogen  elimination   in,  893, 

894 
protein  in,  894 

metabolism     in,    893, 
894 
time   interval    to  death,   892 
urea  content  in,  897 
urinary  constituents  in  starva- 
tion, 899 
water  in,  896 
Steapsin,  476,  501-503 

effect  upon,  of  bile,  51 1 
Steapsinogen,  478 

activation  of,  by  bile,  511 
Stearine,  234,  235 


1022 


GENERAL  INDEX 


-Steensma's   modification   of    Gunzburg's 

test,  487 
Stenson's  test,  590 
Stentorin  blue,  844 
Stercobilin,  428,  429,  529,  743 

in  feces,  532 
Stercorin,  448 
Stethal,  239 

Stoke's  reduction  fluid,  282 
Stomach,  absorption  of  cleavage  products 
in,  484 
action  of  salivary  diastase  in,  480 
contents.     See  Chymus. 
digestion     and     absorption     of 

various  food  in,  513,  514 
fistulas,  365,  460 
gases  in,  485,  486     ' 
glands,  458 

lactic  acid  fermentation  in,  485 
movement   of   food    in,    during 

digestion,  479,  480 
movement  within,  during  diges- 
tion, 479 
self-digestion  of,  486 
Stone-cystine,  148,  150 
Stroma-fibrin,  275 

of   blood    corpuscles,    712, 
273 
Stromata,  273,  274 
Strychnine,  and  glycogen  transformation, 

391,  394 
Sturgeon,  spermataozoa  of,  110,  181 
Sturine,  110 

hexone  bases  in,  165 
Stutz's  test  for  proteid,  794 
Subcutaneous  oedema,  fluid  of,  362 
.Sublingual  glands,  448 
saliva,  450 
Submaxillary  glands,  446 
Submicrons,  19 
Substrate,  47 
"Sucre  immediat,"  331 
"  Sucre  virtuel,"  331 

Sugar,    amounts    in    blood    of    different 
animals,  328 
amounts  in  blood,  331 
detection  of  in  urine,  802-808 
fats,  in  liver,  412,  413 
gelatin,  139 
glycogen,  393,  395 
in  aqueous  humor,  361 
in  ascitic  fluids,  359 
in  blood,  329,  330 
in  blood  serum,  265 
in  cerebrospinal  fluid,  360 
in  lymph,  346 

in  transudates  and  exudates,  355 
in  urine,  802-818 
in  venous  blood,  336 
proteins,  in  liver,  409-411 
Sulphaemoglobin,  286 
Sulphate  quantitative  estimation  of,   in 

urine,  765 
Sulphates,  amounts  in  urine,  765 


Sulphates  in    urine,    764-766.     See   also 
Mineral  substance, 
ethereal  in  urine,  765 
quantitative  estimation  of,  765 
Sulphatide,  609 
Sulphocyanides,  occurrence  in  urine,  752, 

775 
Sulphonal   intoxication,    urine,    294,    797 
Sulphur,  compounds  in  urine,  752,  753 

elimination,  •  importance    of    in 

protein  metabolism,  882,  883 
in    proteins,    78,    79.     See    also 

various  proteins, 
in  urine,  752,  874,  875,  890 
methsemoglobin,  286 
Sulphuretted  hydrogen  in  urine,  753 
Suprarenin,  387 

Surface  tension,  in  relation  to  osmosis.  10 
Suspension  colloids,  precipitation  of,  70 
Suspensoids,  properties  of,  15 
Sympathetic  saliva,  449 
Synovia,  360 

Synovial  cavities  around  joints,  fluid  in, 
360 
analysis  of,  362 
fluid,  362 
Synoviamucin,  362 
Synovin,  362 
Synproteose,  135 
Syntonin,  hexone  bases  in  ,165 
Syntoxoids,  71 

Taenia,  389 
Talose,  211 
Tartar,  460 
Tatalbumin,  632 
Taurine,  150,  151 

fate  of  in  organism,  786 
Tears,  619 

Teichmann's  crystals,  292,  796 
Tendon  mucoid,  544 

elementary  composition  of,  551 
Terpenes,  fate  of  in  organism,  785 
Testes,  620 
Tetanolysin,  72 

Tetanotoxine  and  gastric  juice,  485 
Tetraglyclyglycine,  85,  510 
Tetramethylene  diamine,  47.     See  Putras- 

cine. 
Tetrapeptides,  85,  87,  89,  510 
Tetronerythrin,  843,  844 
Tetroses,  197 
Theobromine,  187 
Theophylline,  187 

Thiophene,  behavior  in  animal  body,  784 
Thiotolene,  784 
Thrombin,  48,  256,  315,  317,  324 

action  of,  256 

formation  of,  318 

preparation  of,  257 

properties  of,  256 

pro-,  256 
Thrombogen,  318,  321 

formation  of,  322 


GENERAL   INDEX 


1023 


Thrombokinase.  318,  310 
Thromb< (plastic  substances,  326 
Thrombosin,  320 

-lime,  320 
Thrombozvm,  321 

formation  of,  322 
Thymine,  178,  181,  185,  195 
Thymus,  366-309 

analysis  of,  369 
constituents  of,  368 
functions  of,  369 
Thyreoglobulin,  375,  376 
Thyre< >pint ciil,  375 
Thyroid,  gland.  373-376 

constituents  of,  373 
effect  of  extirpation  of,  374 
Tissue,  connective,  analysis  of,  545 
elastic  analysis  of,  545 
fatty,  558-564 
fibrinogen,  307 
proteins.  906-910 
Tissues,  end  products  of  oxidation  in,  871 
gas  exchange  in,  858-870 
gelatinous.  545 
mucous.  545 
oxidation  in.  858 
physiological    oxidation    in,    871 
theories    for   oxidation    processes 
in.  871,  872 
Tollen's  reaction  for  pentoses,  209 

Roriye  test  for  levulose,  218 
test  for  glucuronic  acid,  223 
Toluene,  fate  of  in  organism,  779 
Toluylenediamine,    poisoning    with,    441 
Tonus,  chemical,  590 
Tooth,  cement,  557 
dentin,  557 
enamel,  557 
structure,  557 
Toxin,  cysts.  69 
Toxins,  46,  66 

a  method  of  measuring,  67 
Toxoid,  67 

proto,  syn,  epi,  67 
Toxons,  67 
Toxophore.  group.  07 
Tradescantiadiscolor,  6 
Transfusion,  fluid  for  mamallian  heart,  72 

composition  of,  72,  73 
Transudates  and   exudates,     constituents 
found  in.  353-355 
distinguishing  feature  in,  356 
formation  of.  352,  353 
osmotic  pressure  in,  356 
pathological,  gases  of,  857 
reaction  of,  356 
Trichohyalin.  837 
Triglycerides.  232 
Triglyclyglycin.  85.  510 

ethyl  ester,  86 
Trimethylamine  in  urine.  7">7 
Trimethylvinylammonium  hydroxide,  246. 

See  Neurine. 
Triolein,  233,  236 


Trioses,  197 

Trioxypurine,  2,  6,  8,  187.     See  Uric  acid. 
Tripahnitin,  2:53,  235 
Tripeptides,  85,  50!),  510 
Triple  phosphates,  in  urinary  concremente 
S3 1-833 
in    urinary    sediments, 
831 
Trisaccharides,  200 
Tristearin,  233,  23  1 
Trommer's  test,  214,  793,  803 
Trypsin  49,  57,  503-509 

action  of,  504,  505 
action  of  charcoal  on,  62 
digestion  action  of  foreign  bodies 

on,  506 
formation  of,  497 
preparation  of,  504 
properties  of,  503 
quantitative  estimation  of,  505 
rapidity  of,  under  various  con- 
ditions, 506 
retardation  of,  63 
tests  for.  505.    See  also  Enzymes, 
upon  other  bodies,  508 
Trvpsinogen,  504 

activation  of,  496,  497,  498 
activators  of.  497 
Tryptic  digestion,  505 

products,  of  507 
Tryptophane,  85,  111,  119,  155-157 

Adamkiewicz    -     Hopkin's 

reaction,  157 
amounts   in   proteins,    106, 

107 
effect  of  yeast  upon,  206 
quantitative,    colorimetric 
method  for,  157 
Trvptophol,  157.  206 
Tubo-ovaria,  628 
Tunicin.  838,  839 
Turacin,  843 
Turacoverdin,  844 
Turpentine,  fate  of  in  organism,  787 
Tyrosinases,  875 
Tvndall's  phenomenon,  40 
Tyrosine,   85,    109,    111,    119,    123,    124, 
152-154.  267 
amounts  in  proteins,   106,   107, 

113,  115,  125 
effect  of  yeast  upon,  206 
importance  of   in  homogentisic 

acid  formations,  737 
tests  for,  153 
Tyrosol,  153,  206 

Uffelmann's  test   for  free  lactic  acid  in 

gastric  juice,  487 
Umikoff's  reaction,  663 
Uracil.  ITS.  IS.",.  101.  195 

Weidel's  reaction  for,  104 
Wheeler  and  Johnson  reaction  for, 
194 
Uranium  salts,  and  glycosuria,  402 


1024 


GENERAL  INDEX 


Urate,  ammonium,  in  urinary  sediment,  830 
Urates,  acid,  in  urinary  sediment,  830 

of  urine,  674 
Ultra  violet  rays,  50 
Umbilical  cord,  54.5 
Urea,  267,  679,  691 

compounds  of,  687 
creatinine,  682 

.formation,  anhydride  theory,  684 
from  ammonium  salts,  6S3 
from  arginine,  682 
in  ascitic  Quids,  359 
in  blood.  333,  334 
in  cerebrospinal  fluid,  360 
in  lymph,  346 
in  muscles,  572 
in  the  organism,  682 
in  transudates  and  exudates,  355 
in  veins,  336 
mesoxalyl,  699 

mother  substances  of,  682,  683 
nitrate,  686 
occurrence  of,  679 
other  organs  besides  liver,  685 
oxalate,  687 
oxidation  theory,  684 
physiological    significance    of,    680 
preparation  of,  679,  687,  688 
properties  of,  686 

quantitative  methods  for  689-691 
quantity  voided,  68 
tests  for,  686 
Urease,  48,  829 
Urein,  691 

Ureotheobromine,  713 
Urethane,  692 
Uric  acid  stones,  832 
Uricolvsis,  704 
Uridine,  180,  185 
Urinary  calculi,  828,  829,  832-836 

calcium  carbonate,  834 
calcium  oxalate,  833 
chemical  investigation  of, 

834-836 
cystine,  834 
fibrin,  834 
phosphate,  833 
scheme   for   chemical, 

analysis  of,  836 
uric  acid,  833 
urostealith,  834 
xanthine,  834 
pigments,  740-748 
sediments,  674,  828,  829 

acid  hippuric  in,  831 
ammonium  magnesium 

phosphate  in,  831 
ammonium    urate  in. 

830 
calcium  carbonate   in, 

831 
calcium  oxalate  in,  830 
calcium  phosphate  in, 
831 


Urinary  sediments,  calcium    sulphate   in, 
831 
cystin  in,  831 
haematoidine  in,  831 
magnesium      triphos- 
phate in,  831 
non-organized,       830, 

831 
triple   phosphates   in, 

831 
tyrosine  in,  831 
urates,    acid     in,    830 
uric  acid  in,  830 
xanthine  in, 
Urine,  670-836 

abnormal  color  of,  due  to  foreign 

substances,  787 
acetoacetic  acid  in,  824,  825 
acetone  bodies  in,  818-828 
acetone  in,  822-824 
acid  fermentation  of,  829 
alcaptonuric,  test  for,  739 
alkaline   due   to    ammonium    car- 
bonate, 676 
fermentation  of,  829 
Almen's  bismuth    test    for  sugar, 

803 
amino-acids  in,  S27 
ammonium  urate  calculi,  833 
average  composition  of,  772 
Bang's   quantitative   methods   for 

sugar,  809-811 
Bertrand's     quantitative     method 

for  sugar,  811 
Bial's  test  for  pentoses,  816 
bile-acids  in,  799,  800 
bile-pigments  in,  800-801 
casual  constituents  of,  772-787 
cholesterin  in,  827 
cloudy,  reasons  for,  674 
color  of,  674 
conjugated    glucuronic    acids    in, 

817,818 
cystine  in,  827,  828 
degree  of  acidity,  675 
detection   of   acetone   and    aceto- 
acetic acid  in,  825 
Bence-Jones  protein  in, 

792 
betaoxybutyric  acid  in, 

826  " 
bile-acids  in.  800 
blood  in,  796 
conjugated  glucuronic 

acids  in,  817, 818 
fructose  in,  814 
hsematoporphvrin      in, 

798 
lactose  in,  815 
pentose  in,  816 
pigments  in,  800,  801 
proteins  in,  787-781 
proteoses  in  791,  792 
sugar  in,  802-808 


GENERAL  INDEX 


1025 


Urine,  determination  of  acidity,  070,677, 
ton  acidity,  677, 

078 
specific     gravity 
678,  079 
division   of    the   nitrogen   of,    681 
Khrlich's  urine  test,  826 
end     products     of     acids     amino 
aromatic,  780-783 
amino-aeids,       774- 

776 
benzene    ring    and 
homologues,  778, 
779 
fatty  series,  773,774 
heterocyclic     com- 
pounds, 778-787 
homocyclic       com- 
pounds, 778-787 
fat  in,  827 

fermentation  test  for  sugar,  805 
fructose  in,  814,  815 
gases  of,  857 
glucose  in,  802-814 
guaiac  test  for  blood  in,  796 
luvmatoporphyrin  in,  797,  798 
Heller-Teichmann's  test  for  blood 

in,  797 
heptose  in,  817 
indican,  728 

inorganic  constituents  of,  758-770 
inosite  in,  818 
isolation  of  sugar  from,  807 
Kjeldahl's  method  for  total  nitro- 
gen, 688 
Knapp's  quantitative  method  for 

sugar,  812 
lactose  in,  815 
laiose  in,  815 
levulose  in,  814,  815 
maltose  in,  815 

medicinal  coloring  matter  in,  801 
melanin  in.  799 
microscopic  investigation  of  blood 

corpuscles,  796 
nitrogen  content  during  starvation, 

897 
Nylander's  test  for  sugar,  803 
odor  of,  674 
organic  phvsiological  constituents 

of,  679-758 
osmotic  pressure  of,  678 
/3-oxybutyric  acid  in,  825-827 
passage  of  sugar  into,  534 
pathological  constituents  of,  787- 

828 
pentoses  in,  816 
percentage  division  of  nitrogenous 

substances,  631 
phenylhydrazine  test  for  sugar,  806 
physical  properties  of,  674r-679 
physico-chemical  methods  in,  772 
phvsiological  constituents  of,  679- 
787 


Urine,  pigments  in,  798,  799 

polarization  of,  for  sugar,  807 
pus  in,  799 

quantitative  composition  of,  770- 
277 
determination   of  su- 
gar, 808-814 
determination  of  pro- 

teid,  793 
determination  of  total 

nitrogen,  0.XK 
estimation  of  acetone 

in,  825 
estimation  of  /3-oxy- 

butyric  acid,  826 
estimation    of    sugar 
by      fermentation, 
812 
estimation    of     sugar 
by        polarization, 
813-814 
method   for  nitrogen 
in  very  small  quan- 
tities of  urine,  689 
quantity  of,  770 

conditions     affecting, 

770 
of  solids  excreted,  770, 
771 
reaction  of,  674,  675 
Rubner's  test  for  sugar,  807 
sedimentatum  lateritium,  674 
specific  gravity  of,  678 
spectroscopic      investigation      for 

blood,  796 
sugar  in,  802-818 
taste  of,  674 
transparency  of,  674 
Trommer's  test  for  sugar,  802 
urea  content  in  starvation 
Urinometer,  678 
Urobilin,  429,  516,  740,  743-746 
detection  of,  747 
formation  of  in  organism,   744 
in  blood  serum,  268 
preparation  of,  746 
properties  of,  745 
quantitative  estimation  of,  747 
tests  for,  745,  746 
Urobilinogen,  740 

detection  of  in  urine,   746 
formation  of  in  organism, 

744,  746.  747 
in  blocd  serum,  268 
preparation  of,  746,  747 
properties  of,  746 
quantitative  estimation  of, 
747 
Urobilinoids,  743 
Urochrome,  preparation  of,  742 

quantitative  estimation  of,742 
Urochrome,  740,  741,  742 
in  urine,  755 
Urocyanin,  741 


1026 


GENERAL  INDEX 


Uroerythrin,  740,  748 

tests  for,  748 
Urofuscohaematin,  798 
Uroglaucin,  741 
Urohaeniatin,  741 
Urohodin,  741 
Urohypertensin,  758 
Urohypotensin,  758 
Uromelanins,  741 
Urophain,  740 
Uropyrryl,  741 
Urorosein,  741 

in  urine,  733 
Urorubin,  741 
Urospectrin,  Saillet's,  797 
Urostealith  calculi,  834 
Urotheobromine,  713 
Urotoxic  coefficient,  758 
Urorubrohsematin,  798 
Uroxanthine,  728 
Uterine  milk,  642 
Uterus,  colloid,  627 

Valine,  111,  140, 141 

amounts  in  proteins,  106, 107, 113, 
115,  125 

Vanillin,  behavior   in   animal  body,    779 

Van  Slyke's  method  for  amino  acids,  79 

Van't  Hoff's  rule,  201 

Velocity  coefficient,  34 

Vernine,  178 

Vernix  caseosa,  845 

Vesiculase,  621,  622 

Viridinine,  47 

Visual  purple,  615-617 

preparation  of,  617 
properties  of,  616 
regeneration  of,  616 

Visual  red,  615 

Vitali's  pus-blood  test,  796 

Vitamine,  905 

Vitellin,  628 

cleavage  products  of,  106 

Vitellolutein,  632 

Vitellorubin,  632 

Vitelloses,  130 

Vitiatine,  578 

in  urine,  758 

Vitreous  humor,  617 

Voit'a  normal  average  diet,  933 

Volhard  and   Lohlein's  quantitative 
method  for  pepsin,  470 

Volhard's  quantitative  method   for 
chlorides,  760 

Walden's  reversion,  89 
Water,  importance  to  life,  71,  72 

lack  of  in  food,  899 
Wear  and  tear  quota,  890,  917 
Weidel's  reaction  for  histidine,  160 
xanthine,  190 


Weiss'  sparing  theory,  396 

Weyl's  creatinine  reaction,695 

Wheeler  and  Johnson  reaction  for  Uracil, 

194 
Whey,  645 

acid,  645 

protein,  650 

sweet,  645 
Witch's  milk,  665 
Wool-fat  constituents  of,  846 
Work  affecting  metabolism,  926-928 
Worm,  Muller's  sugar  test,  803 

Xanthine,  86, 178, 187, 188, 189,  190,  712 
in  ascitic  fluids,  359 
in  urinary  calculi,  833,  834 
in  urinary  sediments,  831 
in  urine,  710 
para,  187 

Seyler's  reaction  for,  190 
Weidel's  reaction  for,  190 

Xantho  creatinine,  578 

in  urine,  698 

Xantho  melanin,  84 

Aanthoproteic  reaction  for  proteins,   99, 
173 

Xanthoprotein,  84 

Xanthophan,  617 

Xanthosine,  180 

Xylene,  fate  of  in  organism,  779 

Xyliton,  842 

Xyloses,  structural  formula  for,  198,  210 

Yeast,  59,  62 

action  of,  on  glucose,  226 
maltase,  266 
Yoghurt,  654,  659 
Yolk,  constituents  of,  628 

fat  of,  630 

membrane,  628 

mineral  bodies  of,  632 

of  hen's  egg,  628 

pigments  of,  631 

phosphatides  of,  630 

proteins  of,  628-630 

spherules,  628,  636 

Zein,  77,  105,  163 
Zinc,  in  the  bile,  433 

in  the  liver,  389 

passage  into  milk,  670 
Zooerythrin,  843 
Zoofulvin,  843 
Zoorubin,  843 

Zuntz   and   Geppert's  method  for  deter- 
mining respiratory  exchange,  869 
Zymase,  41 
Zymogen,  rennin,  474 
Zymogens,  51,  461 
Zymoplastic  substances  in  blood,  315 


COLUMBIA  UNIVERSITY  LIBRARY 

This  book  is  due  on  the  date  indicated  below,  or  at  the 
expiration  of  a  definite  period  after  the  date  of  borrowing, 
as  provided  by  the  rules  of  the  Library  or  by  special  ar- 
rangement with  the  Librarian  in  charge. 

DATE  BORROWED 

DATE  DUE 

DATE  BORROWED 

DATE  DUE 

MEC*' 

i;i$i& 

C28(23S)M100 

JUN  3  0  1920 


=**~  Annex 

/  \  _    /-   x/_ 


